Protein-tyrosine Phosphatase 4A3 (PTP4A3) Promotes Vascular Endothelial Growth Factor Signaling and Enables Endothelial Cell Motility

Protein-tyrosine phosphatase 4A3 (PTP4A3) is highly expressed in multiple human cancers and is hypothesized to have a critical, albeit poorly defined, role in the formation of experimental tumors in mice. PTP4A3 is broadly expressed in many tissues so the cellular basis of its etiological contributions to carcinogenesis may involve both tumor and stromal cells. In particular, PTP4A3 is expressed in the tumor vasculature and has been proposed to be a direct target of vascular endothelial growth factor (VEGF) signaling in endothelial cells. We now provide the first in vivo experimental evidence that PTP4A3 participates in VEGF signaling and contributes to the process of pathological angiogenesis. Colon tumor tissue isolated from Ptp4a3-null mice revealed reduced tumor microvessel density compared with wild type controls. Additionally, vascular cells derived from Ptp4a3-null tissues exhibited decreased invasiveness in an ex vivo wound healing assay. When primary endothelial cells were isolated and cultured in vitro, Ptp4a3-null cells displayed greatly reduced migration compared with wild type cells. Exposure to VEGF led to an increase in Src phosphorylation in wild type endothelial cells, a response that was completely ablated in Ptp4a3-null cells. In loss-of-function studies, reduced VEGF-mediated migration was also observed when human endothelial cells were treated with a small molecule inhibitor of PTP4A3. VEGF-mediated in vivo vascular permeability was significantly attenuated in PTP4A3-deficient mice. These findings strongly support a role for PTP4A3 as an important contributor to endothelial cell function and as a multimodal target for cancer therapy and mitigating VEGF-regulated angiogenesis.     

Characterization of intracellular ice formation in Drosophila melanogaster embryos

Cryomicroscopy and differential scanning calorimetry (DSC) were used to characterize the incidence of intracellular ice formation (IIF) in 12- to 13-hr-old embryos of Drosophila melanogaster (Oregon-R strain P2) as influenced by the state of the eggcase (untreated, dechorionated, or permeabilized), the composition of the suspending medium (with and without cryoprotectants), and the cooling rate. Untreated eggs underwent IIF over a very narrow temperature range when cooled at 4 or 16 degrees C/min with a median temperature of intracellular ice formation (TIIF50) of -28 degrees C. The freezable water volume of untreated eggs was approximately 5.4 nl as determined by DSC. IIF in dechorionated eggs occurred over a much broader temperature range (-13 to -31 degrees C), but the incidence of IIF increased sharply below -24 degrees C, and the cumulative incidence of IIF at -24 degrees C decreased with cooling rate. In permeabilized eggs without cryoprotectants (CPAs), IIF occurred at much warmer temperatures and over a much wider temperature range than in untreated eggs, and the TIIF50 was cooling rate dependent. At low cooling rates (1 to 2 degrees C/min), TIIF50 increased with cooling rate; at intermediate cooling rates (2 to 16 degrees C/min), TIIF50 decreased with cooling rate. The total incidence of IIF in permeabilized eggs was 54% at 1 degree C/min, and volumetric contraction almost always occurred during cooling. Decreasing the cooling rate to 0.5 degree C/min reduced the incidence of IIF to 43%. At a cooling rate of 4 degrees C/min, ethylene glycol reduced the TIIF50 by about 12 degrees C for each unit increase in molarity of CPA (up to 2.0 M) in the suspending medium. The TIIF50 was cooling rate dependent when embryos were preequilibrated with 1.0 M propylene glycol or ethylene glycol, but was not so in 1.0 M DMSO. For embryos equilibrated in 1.5 M ethylene glycol and then held at -5 degrees C for 1 min before further cooling at 1 degree C/min, the incidence of IIF was decreased to 31%. Increasing the duration of the isothermal hold to 10 min reduced the incidence of IIF to 22% and reduced the volume of freezable water in embryos when intracellular ice formation occurred. If the isothermal hold temperature was -7.5 or -10 degrees C, a 10- to 30-min holding time was required to achieve a comparable reduction in the incidence of IIF.     

Discovery of novel 2-(aminoheteroaryl)-thiazole-5-carboxamides as potent and orally active Src-family kinase p56(Lck) inhibitors

A series of substituted 2-(aminoheteroaryl)-thiazole-5-carboxamide analogs have been synthesized as novel, potent inhibitors of the Src-family kinase p56^Lck. Among them, compound 2 displayed superior in vitro potency and excellent in vivo efficacy.     

A critical role of Gbetagamma in tumorigenesis and metastasis of breast cancer

A growing body of evidence indicates that G protein-coupled receptors (GPCRs) are involved in breast tumor progression and that targeting GPCRs may be a novel adjuvant strategy in cancer treatment. However, due to the redundant role of multiple GPCRs in tumor development, it may be necessary to target a common signaling component downstream of these receptors to achieve maximum efficacy. GPCRs transmit signals through heterotrimeric G proteins composed of Gα and Gβγ subunits. Here we evaluated the role of Gβγ in breast tumor growth and metastasis both in vitro and in vivo. Our data show that blocking Gβγ signaling with Gα(t) or small molecule inhibitors blocked serum-induced breast tumor cell proliferation as well as tumor cell migration induced by various GPCRs in vitro. Moreover, induced expression of Gα(t) in MDA-MB-231 cells inhibited primary tumor formation and retarded growth of existing breast tumors in nude mice. Blocking Gβγ signaling also dramatically reduced the incidence of spontaneous lung metastasis from primary tumors and decreased tumor formation in the experimental lung metastasis model. Additional studies indicate that Gβγ signaling may also play a role in the generation of a tumor microenvironment permissive for tumor progression, because the inhibition of Gβγ signaling attenuated leukocyte infiltration and angiogenesis in primary breast tumors. Taken together, our data demonstrate a critical role of Gβγ signaling in promoting breast tumor growth and metastasis and suggest that targeting Gβγ may represent a novel therapeutic approach for breast cancer.     

Natural variation in decision-making behavior in Drosophila melanogaster

There has been considerable recent interest in using Drosophila melanogaster to investigate the molecular basis of decision-making behavior. Deciding where to place eggs is likely one of the most important decisions for a female fly, as eggs are vulnerable and larvae have limited motility. Here, we show that many natural genotypes of D. melanogaster prefer to lay eggs near nutritious substrate, rather than in nutritious substrate. These preferences are highly polymorphic in both degree and direction, with considerable heritability (0.488) and evolvability.Relative preferences are modulated by the distance between options and the overall concentration of ethanol, suggesting Drosophila integrate many environmental factors when making oviposition decisions. As oviposition-related decisions can be efficiently assessed by simply counting eggs, oviposition behavior is an excellent model for understanding information processing in insects. Associating natural genetic polymorphisms with decision-making variation will shed light on the molecular basis of host choice behavior, the evolutionary maintenance of genetic variation, and the mechanistic nature of preference variation in general.     

The seroepidemiology of dengue in a US military population based in Puerto Rico during the early phase of the Zika pandemic

Understanding the burden and risk factors of dengue virus (DENV) infection in Puerto Rico is important for the prevention of dengue in local, traveler and military populations. Using sera from the Department of Defense Serum Repository, we estimated the prevalence and predictors of DENV seropositivity in those who had served in Puerto Rico, stratified by birth or prior residence (“birth/residence”) in dengue-endemic versus non-endemic regions. We selected sera collected in early 2015 from 500 U.S. military members, a time-point also permitting detection of early cryptic Zika virus (ZIKV) circulation. 87.2% were born or resided in a DENV-endemic area before their military service in Puerto Rico. A high-throughput, flow-cytometry-based neutralization assay was employed to screen sera for ZIKV and DENV neutralizing antibodies, and confirmatory testing was done by plaque-reduction neutralization test (PRNT). We identified one Puerto Rico resident who seroconverted to ZIKV by June 2015, suggesting cryptic ZIKV circulation in Puerto Rico at least 4 months before the first reported cases. A further six PRNT-positive presumptive ZIKV infections which were resolved as DENV infections only by the use of paired sera. We noted 66.8% of the total study sample was DENV seropositive by early 2015. Logistic regression analysis indicated that birth/residence in a dengue non-endemic region (before military service in Puerto Rico) was associated with a lower odds of DENV exposure by January—June 2015 (aOR = 0.28, p = 0.001). Among those with birth/residence in a non-endemic country, we noted moderate evidence to support increase in odds of DENV exposure for each year of military service in Puerto Rico (aOR = 1.58, p = 0.06), but no association with age. In those with birth/residence in dengue-endemic regions (before military service in Puerto Rico), we noted that age (aOR = 1.04, p = 0.02), rather than duration of Puerto Rico service, was associated with dengue seropositivity, suggesting earlier lifetime DENV exposure. Our findings provide insights into the burden and predictors of DENV infection in local, traveler and military populations in Puerto Rico. Our study also highlights substantial PRNT ZIKV false-positivity when paired sera are not available, even during periods of very low ZIKV prevalence.     

Epigenetic and pharmacological control of pigmentation via Bromodomain Protein 9 (BRD9)

Lineage-specific differentiation programs are activated by epigenetic changes in chromatin structure. Melanin-producing melanocytes maintain a gene expression program ensuring appropriate enzymatic conversion of metabolites into the pigment, melanin, and transfer to surrounding cells. During neuroectodermal development, SMARCA4 (BRG1), the catalytic subunit of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes, is essential for lineage specification. SMARCA4 is also required for development of multipotent neural crest precursors into melanoblasts, which differentiate into pigment-producing melanocytes. In addition to the catalytic domain, SMARCA4 and several SWI/SNF subunits contain bromodomains which are amenable to pharmacological inhibition. We investigated the effects of pharmacological inhibitors of SWI/SNF bromodomains on melanocyte differentiation. Strikingly, treatment of murine melanoblasts and human neonatal epidermal melanocytes with selected bromodomain inhibitors abrogated melanin synthesis and visible pigmentation. Using functional genomics, iBRD9, a small molecule selective for the bromodomain of BRD9 was found to repress pigmentation-specific gene expression. Depletion of BRD9 confirmed a requirement for expression of pigmentation genes in the differentiation program from melanoblasts into pigmented melanocytes and in melanoma cells. Chromatin immunoprecipitation assays showed that iBRD9 disrupts the occupancy of BRD9 and the catalytic subunit SMARCA4 at melanocyte-specific loci. These data indicate that BRD9 promotes melanocyte pigmentation whereas pharmacological inhibition of BRD9 is repressive.     

Biological and genetic characterisation of Phoma macrostoma isolates with bioherbicidal activity

The fungus Phoma macrostoma Mont. isolate 94-44B was registered as a bioherbicide for control of broadleaved weeds in Canada and the USA in 2011 and 2012, respectively. To obtain the registrations, the fungus had to be characterised both biologically and genetically. The objectives of this study were to demonstrate that bioherbicidal activity was associated with specific genetic markers and to determine whether bioherbicidal activity was a general trait of the species or only selected isolates. A collection of 64 isolates of P. macrostoma was established. A greenhouse bioassay and bioherbicidal-specific primers were used to determine bioherbicidal activity of all isolates. Only isolates originating from Canada thistle demonstrated the ability to reduce dandelion seedlings and display the 853 bp amplicon for the bioherbicidal-specific primer. Bioherbicidal isolates were consistently differentiated from all other isolates with two main genotypic groupings (I and II) arising from internal transcribed spacer (ITS) and amplified fragment length polymorphisms (AFLP) sequence analyses. Using AFLP, two biotypes of bioherbicidal isolates were also differentiated by the presence or absence of an AFLP marker at a single polymorphic locus. The genetic divergence among the bioherbicidal and nonbioherbicidal isolates of P. macrostoma was only 2.21% which was lower than that reported for other related Phoma sp. Other than the bioherbicidal trait, there was no apparent affiliation of the genetics with known varietal types, host or geographic origin. ITS sequence analysis and AFLP fingerprinting may be used as tools to detect bioherbicidal isolates of P. macrostoma.