Spatial-resolution impacts on local infrasound propagation.

The accuracy of input meteorological data can significantly impact the successful prediction of infrasound propagation at local to near-regional distances. These meteorological inputs are often derived from weather model simulations when event-specific measurements are not available, but the ideal spatial resolutions of these simulations have not been determined. This study seeks to identify the ideal horizontal resolutions for input meteorological data via infrasound simulations conducted with both range-dependent and -independent inputs. Outputs from the Weather Research and Forecasting (WRF) model at 1, 3, 5, and 15 km horizontal resolutions enable these investigations. The parabolic equation propagation model is used to calculate transmission loss for an impulsive signal and is compared against experimental data obtained from a series of 1 lb spherical, suspended C4 shots recorded on the infrasound array on the Waterways Experiment Station in Vicksburg, MS, occurring throughout the diurnal cycle with an overall propagation distance of 14.5 km. Simulations for morning and nighttime correctly predict signal detection and non-detection. Transitional times of day (dawn, evening) were partially successful. Changing horizontal resolutions in WRF incurred greater differences in prediction results than use of range-dependence vs -independence. No clear picture emerged regarding the optimal horizontal resolution for meteorological inputs.

Numerical modeling of mesoscale infrasound propagation in the Arctic.

The impacts of characteristic weather events and seasonal patterns on infrasound propagation in the Arctic region are simulated numerically. The methodology utilizes wide-angle parabolic equation methods for a windy atmosphere with inputs provided by radiosonde observations and a high-resolution reanalysis of Arctic weather. The calculations involve horizontal distances up to 200 km for which interactions with the troposphere and lower stratosphere dominate. Among the events examined are two sudden stratospheric warmings, which are found to weaken upward refraction by temperature gradients while creating strongly asymmetric refraction from disturbances to the circumpolar winds. Also examined are polar low events, which are found to enhance negative temperature gradients in the troposphere and thus lead to strong upward refraction. Smaller-scale and topographically driven phenomena, such as low-level jets, katabatic winds, and surface-based temperature inversions, are found to create frequent surface-based ducting out to 100 km. The simulations suggest that horizontal variations in the atmospheric profiles, in response to changing topography and surface property transitions, such as ice boundaries, play an important role in the propagation.

Discovery and characterization of a neutralizing pan-ELR+CXC chemokine monoclonal antibody.

CXCR1 and CXCR2 signaling play a critical role in neutrophil migration, angiogenesis, and tumorigenesis and are therefore an attractive signaling axis to target in a variety of indications. In human, a total of seven chemokines signal through these receptors and comprise the ELR+CXC chemokine family, so named because of the conserved ELRCXC N-terminal motif. To fully antagonize CXCR1 and CXCR2 signaling, an effective therapeutic should block either both receptors or all seven ligands, yet neither approach has been fully realized clinically. In this work, we describe the generation and characterization of LY3041658, a humanized monoclonal antibody that binds and neutralizes all seven human and cynomolgus monkey ELR+CXC chemokines and three of five mouse and rat ELR+CXC chemokines with high affinity. LY3041658 is able to block ELR+CXC chemokine-induced Ca2+ mobilization, CXCR2 internalization, and chemotaxis in vitro as well as neutrophil mobilization in vivo without affecting other neutrophil functions. In addition to the in vitro and in vivo activity, we characterized the epitope and structural basis for binding in detail through alanine scanning, crystallography, and mutagenesis. Together, these data provide a robust preclinical characterization of LY3041658 for which the efficacy and safety is being evaluated in human clinical trials for neutrophilic skin diseases.

An Endothelial Cell/Mesenchymal Stem Cell Coculture Cord Formation Assay to Model Vascular Biology In Vitro.

Blood vessels are crucial components for normal tissue development and homeostasis, so it is not surprising that endothelial dysfunction and dysregulation results in a variety of different pathophysiological conditions. The large number of vascular-related disorders and the emergence of angiogenesis as a major hallmark of cancer has led to significant interest in the development of drugs that target the vasculature. While several in vivo models exist to study developmental and pathological states of blood vessels, few in vitro assays have been developed that capture the significant complexity of the vascular microenvironment. Here, we describe a high content endothelial colony forming cells (ECFC)/adipose-derived stem cell (ADSC) coculture assay that captures many elements of in vivo vascular biology and is ideal for in vitro screening of compounds for pro- or anti-angiogenic activities.

Low frequency acoustic pulse propagation in temperate forests.

Measurements of acoustic pulse propagation for a 30-m path were conducted in an open field and in seven different forest stands in the northeastern United States consisting of deciduous, evergreen, or mixed tree species. The waveforms recorded in forest generally show the pulse elongation characteristic of propagation over a highly porous ground surface, with high frequency scattered arrivals superimposed on the basic waveform shape. Waveform analysis conducted to determine ground properties resulted in acoustically determined layer thicknesses of 4-8 cm in summer, within 2 cm of the directly measured thickness of the litter layers. In winter the acoustic thicknesses correlated with the site-specific snow cover depths. Effective flow resistivity values of 50-88 kN s m(-4) were derived for the forest sites in summer, while lower values typical for snow were found in winter. Reverberation times (T60) were typically around 2 s, but two stands (deciduous and pruned spruce planted on a square grid) had lower values of about 1.2 s. One site with a very rough ground surface had very low summer flow resistivity value and also had the longest reverberation time of about 3 s. These measurements can provide parameters useful for theoretical predictions of acoustic propagation within forests.

An in vitro cord formation assay identifies unique vascular phenotypes associated with angiogenic growth factors.

Vascular endothelial growth factor (VEGF) plays a dominant role in angiogenesis. While inhibitors of the VEGF pathway are approved for the treatment of a number of tumor types, the effectiveness is limited and evasive resistance is common. One mechanism of evasive resistance to inhibition of the VEGF pathway is upregulation of other pro-angiogenic factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF). Numerous in vitro assays examine angiogenesis, but many of these assays are performed in media or matrix with multiple growth factors or are driven by VEGF. In order to study angiogenesis driven by other growth factors, we developed a basal medium to use on a co-culture cord formation system of adipose derived stem cells (ADSCs) and endothelial colony forming cells (ECFCs). We found that cord formation driven by different angiogenic factors led to unique phenotypes that could be differentiated and combination studies indicate dominant phenotypes elicited by some growth factors. VEGF-driven cords were highly covered by smooth muscle actin, and bFGF-driven cords had thicker nodes, while EGF-driven cords were highly branched. Multiparametric analysis indicated that when combined EGF has a dominant phenotype. In addition, because this assay system is run in minimal medium, potential proangiogenic molecules can be screened. Using this assay we identified an inhibitor that promoted cord formation, which was translated into in vivo tumor models. Together this study illustrates the unique roles of multiple anti-angiogenic agents, which may lead to improvements in therapeutic angiogenesis efforts and better rational for anti-angiogenic therapy.

A method to assess target gene involvement in angiogenesis in vitro and in vivo using lentiviral vectors expressing shRNA.

Current methods to study angiogenesis in cancer growth and development can be difficult and costly, requiring extensive use of in vivo methodologies. Here, we utilized an in vitro adipocyte derived stem cell and endothelial colony forming cell (ADSC/ECFC) co-culture system to investigate the effect of lentiviral-driven shRNA knockdown of target genes compared to a non-targeting shRNA control on cord formation using High Content Imaging. Cord formation was significantly reduced following knockdown of the VEGF receptor VEGFR2 in VEGF-driven cord formation and the FGF receptor FGFR1 in basic FGF (bFGF)-driven cord formation. In addition, cord formation was significantly reduced following knockdown of the transcription factor forkhead box protein O1 (FOXO1), a protein with known positive effects on angiogenesis and blood vessel stabilization in VEGF- and bFGF-driven cord formation. Lentiviral shRNA also demonstrated utility for stable knockdown of VEGFR2 and FOXO1 in ECFCs, allowing for interrogation of protein knockdown effects on in vivo neoangiogenesis in a Matrigel plug assay. In addition to interrogating the effect of gene knockdown in endothelial cells, we utilized lentiviral shRNA to knockdown specificity protein 1 (SP1), a transcription factor involved in the expression of VEGF, in U-87 MG tumor cells to demonstrate the ability to analyze angiogenesis in vitro in a tumor-driven transwell cord formation system and in tumor angiogenesis in vivo. A significant reduction in tumor-driven cord formation, VEGF secretion, and in vivo tumor angiogenesis was observed upon SP1 knockdown. Therefore, evaluation of target gene knockdown effects in the in vitro co-culture cord formation assay in the ADSC/ECFC co-culture, ECFCs alone, and in tumor cells translated directly to in vivo results, indicating the in vitro method as a robust, cost-effective and efficient in vitro surrogate assay to investigate target gene involvement in endothelial or tumor cell function in angiogenesis.

Use of a porous material description of forests in infrasonic propagation algorithms.

Infrasound can propagate very long distances and remain at measurable levels. As a result infrasound sensing is used for remote monitoring in many applications. At local ranges, on the order of 10 km, the influence of the presence or absence of forests on the propagation of infrasonic signals is considered. Because the wavelengths of interest are much larger than the scale of individual components, the forest is modeled as a porous material. This approximation is developed starting with the relaxation model of porous materials. This representation is then incorporated into a Crank-Nicholson method parabolic equation solver to determine the relative impacts of the physical parameters of a forest (trunk size and basal area), the presence of gaps/trees in otherwise continuous forest/open terrain, and the effects of meteorology coupled with the porous layer. Finally, the simulations are compared to experimental data from a 10.9 kg blast propagated 14.5 km. Comparison to the experimental data shows that appropriate inclusion of a forest layer along the propagation path provides a closer fit to the data than solely changing the ground type across the frequency range from 1 to 30 Hz.

Influence of a forest edge on acoustical propagation: experimental results.

Acoustic propagation through a forest edge can produce complicated pressure time histories because of scattering from the trees and changes in the microclimate and ground parameters of the two regions. To better understand these effects, a field experiment was conducted to measure low-frequency acoustic pulses propagating in an open field, a forest, and passing through a forest edge in both directions. Waveforms measured in the open field were simple impulses with very low scattering, whereas waveforms at the edge and within the forest had stronger reverberations after the direct arrival. The direct wave pulse shapes increased in duration in accordance with the path length in the forest, which had an effective flow resistivity 12 to 13 that of the grassy open field. The measurements exhibit different rates of attenuation in the two regions, with relatively lower attenuation in the open field than higher rates in the forest. Decay of SEL transmitted into the forest was 4 dB more per tenfold distance than for outbound transmission. Stronger attenuation in the 1-2 kHz range occurs when propagating into the forest. While the measured meteorological profiles revealed three distinct microclimates, meteorological effects are not sufficient to explain the apparent non-reciprocal propagation.

Development and characterization of a high-throughput in vitro cord formation model insensitive to VEGF inhibition.

BACKGROUND: Anti-VEGF therapy reduces tumor blood vessels, however, some vessels always remain. These VEGF insensitive vessels may help support continued tumor growth and metastases. Many in vitro assays examining multiple steps of the angiogenic process have been described, but the majority of these assays are sensitive to VEGF inhibition. There has been little focus on the development of high-throughput, in vitro assays to model the vessels that are insensitive to VEGF inhibition. METHODS: Here, we describe a fixed end-point and kinetic, high-throughput stem cell co-culture model of cord formation. RESULTS: In this system, cords develop within 24 hours, at which point they begin to lose sensitivity to VEGF inhibitors, bevacizumab, and ramucirumab. Consistent with the hypothesis that other angiogenic factors maintain VEGF-independent vessels, pharmacologic intervention with a broad spectrum anti-angiogenic antagonist (suramin), a vascular disrupting agent (combretastatin), or a combination of VEGF and Notch pathway inhibitors reduced the established networks. In addition, we used our in vitro approach to develop an in vivo co-implant vasculogenesis model that connects with the endogenous vasculature to form functional blood vessels. Similar to the in vitro system, over time these vessels become insensitive to VEGF inhibition. CONCLUSION: Together, these models may be used to identify novel drugs targeting tumor vessels that are not sensitive to VEGF inhibition.

Blast noise characteristics as a function of distance for temperate and desert climates.

Variability in received sound levels were investigated at distances ranging from 4 m to 16 km from a typical blast source in two locations with different climates and terrain. Four experiments were conducted, two in a temperate climate with a hilly terrain and two in a desert climate with a flat terrain, under a variety of meteorological conditions. Sound levels were recorded in three different directions around the source during the summer and winter seasons in each location. Testing occurred over the course of several days for each experiment during all 24 h of the day, and meteorological data were gathered throughout each experiment. The peak levels (L(Pk)), C-weighted sound exposure levels (CSEL), and spectral characteristics of the received sound pressure levels were analyzed. The results show high variability in L(Pk) and CSEL at distances beyond 2 km from the source for each experiment, which was not clearly explained by the time of day the blasts occurred. Also, as expected, higher frequency energy is attenuated more drastically than the lower frequency energy as the distance from the source increases. These data serve as a reference for long-distance blast sound propagation.

Somatic mutations in CCK2R alter receptor activity that promote oncogenic phenotypes.

The roles of cholecystokinin 2 receptor (CCK2R) in numerous physiologic processes in the gastrointestinal tract and central nervous system are well documented. There has been some evidence that CCK2R alterations play a role in cancers, but the functional significance of these alterations for tumorigenesis is unknown. We have identified six mutations in CCK2R among a panel of 140 colorectal cancers and 44 gastric cancers. We show that these mutations increase receptor activity, activate multiple downstream signaling pathways, increase cell migration, and promote angiogenesis. Our findings suggest that somatic mutations in CCK2R may promote tumorigenesis through deregulated receptor activity and highlight the importance of evaluating CCK2R inhibitors to block both the normal and mutant forms of the receptor.

Influence of scattering, atmospheric refraction, and ground effect on sound propagation through a pine forest.

Sound propagation through a forest is affected by the microclimate in the canopy, scattering by trunks and stems, and ground reflection. Each of these effects is such a strong contributor to the attenuation of sound that mutual interactions between the phenomena could become important. A sound propagation model for use in a forest has been developed that incorporates scattering from trunks and branches and atmospheric refraction by modifying the effective wave number in the Green's function parabolic equation model. The ground effect for a hard-backed pine straw layer is approximated as a local reaction impedance condition. Comparisons to experimental data are made for frequencies up to 4,200 Hz. Cumulative influences of the separate phenomena are examined. The method developed in this paper is compared to previously published methods. The overall comparison with spectral transmission data is good, suggesting that the model captures the necessary details.

VCC-1, a novel chemokine, promotes tumor growth.

We have identified a novel human gene by transcriptional microarray analysis, which is co-regulated in tumors and angiogenesis model systems with VEGF expression. Isolation of cDNA clones containing the full-length VCC-1 transcript from both human and mouse shows a 119 amino acid protein with a 22 amino acid cleavable signal sequence in both species. Comparison of the protein product of this gene with hidden Markov models of all known proteins shows weak but significant homology with two known chemokines, SCYA17 and SCYA16. Northern analysis of human tissues detects a 1 kb band in lung and skeletal muscle. Murine VCC-1 expression can also be detected in lung as well as thyroid, submaxillary gland, epididymis, and uterus tissues by slot blot analysis. By quantitative real time RT-PCR 71% of breast tumors showed 3- to 24-fold up-regulation of VCC-1. In situ hybridization of breast carcinomas showed strong expression of the gene in both normal and transformed mammary gland ductal epithelial cells. In vitro, human microvascular endothelial cells grown on fibronectin increase VCC-1 expression by almost 100-fold. In addition, in the mouse angioma endothelial cell line PY4.1 the gene was over-expressed by 28-fold 6 h after induction of tube formation while quiescent and proliferating cells showed no change. VCC-1 expression is also increased by VEGF and FGF treatment, about 6- and 5-fold, respectively. Finally, 100% of mice injected with NIH3T3 cells over-expressing VCC-1 develop rapidly progressing tumors within 21 days while no growth is seen in any control mice injected with NIH3T3 cells containing the vector alone. These results strongly suggest that VCC-1 plays a role in angiogenesis and possibly in the development of tumors in some tissue types.

Detection of differentially expressed HES-6 gene in metastatic colon carcinoma by combination of suppression subtractive hybridization and cDNA library array.

The molecular mechanisms involved in the progression of colon carcinomas from a primary to a metastatic tumor have been only partially elucidated and poorly understood. This study combines suppression subtractive hybridization and cDNA array hybridization to identify genes with expression differences between a primary human colon tumor cell line (HT29) and three isogenic lung tumor metastases. The positive clones isolated in this screen were further validated and quantitated with real-time reverse transcription polymerase chain reactions. HES-6 was identified as up-regulated in each of the individual tumor metastases, as well as in a panel of primary human tumors derived from the lung, breast and kidney. These findings demonstrate that it is possible to utilize longitudinal samples from an in vivo model of colon carcinoma to identify genes up-regulated in metastases and that HES-6 may be an important marker of a range of primary cancers as well as metastatic colon carcinoma.