How a G protein binds a membrane

Heterotrimeric G proteins interact with receptors and effectors at the membrane-cytoplasm interface. Structures of soluble forms have not revealed how they interact with membranes. We have used electron crystallography to determine the structure in ice of a helical array of the photoreceptor G protein, transducin, bound to the surface of a tubular lipid bilayer. The protein binds to the membrane with a very small area of contact, restricted to two points, between the surface of the protein and the surface of the lipids. Fitting the x-ray structure into the membrane-bound structure reveals one membrane contact near the lipidated Ggamma C terminus and Galpha N terminus, and another near the Galpha C terminus. The narrowness of the tethers to the lipid bilayer provides flexibility for the protein to adopt multiple orientations on the membrane, and leaves most of the G protein surface area available for protein-protein interactions.     

From a homeostatic to a homeodynamic self

Life as an autonomous homeostatic system is discussed. A mechanism that drives a homeostatic state to an autonomous self-moving state is examined with two computational cell models. The mechanism is met with Ashby's ultrastability, where random parameter searching is activated when a system breaks a viability constraint. Such a random search process is replaced by the membrane shape in the first model and by chaotic population dynamics in the second model. Emergence of sensors, motors and the recursive coupling between them is shown to be a natural outcome of an autonomous homeostatic system.     

Testing a Primary and a Secondary Endpoint in a Group Sequential Design

Summary We consider a clinical trial with a primary and a secondary endpoint where the secondary endpoint is tested only if the primary endpoint is significant. The trial uses a group sequential procedure with two stages. The familywise error rate (FWER) of falsely concluding significance on either endpoint is to be controlled at a nominal level α. The type I error rate for the primary endpoint is controlled by choosing any α‐level stopping boundary, e.g., the standard O'Brien–Fleming or the Pocock boundary. Given any particular α‐level boundary for the primary endpoint, we study the problem of determining the boundary for the secondary endpoint to control the FWER. We study this FWER analytically and numerically and find that it is maximized when the correlation coefficient ρ between the two endpoints equals 1. For the four combinations consisting of O'Brien–Fleming and Pocock boundaries for the primary and secondary endpoints, the critical constants required to control the FWER are computed for different values of ρ. An ad hoc boundary is proposed for the secondary endpoint to address a practical concern that may be at issue in some applications. Numerical studies indicate that the O'Brien–Fleming boundary for the primary endpoint and the Pocock boundary for the secondary endpoint generally gives the best primary as well as secondary power performance. The Pocock boundary may be replaced by the ad hoc boundary for the secondary endpoint with a very little loss of secondary power if the practical concern is at issue. A clinical trial example is given to illustrate the methods.     

Parameter determination for a computer simulation model of a diver and a springboard

This study used kinematic data on springboard diving performances to estimate viscoelastic parameters of a planar model of a springboard and diver with wobbling masses in the trunk, thigh, and calf segments and spring dampers acting at the heel, ball, and toe of the foot segment. A subject-specific angle-driven eight-segment model was used with an optimization algorithm to determine viscoelastic parameter values by matching simulations to four diving performances. Using the parameters determined from the matching of a single dive in a simulation of another dive resulted in up to 31% difference between simulation and performance, indicating the danger of using too small a set of kinematic data. However, using four dives in a combined matching process to obtain a common set of parameters resulted in a mean difference of 8.6%. Because these four dives included very different rotational requirements, it is anticipated that the combined parameter set can be used with other dives from these two groups.     

Determining a maximum-tolerated schedule of a cytotoxic agent

Most phase I clinical trials are designed to determine a maximum-tolerated dose (MTD) for one initial administration or treatment course of a cytotoxic experimental agent. Toxicity usually is defined as the indicator of whether one or more particular adverse events occur within a short time period from the start of therapy. However, physicians often administer an agent to the patient repeatedly and monitor long-term toxicity due to cumulative effects. We propose a new method for such settings. It is based on the time to toxicity rather than a binary outcome, and the goal is to determine a maximum-tolerated schedule (MTS) rather than a conventional MTD. The model and method account for a patient's entire sequence of administrations, with the overall hazard of toxicity modeled as the sum of a sequence of hazards, each associated with one administration. Data monitoring and decision making are done continuously throughout the trial. We illustrate the method with an allogeneic bone marrow transplantation (BMT) trial to determine how long a recombinant human growth factor can be administered as prophylaxis for acute graft-versus-host disease (aGVHD), and we present a simulation study in the context of this trial.     

Antagonistic coevolution between a bacterium and a bacteriophage

Antagonistic coevolution between hosts and parasites is believed to play a pivotal role in host and parasite population dynamics, the evolutionary maintenance of sex and the evolution of parasite virulence. Furthermore, antagonistic coevolution is believed to be responsible for rapid differentiation of both hosts and parasites between geographically structured populations. Yet empirical evidence for host-parasite antagonistic coevolution, and its impact on between-population genetic divergence, is limited. Here we demonstrate a long-term arms race between the infectivity of a viral parasite (bacteriophage; phage) and the resistance of its bacterial host. Coevolution was largely driven by directional selection, with hosts becoming resistant to a wider range of parasite genotypes and parasites infective to a wider range of host genotypes. Coevolution followed divergent trajectories between replicate communities despite establishment with isogenic bacteria and phage, and resulted in bacteria adapted to their own, compared with other, phage populations.     

Transformation of a psychrotrophic bacterium with a plasmid from a mesophile

The psychrotrophic bacteriumBacillus psychrophilus was transformed with the broadhost-range plasmid pC194. The ability of the transformant to express chloramphenicol (CAM) resistance and the possible effects of such expression on the physiology of the psychrotroph were examined. The transformant exhibited growth rates, filament formation at elevated temperatures, synthesis of cold shock proteins and cold acclimation proteins, similar to the parentalB. psychrophilus.     

Eo: a history of a mutation

Eighteen mouse t haplotype-carrying strains were found not to express cell-surface E molecules controlled by class II genes of the H-2 complex (= Eo strains). Northern and Southern blot analysis of these and other, non-t strains that also fail to express the E molecule, has revealed two kinds of defect. Three strains (CRO437, tw2, and presumably to) were found to transcribe the E alpha gene, but they were not able to convert the message into a functional protein. All other Eo strains fail to transcribe the E alpha gene because of a deletion encompassing the promoter region, the RNA initiation site, and the first exon. The length of the deletion is approximately 650 +/- 50 bp. These two defects closely resemble those found previously in standard inbred strains carrying the H-2f, H-2q (failure of E mRNA to be expressed functionally), H-2b, and H-2s (deletion of a part of the E alpha gene) haplotypes. In particular, the location and length of the E alpha deletion appear to be the same in the strains carrying this mutation. The E alpha deletion is in linkage disequilibrium with certain alleles at other H-2 loci in some of the strains. These observations, combined with the growing evidence that H-2 haplotypes associated with t chromosomes derive from a single ancestral haplotype, suggest that the E alpha deletion is an old mutation and that it has been disseminated in mouse populations by the t chromosomes.     

Cellular events in the reestablishment of a symbiosis between a marine dinoflagellate and a coelenterate

Summary Within 24 h after the initial phagocytotic uptake of freshly isolated (from host tissue) symbiotic algae (Symbiodinium microadriaticum) by the endodermal cells of the polyp (scyphistoma) stage of the jellyfish Cassiopeia xamachana, the algal population was observed to decline despite evidence of algal cell division. Analyses of the frequency of phago-lysosome fusion as an indicator of possible attempts of the host to digest the algae indicated that, although phago-lysosome fusion did occur, the low frequency of occurrence is inconsistent with the interpretation that the animals digested the algae. Animal cell lysosomes were located predominantly at the apices of the endodermal cells, and the symbiotic algae were transported toward the bases of the endodermal cells.Within 3 days after initial infection, most endodermal cells with algae ceased to be phagocytotically active (with respect to the uptake of carmine particles). Many of these endodermal cells soon migrated into the mesoglea to become what are traditionally referred to as amoebocytes. Within amoebocytes the algae proliferated. The onset of strobilation by the scyphistomae was directly correlated with the increase in the algal population within these amoebocytes.     

Ventilation of a rat with a large-animal respirator

Rats were effectively ventilated with 100% O2 mixed with room air utilizing a modified tracheostomy tube and a Bird Mark 7 respirator to maintain arterial blood gases within normal limits. A 3-cm segment of rubber pilot tubing was attached to a 15-mm respiratory connector and a 3-cm piece of polyethylene catheter tubing was fitted snugly into the other end. The catheter was inserted and secured into the trachea of 250- to 500-g Sprague-Dawley rats with the adaptor hose of the respirator fitted onto the 15-mm connector following tracheostomy. Manometer and inspiratory flow rate controls of the respirator were set to their minimum operating position. Appropriate rate control adjustments were made when necessary as determined by arterial blood gas measurements. By use of the above ventilation system, adequate arterial blood gases of anesthesized rats can be maintained for greater than 3 h.     

Matching a surface to a given sectional curve

We describe an algorithm to position a rigid surface so as to make its cross-section by a given plane match a given curve in that plane, a problem relevant to model-based medical imaging. After building an atlas of cross-sections of the surface and searching it for a best position to start from, each iteration of the algorithm (1) determines a vector field along the intersection curve that will improve its matching with the target curve, and (2) computes and applies a small displacement of the surface whose effect on the intersection will approximate best the required vector field. Computations use least-square techniques, an exponential formula for Lie groups of transformations, and generic properties of cross-sections. Experiments with an implementation are reported and theoretical tools for justifying and improving the algorithm, some of them based on Catastrophe Theory, are outlined.     

Primary hypothyroidism in a child mimicking a pituitary macroadenoma

We report the case of an 11-year-old girl with primary autoimmune hypothyroidism causing secondary pituitary enlargement. She presented with headaches and a pituitary mass on MRI thought to be due to a pituitary macroadenoma. Resolution of the pituitary mass and symptoms occurred with thyroxine therapy. It is mandatory to rule out primary hypothyroidism as a cause of pituitary enlargement before surgery is considered.     

Aspergillus myositis in a patient with a myelodysplastic syndrome

We present the case of an elderly man who, while being treated with corticosteroids for a myelodysplastic syndrome, developed myositis of the calf due to Aspergillus fumigatus. Despite therapy with amphotericin B the myositis failed to resolve and he died. At autopsy, a localized necrotizing myositis of the right calf was found with no evidence of disseminated Aspergillus infection. Myositis in the setting of disseminated candidiasis or cryptococcosis has been previously reported. This case is unique in that it is the first reported case of localized fungal myositis and of myositis caused by Aspergillus.     

Pollinator rarity as a threat to a plant with a specialized pollination system

An increasing diversity of highly specialized pollination systems are being discovered, many of which are likely to be vulnerable to anthropogenic landscape modification. Here, we investigate if a specialized pollination system limits the persistence of Caladenia huegelii (Orchidaceae), an endangered species pollinated by sexual deception of thynnine wasps. Once locally common in part of its geographical range, C. huegelii is now largely restricted to small habitat remnants in urban areas. Pollinator surveys coupled with DNA barcoding detected a single pollinator taxon, a small form of Macrothynnus insignis. Phylogenetic analysis revealed that small M. insignis from within the range of C. huegelii are strongly divergent from other wasp populations, suggesting that some reproductive isolation may exist. Although common in intact landscapes outside the range of C. huegelli, small M. insignis individuals were recorded at only 4% of sites in suitable C. huegelii habitat. Accordingly, reproductive success in C. huegelii was low compared with related Caladenia spp., with 33–60% of populations failing to set fruit in any given year. As such, populations are likely to now persist primarily through individual plant longevity rather than reproduction. Due to the low reproductive success of C. huegelii, ongoing human intervention will almost certainly be needed to sustain the species. Future research will need to focus on optimizing hand pollination to maintain reproduction and high seed fitness. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 511–525.     

Characterization of a neuraminidase-deficient influenza a virus as a potential gene delivery vector and a live vaccine

We recently identified a packaging signal in the neuraminidase (NA) viral RNA (vRNA) segment of an influenza A virus, allowing us to produce a mutant virus [GFP(NA)-Flu] that lacks most of the NA open reading frame but contains instead the gene encoding green fluorescent protein (GFP). To exploit the expanding knowledge of vRNA packaging signals to establish influenza virus vectors for the expression of foreign genes, we studied the replicative properties of this virus in cell culture and mice. Compared to wild-type virus, GFP(NA)-Flu was highly attenuated in normal cultured cells but was able to grow to a titer of >10(6) PFU/ml in a mutant cell line expressing reduced levels of sialic acid on the cell surface. GFP expression from this virus was stable even after five passages in the latter cells. In intranasally infected mice, GFP was detected in the epithelial cells of nasal mucosa, bronchioles, and alveoli for up to 4 days postinfection. We attribute the attenuated growth of GFP(NA)-Flu to virion aggregation at the surface of bronchiolar epithelia. In studies to test the potential of this mutant as a live attenuated influenza vaccine, all mice vaccinated with >/==" BORDER="0">10(5) PFU of GFP(NA)-Flu survived when challenged with lethal doses of the parent virus. These results suggest that influenza virus could be a useful vector for expressing foreign genes and that a sialidase-deficient virus may offer an alternative to the live influenza vaccines recently approved for human use.     

Defining a monophyletic Cardinalini: a molecular perspective

Within the New World nine-primaried oscine assemblage, feeding morphology and behavior have long been used as a guideline for assigning membership to subgroups. For example, birds with stout, conical bills capable of crushing heavy seeds have generally been placed within the tribe Cardinalini (cardinal-grosbeaks). Many workers have tried to characterize this group more definitively, using a variety of morphological characters; however, the characters used often conflicted with one another. Previous molecular studies addressing the monophyly of Cardinalini have had only limited sampling within the group. In this study, we analyze mtDNA sequence data from all genera and 34 of the 42 Cardinalini species (sensu [Sibley, C.G., Monroe, B.L., 1990. Distribution and Taxonomy of the Birds of the World, Yale University Press, New Haven, CT]) to address the monophyly of the group and to reconstruct the most complete phylogeny of this tribe published to date. We found strong support for a redefined Cardinalini that now includes some members previously placed within Thraupini (tanagers; the genera Piranga, Habia, Chlorothraupis, and Amaurospiza) and some members previously placed within the Parulini (wood-warblers; the genus Granatellus). In addition, some genera traditionally considered members of the Cardinalini are shown to have affinities with other groups (the genera Porphyrospiza, Parkerthraustes, and Saltator). Our redefined Cardinalini contains 48 species, six more than are listed in Sibley and Monroe's (1990) taxonomy of the group. Within the nine-primaried oscine assemblage, the Cardinalini are more closely related to the Thraupini (tanagers) than they are to the Emberizini (sparrows), Parulini (wood-warblers), or Icterini (blackbirds), consistently forming a monophyletic group with Thraupini across all analyses. The reconfigured Cardinalini is comprised of five well-supported, major clades: (1) a "masked" clade (Piranga, Cardinalis, Caryothraustes, Periporphyrus, and Rhodothraupis), (2) a "blue" clade (Amaurospiza, Cyanocompsa, Cyanoloxia, Passerina, and Spiza), (3) a clade containing the genera Habia and Chlorothraupis, (4) a clade containing all species of Granatellus, and (5) a clade containing only species of Pheucticus. Diversification of these five lineages from one another occurred relatively rapidly during the mid-Pliocene, around 5 or 8 million years ago. Each of these major clades includes both North and South American species; thus, a complex biogeographic history is inferred for the group.     

Active subtilisin-like protease from a hyperthermophilic archaeon in a form with a putative prosequence

The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly(-82) to Gly(316)), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca(2+) ion with an optimal pH and temperature of pH 9.5 and 80 degrees C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80 degrees C, 20 min at 90 degrees C, and 7 min at 100 degrees C.     

Drugging a Stem Cell Compartment Using Wnt3a Protein as a Therapeutic

The therapeutic potential of Wnt proteins has long been recognized but challenges associated with in vivo stability and delivery have hindered their development as drug candidates. By exploiting the hydrophobic nature of the protein we provide evidence that exogenous Wnt3a can be delivered in vivo if it is associated with a lipid vesicle. Recombinant Wnt3a associates with the external surface of the lipid membrane; this association stabilizes the protein and leads to prolonged activation of the Wnt pathway in primary cells. We demonstrate the consequences of Wnt pathway activation in vivo using a bone marrow engraftment assay. These data provide validation for the development of WNT3A as a therapeutic protein.     

Myocilin, a component of a membrane-associated protein complex driven by a homologous Q-SNARE domain

Myocilin is a widely expressed protein with no known function; however, mutations in myocilin appear to manifest uniquely as ocular hypertension and the blinding disease of glaucoma. Using the protein homology/analogy recognition engine (Phyre), we find that the olfactomedin domain of myocilin is similar in sequence motif and structure to a six-blade, kelch repeat motif based on the known crystal structures of such proteins. Additionally, using sequence analysis, we identify a coiled-coil segment of myocilin with homology to human Q-SNARE proteins (inset). Using COS-7 cells expressing full-length human myocilin and a version lacking the C-terminal olfactomedin domain, we identified a membrane-associated protein complex containing myocilin by hydrodynamic analysis. The myocilin construct that included the coiled-coil but lacked the olfactomedin domain formed complexes similar to the full-length protein, indicating that the coiled-coil domain of myocilin is sufficient for myocilin binding to the large detergent-resistant complex. In human retina and retinal pigment epithelium, which express myocilin, we detected the protein in a large, sodium dodecyl sulfate-resistant, membrane-associated complex. We characterized myocilin in human tissues as either a 15 S complex with an M(r) of 405000-440000 yielding a slightly elongated globular shape similar to that of known SNARE complexes or a 6.4 S dimer with an M(r) of 108000. By identifying the Q-SNARE homology within the second coil of myocilin and documenting its participation in a SNARE-like complex, we provide evidence of a SNARE domain-containing protein associated with a human disease.