Characterization of an aerosolized nanoparticle beam beyond the diffraction limit through strong field ionization.

The study of nanomaterials is an active area of research for technological applications as well as fundamental science. A common method for studying properties of isolated nanoparticles is by an in-vacuum particle beam produced via an aerodynamic lens. Despite being common practice, characterization of such beams has proven difficult as light scattering detection techniques fail for particles with sizes beyond the diffraction limit. Here we present a new technique for characterizing such nanoparticle beams using strong field ionization. By focusing an ultrafast, mJ-level laser into the particle beam, a nanoparticle within the laser focus is ionized and easily detected by its ejected electrons. This method grants direct access to the nanoparticle density at the location of the focus, and by scanning the focus through the transverse and longitudinal profiles of the particle beam we attain the 3-dimensional particle density distribution for a cylindrically symmetric beam. Further, we show that strong field ionization is effective in detecting spherical nanoparticles as small as 10 nm in diameter. Additionally, this technique is an effective tool in optimizing the particle beam for specific applications. As an example we show that the particle beam density and width can be manipulated by restricting the gas flow into the aerodynamic lens.

Genome-wide SNPs resolve phylogenetic relationships in the North American spruce budworm (Choristoneura fumiferana) species complex.

High throughput sequencing technologies have revolutionized the potential to reconcile incongruence between gene and species trees, and numerous approaches have been developed to take advantage of these advances. Genotyping-by-sequencing is becoming a regular tool for gathering phylogenetic data, yet comprehensive evaluations of phylogenetic methods using these data are sparse. Here we use multiple phylogenetic and population genetic methods for genotyping-by-sequencing data to assess species relationships in a group of forest insect pests, the spruce budworm (Choristoneura fumiferana) species complex. With few exceptions, all methods agree on the same relationships, most notably placing C. pinus as basal to the remainder of the group, rather than C. fumiferana as previously suggested. We found strong support for the monophyly of C. pinus, C. fumiferana, and C. retiniana, but more ambiguous relationships and signatures of introgression in a clade of western lineages, including C. carnana, C. lambertiana, C. occidentalis occidentalis, C. occidentalis biennis, and C. orae. This represents the most taxonomically comprehensive genomic treatment of the spruce budworm species group, which is further supported by the broad agreement among multiple methodologies.

CIB1 contributes to oncogenic signalling by Ras via modulating the subcellular localisation of sphingosine kinase 1.

CIB1 (calcium and integrin binding protein 1) is a small intracellular protein with numerous interacting partners, and hence has been implicated in various cellular functions. Recent studies have revealed emerging roles of CIB1 in regulating cancer cell survival and angiogenesis, although the mechanisms involved have remained largely undefined. In investigating the oncogenic function of CIB1, we initially found that CIB1 is widely up-regulated across a diverse range of cancers, with this upregulation frequently correlating with oncogenic mutations of KRas. Consistent with this, we found that ectopic expression of oncogenic KRas and HRas in cells resulted in elevated CIB1 expression. We previously described the Ca2+-myristoyl switch function of CIB1, and its ability to facilitate agonist-induced plasma membrane localisation of sphingosine kinase 1 (SK1), a location where SK1 is known to elicit oncogenic signalling. Thus, we examined the role this may play in oncogenesis. Consistent with these findings, we demonstrated here that over-expression of CIB1 by itself is sufficient to drive localisation of SK1 to the plasma membrane and enhance the membrane-associated enzymatic activity of SK1, as well as its oncogenic signalling. We subsequently demonstrated that elevated levels of CIB1 resulted in full neoplastic transformation, in a manner dependent on SK1. In agreement with our previous findings that SK1 is a downstream mediator of oncogenic signalling by Ras, we found that targeting CIB1 also inhibited neoplastic growth of cells induced by oncogenic Ras, suggesting an important pro-tumorigenic role for CIB1. Thus, we have demonstrated for the first time a role for CIB1 in neoplastic transformation, and revealed a novel mechanism facilitating oncogenic signalling by Ras and SK1.

Sustained inhibition of STAT5, but not JAK2, is essential for TKI-induced cell death in chronic myeloid leukemia.

Kinase inhibitors block proliferative signals in BCR-ABL1+ leukemic cells, but their capacity to induce apoptosis is poorly understood. Initial studies suggested that very brief exposure to kinase inhibitors was sufficient to induce apoptosis in chronic myeloid leukemia (CML) cells. However, flaws in this experimental model have subsequently been identified, leading to the conclusion that apoptosis only occurs with sustained low-level kinase inhibition. Thus, the minimum duration of complete kinase inhibition required to commit CML cells to death is unknown. Here we confirm that <1 h is insufficient to induce significant commitment to death in BCR-ABL1+ cell lines and in primary CD34+ progenitor cells, and establish that commitment to cell death only occurs if kinase inhibition is maintained for 4 h or more. Remarkably, signal transducer and activator of transcription 5 (STAT5) inhibition in combination with transient (<1 h) tyrosine kinase inhibitor (TKI) exposure proved lethal for CML progenitors, despite the reactivation of Bcr-Abl after 1 h. JAK kinase inhibition did not induce cell death in combination with transient TKI exposure. Thus, STAT5 appears to be a critical determinant of the time-dependent sensitivity of CML progenitor cells to TKI treatment in a Bcr-Abl-dependent, but JAK-independent, manner. We conclude that combining kinase inhibition with STAT5 inhibition represents a promising therapeutic approach in BCR-ABL1+ leukemias.

Enhanced expression of transferrin receptor 1 contributes to oncogenic signalling by sphingosine kinase 1.

Sphingosine kinase 1 (SK1) is a lipid kinase that catalyses the formation of sphingosine-1-phosphate (S1P). Considerable evidence has implicated elevated cellular SK1 in tumour development, progression and disease severity. In particular, SK1 has been shown to enhance cell survival and proliferation and induce neoplastic transformation. Although S1P has been found to have both cell-surface G-protein-coupled receptors and intracellular targets, the specific downstream pathways mediating oncogenic signalling by SK1 remain poorly defined. Here, using a gene expression array approach, we have demonstrated a novel mechanism whereby SK1 regulates cell survival, proliferation and neoplastic transformation through enhancing expression of transferrin receptor 1 (TFR1). We showed that elevated levels of SK1 enhanced total as well as cell-surface TFR1 expression, resulting in increased transferrin uptake into cells. Notably, we also found that SK1 activation and localization to the plasma membrane, which are critical for its oncogenic effects, are necessary for regulation of TFR1 expression specifically through engagement of the S1P G-protein coupled receptor, S1P2. Furthermore, we showed that blocking TFR1 function with a neutralizing antibody inhibits SK1-induced cell proliferation, survival and neoplastic transformation of NIH3T3 fibroblasts. Similar effects were observed following antagonism of S1P2. Together these findings suggest that TFR1 has an important role in SK1-mediated oncogenesis.

Pattern formation in a model for mountain pine beetle dispersal: linking model predictions to data.

Pattern formation occurs in a wide range of biological systems. This pattern formation can occur in mathematical models because of diffusion-driven instability or due to the interaction between reaction, diffusion, and chemotaxis. In this paper, we investigate the spatial pattern formation of attack clusters in a system for Mountain Pine Beetle. The pattern formation (aggregation) of the Mountain Pine Beetle in order to attack susceptible trees is crucial for their survival and reproduction. We use a reaction-diffusion equation with chemotaxis to model the interaction between Mountain Pine Beetle, Mountain Pine Beetle pheromones, and susceptible trees. Mathematical analysis is utilized to discover the spacing in-between beetle attacks on the susceptible landscape. The model predictions are verified by analysing aerial detection survey data of Mountain Pine Beetle Attack from the Sawtooth National Recreation Area. We find that the distance between Mountain Pine Beetle attack clusters predicted by our model closely corresponds to the observed attack data in the Sawtooth National Recreation Area. These results clarify the spatial mechanisms controlling the transition from incipient to epidemic populations and may lead to control measures which protect forests from Mountain Pine Beetle outbreak.

The role of temperature variability in stabilizing the mountain pine beetle-fungus mutualism.

As global climate patterns continue to change and extreme weather events become increasingly common, it is likely that many ecological interactions will be affected. One such interaction is the multipartite symbiosis that exists between the mountain pine beetle and two species of fungi, Grosmannia clavigera and Ophiostoma montium. In this mutualism, the fungi provide nutrition to the beetle, while the fungi benefit by being dispersed to new host trees. Multi-partite mutualisms are predicted to be unstable due to strong direct competition among symbionts or natural selection for superior over inferior mutualists. However, this mutualism has remained stable over long periods of evolutionary time. In this paper, we developed a temperature-based model for the spread of fungi within a tree and connected it to an existing model for mountain pine beetle development. Using this integrated model for fungal growth, we explored the possibility that temperature variability is a stabilizing mechanism for the mountain pine beetle-fungi mutualism. Of the three types of temperature variability we tested: intra-year, inter-year and variability due to transitioning between different thermal habitats (thermal migration), we found that thermal migration was the most robust stabilizing mechanism. Additionally, we found that the MPB attack density or spacing between fungal lesions also had a significant effect on the stability of the system. High attack densities or close lesion spacings also tended to stabilize the system, regardless of temperature.

Evolution stabilises the synchronising dynamics of poikilotherm life cycles.

Temperature is the most significant factor controlling developmental timing of most temperate poikilotherms. In the face of climate change, a crucial question is how will poikilothermic organisms evolve when faced with changing thermal environments? In this paper, we integrate models for developmental timing and quantitative genetics. A simple model for determining developmental milestones (emergence times, egg hatch) is introduced, and the general quantitative genetic recursion for the mean value of developmental parameters presented. Evolutionary steps proportional to the difference between current median parameters and parameters currently selected for depend on the fitness, which is assumed to depend on emergence density. Asymptotic states of the joint model are determined, which turn out to be neutrally stable (marginal) fixed points in the developmental model by itself, and an associated stable emergence distribution is also described. An asymptotic convergence analysis is presented for idealized circumstances, indicating basic stability criteria. Numerical studies show that the stability analysis is quite conservative, with basins of attraction to the asymptotic states that are much larger than expected. It is shown that frequency-dependent selection drives oscillatory dynamics and that the asymptotic states balance the asymmetry of the emergence distribution and the fitness function.

Blocking cytokine signaling along with intense Bcr-Abl kinase inhibition induces apoptosis in primary CML progenitors.

In chronic myeloid leukemia (CML) cell lines, brief exposure to pharmacologically relevant dasatinib concentrations results in apoptosis. In this study, we assess the impact of intensity and duration of Bcr-Abl kinase inhibition on primary CD34(+) progenitors of chronic phase CML patients. As CML cells exposed to dasatinib in vivo are in a cytokine-rich environment, we also assessed the effect of cytokines (six growth factors cocktail or granulocyte-macrophage colony-stimulating factor (CSF) or granulocyte-CSF) in combination with dasatinib. In the presence of cytokines, short-term intense Bcr-Abl kinase inhibition (>or=90% p-Crkl inhibition) with 100 nM dasatinib did not reduce CD34(+) colony-forming cells (CFCs). In contrast, without cytokines, short-term exposure to dasatinib reduced CML-CD34(+) CFCs by 70-80%. When cytokines were added immediately after short-term exposure to dasatinib, CML-CD34(+) cells remained viable, suggesting that oncogene dependence of these cells can be overcome by concomitant or subsequent exposure to cytokines. Additional inhibition of Janus tyrosine kinase (Jak) activity re-established the sensitivity of CML progenitors to intense Bcr-Abl kinase inhibition despite the presence of cytokines. These findings support the contention that therapeutic strategies combining intense Bcr-Abl kinase inhibition and blockade of cytokine signaling pathways can be effective for eradication of CML progenitors.

'Up and coming' treatments for premature ejaculation: progress towards an approved therapy.

It is generally accepted that premature ejaculation (PE) is a more common problem than erectile dysfunction, although at present the options currently available for the treatment of PE are limited to behavioural psychotherapy and 'off-label' prescribing of pharmacological therapies. A sexual complaint with such a high prevalence together with an increasing understanding of the psychosocial consequences of PE has naturally stimulated the interest of the pharmaceutical industry and the first products designed specifically for the treatment of PE are either in late-stage clinical development or are already under regulatory review. Most of the new treatments for PE have been developed for 'on-demand' use, which may prove to offer the most favourable risk: benefit profile as well as the flexibility to adapt to differing frequencies of sexual activity. This paper reviews a number of emerging therapies in various stages of development that show potential for use in the treatment of PE.

A critical analysis of the literature on the Internet and consumer health information.

A critical review of the published literature investigating the Internet and consumer health information was undertaken in order to inform further research and policy. A qualitative, narrative method was used, consisting of a three-stage process of identification and collation, thematic coding, and critical analysis. This analysis identified five main themes in the research in this area: (1) the quality of online health information for consumers; (2) consumer use of the Internet for health information; (3) the effect of e-health on the practitioner-patient relationship; (4) virtual communities and online social support and (5) the electronic delivery of information-based interventions. Analysis of these themes revealed more about the concerns of health professionals than about the effect of the Internet on users. Much of the existing work has concentrated on quantifying characteristics of the Internet: for example, measuring the quality of online information, or describing the numbers of users in different health-care settings. There is a lack of qualitative research that explores how citizens are actually using the Internet for health care.

Modeling Spatial Characteristics in the Biological Control of Fungi at Leaf Scale: Competitive Substrate Colonization by Botrytis cinerea and the Saprophytic Antagonist Ulocladium atrum.

ABSTRACT A spatially explicit model describing saprophytic colonization of dead cyclamen leaf tissue by the plant-pathogenic fungus Botrytis cinerea and the saprophytic fungal antagonist Ulocladium atrum was constructed. Both fungi explore the leaf and utilize the resources it provides. Leaf tissue is represented by a two-dimensional grid of square grid cells. Fungal competition within grid cells is modeled using Lotka-Volterra equations. Spatial expansion into neighboring grid cells is assumed proportional to the mycelial density gradient between donor and receptor cell. Established fungal biomass is immobile. Radial growth rates of B. cinerea and U. atrum in dead cyclamen leaf tissue were measured to determine parameters describing the spatial dynamics of the fungi. At temperatures from 5 to 25 degrees C, B. cinerea colonies expanded twice as rapidly as U. atrum colonies. In practical biological control, the slower colonization of space by U. atrum thus needs to be compensated by a sufficiently dense and even distribution of conidia on the leaf. Simulation results confirm the importance of spatial expansion to the outcome of the competitive interaction between B. cinerea and U. atrum at leaf scale. A sensitivity analysis further emphasized the importance of a uniform high density cover of vital U. atrum conidia on target leaves.

Inhibition of angiogenesis by antibody blocking the action of proangiogenic high-molecular-weight kininogen.

Previously we demonstrated that domain 5 (D5) of high-molecular-weight kininogen (HK) inhibits neovascularization in the chicken chorioallantoic membrane (CAM) assay and further found that kallikrein cleaved HK (HKa) inhibited FGF2-and VEGF-induced neovascularization, and thus was antiangiogenic. In this study, we sought to demonstrate whether uncleaved HK stimulates neovascularization and thus is proangiogenic. The chick chorioallantoic membrane was used as an in ovo assay of angiogenesis. Low-molecular-weight kininogen stimulates angiogenesis, indicating that D5 is not involved. Bradykinin stimulates neovascularization equally to HK and LK and is likely to be responsible for the effect of HK. A murine monoclonal antibody to HK (C11C1) also recognizes a similar component in chicken plasma as detected by surface plasmon resonance. Angiogenesis induced by FGF2 and VEGF is inhibited by this monoclonal antibody and is a more potent inhibitor of neovascularization induced by VEGF than an integrin alphavbeta3 antibody (LM 609). Our postulate that C11C1 inhibits the stimulation of angiogenesis by HK was confirmed when either C11C1 or D5 completely inhibited angiogenesis in the CAM induced by HK. Growth of human fibrosarcoma (HT-1080) on the CAM was inhibited by GST-D5 and C11C1. These results indicate HK is proangiogenic probably by releasing bradykinin and that a monoclonal antibody directed to HK could serve as an antiangiogenic agent with a potential for inhibiting tumor angiogenesis and other angiogenesis-mediated disorders.

IP(3) receptor function and localization in myotubes: an unexplored Ca(2+) signaling pathway in skeletal muscle.

We present evidence for an unexplored inositol 1,4,5-trisphosphate-mediated Ca(2+) signaling pathway in skeletal muscle. RT-PCR methods confirm expression of all three known isotypes of the inositol trisphosphate receptor in cultured rodent muscle. Confocal microscopy of cultured mouse muscle, doubly labeled for inositol receptor type 1 and proteins of known distribution, reveals that the receptors are localized to the I band of the sarcoplasmic reticulum, and this staining is continuous with staining of the nuclear envelope region. These results suggest that the receptors are positioned to mediate a slowly propagating Ca(2+) wave that follows the fast Ca(2+) transient upon K(+) depolarization. This slow wave, imaged using fluo-3, resulted in an increase in nucleoplasmic Ca(2+) lasting tens of seconds, but not contraction; the slow wave was blocked by both the inositol trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate and the phospholipase C inhibitor U-73122. To test the hypothesis that these slow Ca(2+) signals are involved in signal cascades leading to regulation of gene expression, we assayed for early effects of K(+) depolarization on mitogen-activated protein kinases, specifically extracellular-signal related kinases 1 and 2 and the transcription factor cAMP response element-binding protein (CREB). Within 30-60 seconds following depolarization, phosphorylation of both the kinases and CREB was evident and could be inhibited by 2-aminoethoxydiphenyl borate. These results suggest a signaling system mediated by Ca(2+) and inositol trisphosphate that could regulate gene expression in muscle cells.

Low seasonal temperatures promote life cycle synchronization.

In this paper we discuss how seasonal temperature variation and life-stage specific developmental thresholds that cause quiescence can synchronize the seasonal development of exothermic organisms. Using a simple aging model it is shown that minimal seasonal temperature variation and periods of quiescence during extreme temperature conditions are sufficient to establish stable, univoltine ovipositional cycles. Quiescence induced by life-stage specific developmental thresholds, in fact, promotes synchronous oviposition and emergence. The mountain pine beetle, an important insect living in extreme temperature conditions and showing no evidence of diapause, invites direct application of this model. Simulations using mountain pine beetle parameters are used to determine temperature regimes for which stable ovipositional cycles exist.

Maternal and child health nutrition training builds leadership skills.

For more than 30 years, the Maternal and Child Health Bureau (MCHB) has funded specialized professional training in nutrition with an emphasis on the development of leadership skills. A survey was conducted of individuals who received Maternal and Child Health nutrition training at the Center for Human Development and Disability (CHDD) at the University of Washington. Those surveyed were asked to reflect on the impact of the training on their career pathway and to report on their leadership activities. Of the 91 respondents, 83 (92%) indicated that the training supported a career pathway that emphasized maternal and child health or children with special healthcare needs. Eighty-five former trainees (93% of respondents) reported leadership activities at the local level, 61 (67%) reported leadership activities at the state level, and 46 (51%) reported leadership activities at the national level. The results of the survey indicate that specialized, intensive training in the field of nutrition develops and supports leadership skills.

Loss of heterozygosity mapping at chromosome arm 16q in 712 breast tumors reveals factors that influence delineation of candidate regions.

Loss of heterozygosity (LOH) at the long arm of chromosome 16 occurs in at least half of all breast tumors and is considered to target one or more tumor suppressor genes. Despite extensive studies by us and by others, a clear consensus of the boundaries of the smallest region of overlap (SRO) could not be identified. To find more solid evidence for SROs, we tested a large series of 712 breast tumors for LOH at 16q using a dense map of polymorphic markers. Strict criteria for LOH and retention were applied, and results that did not meet these criteria were excluded from the analysis. We compared LOH results obtained from samples with different DNA isolation methods, ie., from microdissected tissue versus total tissue blocks. In the latter group, 16% of the cases were excluded because of noninterpretable LOH results. The selection of polymorphic markers is clearly influencing the LOH pattern because a chromosomal region seems more frequently involved in LOH when many markers from this region are used. The LOH detection method, i.e., radioactive versus fluorescence detection, has no marked effect on the results. Increasing the threshold window for retention of heterozygosity resulted in significantly more cases with complex LOH, i.e., several alternating regions of loss and retention, than seen in tumors with a small window for retention. Tumors with complex LOH do not provide evidence for clear-cut SROs that are repeatedly found in other samples. On disregarding these complex cases, we could identify three different SROs, two at band 16q24.3 and one at 16q22.1. In all three tumor series, we found cases with single LOH regions that designated the distal region at 16q24.3 and the region at 16q22.1. Comparing histological data on these tumors did not result in the identification of a particular subtype with LOH at 16q or a specific region involved in LOH. Only the rare mucinous tumors had no 16q LOH at all. Furthermore, a positive estrogen content is prevalent in tumors with 16q LOH, but not in tumors with LOH at 16q24.3 only.

Brain mitochondrial responses to postischemic reperfusion: a role for calcium and hydrogen peroxide?

During early recirculation following global brain ischemia, mitochondria are exposed to markedly elevated Ca(2+) concentrations and a short-lived production of reactive oxygen species, including hydrogen peroxide (H(2)O(2)). A brief increase in mitochondrial Ca(2+) and a subsequent increase in mitochondrial glutathione content have been observed. In the present study, we have confirmed the increase in mitochondrial glutathione in a rat model of global forebrain ischemia. This change was not inhibited by treatment of the rats with FK506, contrasting with our previous finding that cyclosporin A partially blocked the increase. These results suggest that induction of the mitochondrial permeability transition may be necessary for the increase in glutathione content in these organelles. To further investigate possible mitochondrial responses during early postischemic reperfusion, mitochondria isolated from normal brain were exposed to Ca(2+) and H(2)O(2), under conditions similar to those in intact cells. Respiratory activity was substantially modified when the mitochondria were exposed to Ca(2+) and H(2)O(2) together. Two distinct and largely noninteracting mechanisms apparently accounted for the responses to these agents. The effects of Ca(2+), but not H(2)O(2), were inhibited by cyclosporin A, again implicating the permeability transition in some of the mitochondrial changes.

Seasonal temperature alone can synchronize life cycles.

In this paper we discuss the effects of yearly temperature variation on the development and seasonal occurrence of poikiliothermic organisms with multiple life stages. The study of voltinism in the mountain pine beetle (Dendroctonus ponderosae Hopkins), an important forest insect living in extreme temperature environments and exhibiting no diapause, provides a motivational example. Using a minimal model for the rates of aging it is shown that seasonal temperature variation and minimal stage-specific differences in rates of aging are sufficient to create stable uni- and multi-voltine oviposition cycles. In fact, these cycles are attracting and therefore provide an exogenous mechanism for synchronizing whole populations of organisms. Structural stability arguments are used to extend the results to more general life systems.