All data are within the paper and supporting files.

Introduction {#sec001}
============

Although initially thought to be homogeneous, early proof of heterogeneous physiology in tumor vessels were described with low oxygen tensions (\<10 mmHg pO~2~) found in selected tumor blood vessels \[[@pone.0135607.ref001]--[@pone.0135607.ref005]\]. This low oxygen or hypoxic environment leads to stabilization of the transcriptional regulating protein hypoxia inducible factor (HIF-1α), meditating downstream signaling of pro-angiogenic proteins and small molecules which have been implicated in driving angiogenesis and malignant progression \[[@pone.0135607.ref006], [@pone.0135607.ref007]\]. In recent years, the hypoxic, perivascular niche has been extensively studied and demonstrated to harbor cancer stem cells, as well as generally promoting metastatic spread \[[@pone.0135607.ref008]--[@pone.0135607.ref011]\]. However, the majority of these investigations have been largely *in vitro* or focused on genetically manipulated HIF-1 protein knockout mouse models \[[@pone.0135607.ref006], [@pone.0135607.ref007]\] where the true details of hypoxia and cell type(s) involved are not well documented.

In addition to hypoxia-related tumor progression, hypoxia-induced protection against standard cytotoxic therapies leading to suboptimal response and eventual tumor recurrence has been well documented \[[@pone.0135607.ref012]--[@pone.0135607.ref014]\]. Mounting evidence suggests high-dose radiotherapy response in solid tumors is mediated by indirect death resulting from direct radiation-induced tumor endothelial cell death \[[@pone.0135607.ref015]\]. A low pO~2~ environment protects cells from radiation-induced cell death by reducing the oxygen fixation of DNA strand breaks. In the presence of O~2~, free radicals formed following ionizing radiation potentiate DNA damage resulting in cellular death while hypoxic environments attenuate this effect \[[@pone.0135607.ref016]\]. Applying hypoxic radioprotection dogma to the idea of vascular-damaging doses of radiation or other vascular-targeted therapies is a field in large part unstudied. Despite compelling evidence demonstrating the impact of the hypoxic tumor vessel niche on tumor progression, clinical techniques evaluating the oxygenation level in tumor vessels or tumor endothelial cells themselves remains elusive, limiting the ability to study the impact on therapeutic response.

The bioreductive compound pimonidazole is a substituted 2-nitroimidazole that is preferentially reduced in viable hypoxic cells forming an artificial tumor target that can be detected by flow cytometry and immunohistochemistry \[[@pone.0135607.ref017]\]. Irreversible reduction of pimonidazole allows cells which are intermittently hypoxic to also be detected by the aforementioned techniques, and thus reoxygenation is not a limiting factor. Unlike naturally occurring tumor-associated antigens \[[@pone.0135607.ref018]\], hypoxia marker antigens are not susceptible to the inherent heterogeneous tumor cell genetics or changes in protein expression and genetic drift during disease progression or therapeutic intervention. These aspects coupled with the observation of pimonidazole dependent complement cell lysis suggesting pimonidazole antigens exist on the cell membrane led to the current investigation \[[@pone.0135607.ref019]\]. Therefore, in the present study, we tested the utility of pimonidazole antigen-like targets formed in the luminal surface of tumor blood vessels as a target for intravenously-injected molecular contrast agents that may avoid many of the pitfalls that face traditional hypoxia imaging and quantification. We describe a novel hypoxemia (vascular hypoxia) detection method based on high-frequency ultrasound imaging of vascular restricted α-pimonidazole targeted-microbubbles, targeting the hypoxia marker pimonidazole in mouse mammary gland carcinoma models.

Materials and Methods {#sec002}
=====================

Cell lines {#sec003}
----------

Murine endothelial cells (2H11) and murine breast carcinoma cells (4T1 & SCK) were cultured at 5% CO~2~ and 37°C. Cell lines were maintained in the following media conditions: 2H11---DMEM high glucose (4.5 g/L) + 10% FBS, 4T1---DMEM F-12 (Gibco) + 10% BCS, and SCK---RPMI1 1640 + 10% BCS. 2H11 cells were purchased from ATCC, 4T1 cells were obtained from Dr. Michael Borrelli (UAMS) and SCK cells were obtained from Dr. Chang Song (U of Minn.) \[[@pone.0135607.ref020], [@pone.0135607.ref021]\]. All cell lines were tested and negative for mycoplasma.

Generating pimonidazole-targeting microbubbles {#sec004}
----------------------------------------------

Commercially available microbubbles, on average 2.5 μm in diameter with 1 x 10^5^ streptavidin biding sites, allow for molecular targeted imaging (FUJIFILM Visualsonics, Inc.) \[[@pone.0135607.ref001]\]. In brief, streptavidin molecules coat a lipid shell containing a gas filled hollow core of perfluorobutane/nitrogen (C~4~F~10~/N~2~). Here, 20 μg of biotinylated anti-pimonidazole antibody \[[@pone.0135607.ref022]\] was incubated for 15 minutes with streptavidin-coated microbubbles to create pimonidazole targeting microbubbles, MBα-pimo (stock concentration 2 x 10^9^/mL) ([Fig 1A](#pone.0135607.g001){ref-type="fig"}). Microbubbles are stable up to 3 hours after preparation and have a clearance rate of 10--20 minutes in mice.

![Pimonidazole targeting microbubbles.\
**(A)** A graphic representation of the microbubbles and conditions used. *Left*, unlabeled microbubbles (MB); middle, pimonidazole-targeting MB (MBα-pimo); and *right*, MBα-pimo without pimonidazole present in the circulation. **(B)** MBα-pimo binds hypoxic 2H11 endothelial cells only in the presence of pimonidazole. MBα-pimo does not bind endothelial cells (*left*), unless pimonidazole is added (*middle*). (*right*), MBα-pimo binding to the cell surface of hypoxic endothelial cells, magnification 40X.](pone.0135607.g001){#pone.0135607.g001}

*In vitro* microbubble binding experiments {#sec005}
------------------------------------------

2H11 endothelial cells growing exponentially were plated in 6 --well culture plates at 3 x 10^5^ cells/well. Three washes with PBS were performed to remove serum prior to adding serum-free media. One row of wells (3 wells) was incubated with 75 μg/mL pimonidazole (equivalent to the *in vivo* dose regimen) in serum free media while the other row received serum free media and no pimonidazole. Hypoxic conditions were generated 24 hours after sub-culturing in 6-wells plates. Hypoxia was induced by incubating cells for 2 hours at 37°C in an anaerobic chamber (Forma Scientific Inc.) were a gas mix (5% CO~2~, 10% H~2~, 85% N~2~) produced oxygen concentrations within the tissue culture dishes at or below 10 mmHg. This was confirmed with flow cytometry by pimonidazole saturation of cells incubated under these conditions \[[@pone.0135607.ref023], [@pone.0135607.ref024]\].

Following hypoxia incubation with and without pimonidazole, 6-well plates were removed from the anaerobic chamber and the following procedure followed. All wells were washed with PBS (3X) followed by 1 mL of serum free media added with 10 μL of MBα-pimo. MBs were allowed to incubate with cells for 5 minutes, after which a gentle wash with serum free media and immediate phase-contrast imaging was performed on an Olympus IX71.

Murine mammary gland carcinoma models {#sec006}
-------------------------------------

Two murine mammary gland carcinomas models (4T1, SCK) were generated in 5--12 week old female Balb/c and AJ mice (Charles River Laboratories) as described previously \[[@pone.0135607.ref020], [@pone.0135607.ref025]\]. MMTV-Wnt-1-Tg mice develop spontaneous mammary gland tumors and were kindly provided by Dr. Rosalia Simmen (UAMS). Subcutaneously implanted tumors in the right rear limb reached an approximate size of 200 mm^3^ in size by day 7, after which tumors underwent contrast-enhanced ultrasound imaging (as described below) and were excised for further histological studies. All animal experiments were performed with the approval of the University of Arkansas for Medical Sciences Animal Use and Care Committee (IACUC).

Immunofluorescence {#sec007}
------------------

Mice received intraperitoneal (i.p.) injections with 75 mg/kg pimonidazole 2 hours prior to euthanasia and tissue collection, as described previously \[[@pone.0135607.ref020], [@pone.0135607.ref026]\]. Pimonidazole, a substituted 2-nitroimidazole (290.7 Da), is preferentially reduced in hypoxic viable cells and forms irreversible protein adducts, and has been optimized for detection with immunohistochemical and fluorescence methods \[[@pone.0135607.ref020], [@pone.0135607.ref026]\]. Normal (kidney, liver, and spleen) and tumor tissues were harvested and snap frozen in OCT for histological sectioning (5 μm) and subsequent staining. Hypoxia was detected by anti-pimonidazole antibody (mouse FITC-MAb 1:50, Hypoxyprobe, Inc.) and tumor blood vessels were detected by primary antibody CD31 (rat anti-CD31 1:100, BD Pharmingen) and secondary antibody anti-rat Alexa 647 (1:100, Invitrogen Molecular Probes) mounted in Vectashield mounting media with DAPI (Vector Labs, Inc.). Imaging was performed using an Olympus IX71 fluorescent microscope workstation or Aperio Scanscope FL.

2D High-frequency ultrasound imaging {#sec008}
------------------------------------

Non-linear imaging of targeted, contrast-enhanced microbubbles within tumor tissue was performed according to the manufacturer's protocol (FUJIFILM Visualsonics, Inc.). Two hours after intraperitoneal (i.p.) administration of 75 mg/kg pimonidazole (Hypoxyprobe, Inc), mice were anesthetized with 1% isoflurane and placed on a heated platform for the duration of the ultrasound imaging session. A 27G catheter was placed in the lateral tail vein and a 50 μL bolus of microbubbles injected intravenously (i.v.) using a syringe pump. After 5 minutes, allowing the microbubbles to circulate and bind, a programed data collection sequence was initiated and captured using a MS250 transducer, 18 MHz (FUJIFILM Visualsonics, Inc.). The program includes an initial data collection sequence during 25 seconds (designated 'pre'), followed by a destruction phase where 100% of the microbubbles within the transducer field are obliterated by a high-mechanical index ultrasound pulse that eliminates both free and bound microbubbles, to complete with a 25 second data collection sequence of imaging after the burst (designated 'post'). Comparisons were made between the steady state prior to and following the microbubble burst sequence. The difference in signal between pre- and post-burst (differential targeted expression, d.T.E.), represents the relative amount and location of microbubbles bound to pimonidazole. Four experimental conditions were investigated in the 4T1 tumor model: nontargeted-microbubbles which lack streptavidin moieties (MB) and pimonidazole-targeting MB (MBα-pimo) within the tumor and with circulating pimonidazole present, tumoral MBα-pimo without pimonidazole present ([Fig 1A](#pone.0135607.g001){ref-type="fig"}), and MBα-pimo measured within the muscle while circulating pimonidazole present (n = 3--11).

3D High-frequency ultrasound imaging, 3D modeling and animation {#sec009}
---------------------------------------------------------------

A modified manufacturer's protocol was used to obtain 3D distribution of tumor vessel hypoxia. Five minutes after i.v. injection of targeted-microbubbles with a 27G 0.5cc insulin syringe (Terumo), 10 repetitive burst sequences were performed over the heart to destroy microbubbles in a highly concentrated region, until baseline contrast signal was observed. Following reduction of circulating unbound targeted-microbubbles, 3D imaging was acquired according to the manufacturer's protocol. At 0.152mm/slice, 110 slices were collected from the tumor pre- and post-burst. Images were extracted and used for subsequent modeling and animation.

The unprocessed microbubble images collected with Visualsonics software were post-processed using a MatLab subtraction algorithm to illustrate the absolute signal difference between pre and post-burst. Deconvolved images were stacked with ImageJ software and used to create a 3D model and video. Additionally, Huygens Essential v2.10 software (Scientific Volume Imaging, B.V.) was used to further process deconvolved stacked images using maximum-intensity projections of the image stacks for 3D image modeling.

Statistical Analysis {#sec010}
--------------------

A one-way ANOVA with post-hoc Holm-Sidak's multiple comparisons test was used for statistical analysis of microbubble data.

Results and Discussion {#sec011}
======================

Pimonidazole targeting microbubbles {#sec012}
-----------------------------------

When pimonidazole is reduced within or on the surface of hypoxic cells, it forms an artificial tumor target \[[@pone.0135607.ref027]\] impervious to the tumor's genetic origin or acquired mutations \[[@pone.0135607.ref019], [@pone.0135607.ref022]\], allowing broad-spectrum application. Here we designed a novel strategy using vascular restricted microbubbles to exploit this potential diagnostic and therapeutic target of vascular hypoxia ([Fig 1](#pone.0135607.g001){ref-type="fig"}). Targeting an artificially created target, the pimonidazole-targeting MB (MBα-pimo) can inherently act as its own control, i.e. in the absence of the target ([Fig 1A](#pone.0135607.g001){ref-type="fig"}). MBα-pimo can bind hypoxic endothelial cells *in vitro* incubated with pimonidazole, whereas the absence of pimonidazole prevents MBα-pimo binding ([Fig 1B](#pone.0135607.g001){ref-type="fig"} and [S1 Fig](#pone.0135607.s001){ref-type="supplementary-material"}).

Vessel hypoxia detection {#sec013}
------------------------

After demonstrating *in vitro* that MBα-pimo was specific to hypoxic endothelial cells incubated with pimonidazole, quantification of the amount of vascular hypoxia in three different murine mammary gland carcinoma models using immunohistochemical staining was performed, i.e. 4T1, SCK and MMTV-Wnt-1 (2). Whereas overall tumor hypoxia, as indicated by pimonidazole positive staining ranged from 5%-- 18% in these models, vascular hypoxia, as indicated by co-localization of tumor vessels (CD31^+^) and hypoxia (pimonidazole^+^) amounted up to 20% of the total stained vasculature in the 4T1 model ([Fig 2F](#pone.0135607.g002){ref-type="fig"}). These findings are similar to earlier reports studying various malignant tissues and hypoxia subtypes \[[@pone.0135607.ref028]\]. In contrast, no vascular hypoxia was detected in normal healthy tissues i.e. kidney, spleen and liver ([Fig 2G, 2H and 2I](#pone.0135607.g002){ref-type="fig"}); suggesting cell surface pimonidazole targets are a promising hypoxia target in malignant tissues ([S2 Fig](#pone.0135607.s002){ref-type="supplementary-material"}).

![Vascular hypoxia in murine breast carcinomas and normal tissue.\
Immunofluorescence analysis of hypoxemia in 4T1 mammary gland carcinoma. **(A)**, 4T1 tumor tissue is stained for tumor vessels (CD31; red). (**B)** Tumor hypoxia (pimonidazole; green) is co-localized (white) in relation to microvasculature in 4T1 tumor tissue **(C)**. Quantification of overall tumor vessels **(D)**, hypoxia **(E)**, and hypoxic tumor vessels **(F)** in 4T1, SCK and MMTV-Wnt-1 carcinomas. Immunofluorescence analysis of vasculature (CD31; red) and hypoxia (pimonidazole; green) in non-diseased kidney **(G)**, spleen **(H)** and liver **(I)** indicates a lack of global and vessel hypoxia in normal tissue.](pone.0135607.g002){#pone.0135607.g002}

High-frequency ultrasound imaging with pimonidazole-targeted microbubbles {#sec014}
-------------------------------------------------------------------------

High-frequency ultrasound was used to image and quantify bound pimonidazole-targeted microbubbles (MBα-pimo) in tumor and normal tissue ([Fig 3](#pone.0135607.g003){ref-type="fig"}).

![Schematic of anti-pimonidazole targeted-microbubbles (MBα-pimo) with ultrasound imaging for detection of vascular hypoxia.\
**(A)** Illustration showing the differential distribution of MBα-pimo in well-oxygenated tumor endothelium (red) compared to hypoxic tumor endothelium (blue) during imaging and intervention by ultrasound. **(B)** Representative quantification graphic of MBα-pimo where the binding occurs over a 5 minute window after IV injection followed by a data collection period of contrast signal, a single ultrasound pulse to burst bound and free MBα-pimo, and a final data collection during the immediate reperfusion window. Subsequently, the difference in signal from the steady state prior to microbubble burst ('pre') and following burst ('post') can be calculated. This differential targeted expression (d.T.E.; linear, a.u.) represents the relative amount of bound MBα-pimo and indirectly indicates the location and amount of vascular hypoxia within the tumor (x-axis scale not linear).](pone.0135607.g003){#pone.0135607.g003}

By creating the artificial targets with injection of pimonidazole, the accumulation of MBα-pimo contrast agent in tumor vessels was significantly enhanced with a nearly 8-fold higher differential targeted expression (d.T.E.) (206.2 ± 45.1 vs. 25.5 ± 12.4 linear a.u., p \< 0.05) than in tumors not receiving pimonidazole. In normal healthy muscle tissue, the pre- and post- signal was not significantly different resulting in a d.T.E. of essentially zero (-0.33 ± 1.4 linear a.u.) despite the presence of circulating pimonidazole. Non-targeted-microbubbles (MB) accumulated within the tumor similar to controls (MBα-pimo in mice lacking pimonidazole injection), however with greater variance (33.3 ± 13.2 linear a.u., [Fig 4C and 4D](#pone.0135607.g004){ref-type="fig"}). Passive accumulation and extravasation into the tumor might contribute to this, although this effect should be inherent in the other conditions and would require inter-endothelial cell junctions much larger than typically reported \[[@pone.0135607.ref029]\]. Relatedly, high interstitial fluid pressure, which is a well-documented physiological feature of solid tumors \[[@pone.0135607.ref030]--[@pone.0135607.ref033]\], might affect contrast-enhanced ultrasound imaging with microbubbles. The high intra-tumoral pressure can lead to reduced tumor blood flow and thus, may influence microbubble imaging through dynamic changes in blood flow kinetics \[[@pone.0135607.ref021]\]. Reduced or sluggish blood flow may also result in trapping or non-specific binding/electrostatic interactions within tumor blood vessels. We were cognizant that other external factors may also exacerbate the influence on physiology (i.e. anesthesia, body temperature) and contribute to the amount of non-specific signal. Nonetheless, the signal generated in the tumor from targeted microbubbles suggests a detectable level of hypoxic vasculature is present in these models and may be translated into human cancer.

![High-frequency ultrasound imaging of targeted-microbubbles detects tumor vessel hypoxia.\
Representative image and quantified data of anti-pimonidazole labeled microbubbles (MBα-pimo) bound in perfused hypoxic tumor vasculature without pimonidazole injection **(A)**, and with pimonidazole injection **(B)** in 4T1 tumor bearing mice. Top image shows the signal before the burst sequence and the bottom image shows after the burst sequence **(A, B)**. **(C)** Quantified data of different experimental conditions using targeting and non-targeting microbubbles (as indicated). **D)** Summary of quantitated data statistically analyzed represented as mean ± SEM, ^\#^p \< 0.05, versus non-targeting MB, MBα-pimo without pimonidazole injection, and MBα-pimo in muscle tissue (ANOVA post-hoc Holm-Sidak).](pone.0135607.g004){#pone.0135607.g004}

While this molecular imaging methodology allows for imaging of tumor vessel hypoxia in a specific 2D plane, it also permits imaging of the whole tumor. By using specialized software (Huygens essential software), a 3D representation of overall vessel hypoxia was generated ([Fig 5B](#pone.0135607.g005){ref-type="fig"}). This permits not only a read-out on the general hypoxic state of the tumor, but also whether there is micro-regional hypoxia within the tumor, i.e. improved resolution over standard hypoxia imaging techniques. Prior to non-linear contrast-enhanced imaging, anatomical visualization was obtained with the B-mode ultrasound ([Fig 5A](#pone.0135607.g005){ref-type="fig"}) followed by imaging of MBα-pimo in 3D. Following image collection, post-processed images reflect the d.T.E ([Fig 5A](#pone.0135607.g005){ref-type="fig"}). Subsequently, the deconvolved images were stacked to build a maximum-intensity 3D model and video ([Fig 5B](#pone.0135607.g005){ref-type="fig"} and [S3 Fig](#pone.0135607.s003){ref-type="supplementary-material"}). The ability to image whole tumor hypoxemia presents an attractive method for longitudinal, clinically meaningful studies on its role in tumor progression and therapeutic response. For example, hypoxia has attenuating anti-tumor effects on radiotherapy and drug efficacy \[[@pone.0135607.ref012]--[@pone.0135607.ref014]\], and while these tempering effects have been reported for various tumor and normal cell types \[[@pone.0135607.ref016]\], there is a paucity of investigations delineating the direct consequences of hypoxia on radiation or drug efficacy in tumor endothelial cells, *in vitro* or *in vivo*. Conversely, there is a substantial amount of literature identifying the vascular condition in response to various treatment therapies. The detection of vascular hypoxia using contrast-enhanced ultrasound as described within would permit a directed and non-invasively applied strategy for pre-clinical and ultimately clinical settings focused on the status of the vasculature before, during or after therapy.

![3D modeling of MBα-pimo distribution in mammary gland carcinoma.\
**A)** Single slice images taken from a 3D imaging sequence in B-mode *(left)*. Single slice images taken from a 3D imaging sequence depicting the differential targeted expression (d.T.E) *(right)*. **B)** Three-dimensional contrast projection of 3D stack image data from hypoxia targeted, MBα-pimo, contrast signal collected in a rear-limb 4T1 tumor. Images (0.152mm/slice) generated using Visualsonics imaging system and post-processed using the Huygens essential software.](pone.0135607.g005){#pone.0135607.g005}

Conclusions {#sec015}
===========

Studies have shown that hypoxic vessels are associated with tumor angiogenesis, progression, stem cell induction/protection, metastasis and possibly revascularization of recurrent tumors \[[@pone.0135607.ref004], [@pone.0135607.ref006]--[@pone.0135607.ref010], [@pone.0135607.ref034]\]. The realization that hypoxic microvasculature exists as early as the developmental stage \[[@pone.0135607.ref004], [@pone.0135607.ref005]\] and plays a key-role in tumor progression and treatment response, brings about new possibilities for the role of hypoxia in cancer biology. Improved radiation delivery techniques allow the potential for dose painting tumor areas found to harbor hypoxic tumor vessels. In addition, creating a new target with an exogenously administered agent of high specificity to the tumor vasculature and tumor microenvironment (e.g. hypoxia) presents a unique and ideal platform for drug delivery to tumor and stromal cells via antibody directed drug conjugates, targeted liposomes and other targeted drug-encapsulated technologies \[[@pone.0135607.ref027]\]. While some "non-specific" binding with non-targeted-microbubbles were noted in this approach, further study and refinement of the microbubble imaging technique should provide insight into the underlying variables involved and could potentially reveal further applications of this imaging technique if correlated with tumor IFP or other diagnostic tumor physiology \[[@pone.0135607.ref032], [@pone.0135607.ref033]\]. The data presented herein suggests tumor vessel hypoxia is a detectable physiological phenomenon utilizing a new method of pimonidazole-targeted contrast-enhanced microbubbles confined to the microcirculation of a tissue. Segmenting subtypes of hypoxia with an imaging technique that possess capillary level resolution, provides exciting new opportunities for longitudinal studies investigating vascular hypoxia in tumor progression and therapeutic response. The method described is based on generating artificial targets able to be 'created' on-demand by injection or ingesting pimonidazole, an already clinical approved agent. Thus, this method does not rely on heterogeneously or transiently expressed biomarkers of any kind, is not affected by genetic variability or constrained to the tumor microenvironment; instead, it is a snapshot of hypoxia occurring in and around vasculature in a variety of ischemic conditions.

Supporting Information {#sec016}
======================

###### Anti-pimonidazole antibody binds cell surface pimonidazole antigens in the presence of hypoxia.

MBα-pimo does not bind endothelial cells without pimonidazole (*left* vs. *right panels*), and hypoxic conditions (*top vs*. *bottom panels*).

(TIF)

######

Click here for additional data file.

###### Evaluation of cell surface anti-pimonidazole antigens by flow cytometry.

Pimonidazole adduct formation on the cell membrane is preferentially formed under hypoxic conditions. Cells negative for 7-AAD, a fluorescent compound with a strong affinity for DNA were deemed viable and non-porous, thus preventing any significant degree of intracellular binding of anti-pimonidazole antibody.

(TIF)

######

Click here for additional data file.

###### 3D video of vascular hypoxia after off-site bursting all free microbubbles in circulation: detection of anti-pimonidazole functionalized microbubbles.

A 3D video was made using a customized Matlab algorithm to subtract the free flowing microbubble signal in order to display the relative amount and distribution of MBα-pimo, or tumor vessel hypoxia. Representative control (no pimonidazole) and pimonidazole-injected tumor bearing mice imaged with MBα-pimo are shown. A static B-mode image from the central region of the tumor is shown for orientation.

(PPTX)

######

Click here for additional data file.

We would like to thank Visualsonics corporation for ongoing consultation and support of their ultrasound imaging system. We also thank Dr. Rosalia Simmen for supplying the MMTV-Wnt-Tg mice. We also thank Dr. Dmitry Nedosekin for assistance with Figures. We acknowledge the Biotelemetry and Ultrasound Imaging Core for Small Animals.

[^1]: **Competing Interests:**The authors have declared that no competing interests exist. A patent, PCT/US2013/045867: Compositions and Methods for Non- Invasive Detection and Treatment of Hypoxic Cells In Vivo, is under review based on the authors' own original research. This does not alter the authors\' adherence to all the PLOS ONE policies on sharing data and materials.

[^2]: Conceived and designed the experiments: NAK RJG. Performed the experiments: NAK RJG AJ-P JL. Analyzed the data: NAK RJG SS KBV. Contributed reagents/materials/analysis tools: JAR. Wrote the paper: NAK RPMD RJG. Revised manuscript for content: MEH RPMD. Consulted on experimental design: KBV.


Q:

Why Are Mining Passwords 'x'

In any of the 'get started mining' type sites, I see that password is always x. In xmr-stak, it seems to suggest putting in 'mostly empty or x' as a password. Does this really mean I should be using some default one-character password, or am I just misreading?

A:

The 'x' is just a placeholder. It is up to the pool to decide how to use it. It is just an optional message that could be passed between a miner and a pool. For example, often pools allow miners to use it to assign individual workers names/IDs.


Historical background {#Sec1}
=====================

In higher organisms, blood and lymphatic vasculature is formed by two distinct processes: vasculogenesis and angiogenesis. Vasculogenesis is the de novo formation of vessels from hematopoietic precursor cells and predominantly takes place in the developing embryo. Angiogenesis, on the other hand, is the formation of vessels from preexisting vasculature by processes such as sprouting, pruning and intussusception \[[@CR1]\] and is an important biological process throughout the life of an organism, both under normal conditions and in disease. Impaired vessel function is the cause of many illnesses such as atherosclerosis, diabetic retinopathy, psoriasis, arthritis, malignant cell growth, neurodegenerative disease and a placental insufficiency, preeclampsia \[[@CR2]\]. Hematopoietic precursor cells are programmed by soluble factors, extracellular matrix (ECM) components as well as cell-cell contacts to mature to their final functional states during vessel formation. Research in this field has been profoundly stimulated by the discovery of a plethora of growth factors that instruct primordial cells to migrate, divide and differentiate and to give rise to endothelial cells that ultimately form blood and lymphatic vessels. Vascular endothelial growth factors, (VEGFs) are among the most important players that regulate vessel formation during embryonic development, in wound healing and in maintaining vessel homeostasis in adult organisms. In addition, impaired vessel function resulting from defects in VEGF ligands or receptors is the cause of many diseases. VEGF was originally described as vascular permeability factor (VPF), an activity released by tumor cells that promotes vascular leakage \[[@CR3]--[@CR12]\]. It is now clear that VPF represented a biological activity attributable to a family of polypeptide growth factors that are encoded by several genes. VEGFs specifically interact with hematopoietic cells, endothelial precursor cells, such as the angioblasts, and with differentiating and mature endothelial cells. The best studied variant is VEGF-A, which activates a plethora of signaling pathways through VEGF receptor-2 (VEGFR-2) and regulates vessel morphogenesis through VEGFR-1. The importance of VEGF-A and its receptors in vascular development has been best illustrated in knockout mice. Both VEGF-A \[[@CR13]\] and VEGFR-2 knockouts \[[@CR14], [@CR15]\] are lethal due to a deficiency in blood vessel formation, while VEGFR-1 knockouts show overgrowth of immature vessels that blocks the development of a fully developed functional vasculature and leads to embryonic death \[[@CR16]\]. The function of VEGFs in vessel formation is complemented by additional factors, such as basic fibroblast growth factor (bFGF) \[[@CR17]\], transforming growth factor β (TGFβ) \[[@CR18]\], platelet-derived growth factors (PDGFs) \[[@CR19]\] and angiopoietins \[[@CR20]\].

VEGF homologs also exist in arthropods, where they regulate hemocyte development upon binding to PDGF/VEGF receptors (PVRs). Apparently, in such simpler organisms, a single growth factor performs the tasks performed by PDGF and VEGF in higher organisms \[[@CR21]--[@CR23]\]. Apart from their role in vessel development and homeostasis, VEGF family proteins play diverse roles in other organs such as the neural system, bones, the hematopoietic system and the reproductive organs, which have been excellently reviewed recently \[[@CR24]\].

Biological function of VEGF receptors and their ligands {#Sec2}
=======================================================

VEGF family proteins {#Sec3}
--------------------

VEGF polypeptides belong to the PDGF family of growth factors. They are dimeric cysteine-linked secreted glycoproteins with an M~r~ of approximately 40 kDa. In mammals, VEGFs are encoded by a family of genes that includes VEGF-A, -B, -C, -D \[[@CR25]\] and PlGF \[[@CR26]\]. Highly related proteins called VEGF-E are encoded by pox viruses of the Orf family \[[@CR27]--[@CR29]\] and additional variants, collectively called VEGF-F, have been isolated from snake venoms \[[@CR30]--[@CR35]\]. Alternative splicing and proteolytic processing of VEGFs give rise to a number of functionally distinct isoforms with different signaling properties \[[@CR36], [@CR37]\]. For example, Bates and colleagues described an interesting variant, VEGF-A165b, closely related to VEGF-A165, that carries sequences encoded by exon 9, instead of exon 8, at the carboxy terminus \[[@CR38], [@CR39]\]. When added together with VEGF-A165 to endothelial cells, this variant inhibited VEGF signaling. The concept emerging from such studies is that multiple isoforms of VEGFs, binding their receptors with similar affinity, yet eliciting distinct signaling properties, are responsible for the bewildering complexity of VEGF-induced signal output.

VEGFs are expressed in response to hypoxia and when cells encounter specific growth and differentiation factors and are produced by many cell types, in particular by hematopoietic, stromal and endothelial cells \[[@CR40]--[@CR42]\]. One of the most prominent examples is the stimulation of VEGF expression by cancer cells that cannot form tumors bigger than a few millimeters unless they produce angiogenic growth factors \[[@CR2], [@CR43]\].

VEGF receptors {#Sec4}
--------------

The biological functions of VEGF polypeptides are mediated upon binding to type III receptor tyrosine kinases (RTKs), VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4) \[[@CR2], [@CR44]--[@CR47]\]. VEGF receptors are closely related to Fms, Kit and PDGF receptors. They consist of seven extracellular immunoglobulin (Ig)-like domains, a transmembrane (TM) domain, a regulatory juxtamembrane domain, an intracellular tyrosine kinase domain interrupted by a short peptide, the kinase insert domain, followed by a sequence carrying several tyrosine residues involved in recruiting downstream signaling molecules. These receptors are expressed on the cell surface of many bone-marrow-derived cells such as hematopoietic cells \[[@CR48]\], macrophages and endothelial cells \[[@CR49]\], on some malignant cells \[[@CR50]\] and on vascular smooth muscle cells (VSMCs) \[[@CR51]\]. Mutation analysis of the extracellular domains of VEGFR-1 and -2 showed that the second and third Ig-like domains constitute the high-affinity ligand-binding domain for VEGF with the first and fourth Ig domains apparently regulating ligand binding and receptor dimerization, respectively \[[@CR52]--[@CR54]\]. VEGFs show distinct patterns of receptor specificity as indicated in figure [1](#Fig1){ref-type="fig"}. VEGF-A binds to VEGFR-1 and -2 and to receptor heterodimers, while VEGF-C and -D bind VEGFR-2 and -3. Receptor-specific interactions have been described for some VEGF variants: PlGF \[[@CR55], [@CR56]\] and VEGF-B \[[@CR57]\] exclusively bind VEGFR-1 and VEGF-E interacts only with VEGFR-2 \[[@CR27], [@CR58]\]. VEGF-F variants interact with either VEGFR-1 or -2, e.g. VR-1 and Vammin bind only to VEGFR-2 \[[@CR32], [@CR33]\]. VEGF-A, -B and PlGF are predominantly required for blood vessel formation, while VEGF-C and -D are essential for the formation of lymphatic vessels \[[@CR59], [@CR60]\]. Figure 1Schematic representation of VEGF family ligands and their receptors.

Regulation of receptor activity {#Sec5}
-------------------------------

RTKs are activated upon ligand-mediated receptor dimerization \[[@CR61]--[@CR63]\]. Earlier published work suggested that ligand binding is not directly responsible for receptor dimerization but may induce conformational changes in the extracellular Ig domain 4 which then promote receptor dimerization \[[@CR64]--[@CR67]\]. Ligand-induced dimerization leads to structural changes transduced to the intracellular kinase domain upon rearrangement of the transmembrane and the juxtamembrane domain. The molecular mechanisms responsible for activation of the intracellular kinase domain of these receptors are poorly understood at present. Kit and PDGF receptors mutated in the trans- or juxtamembrane domains are constitutively active and often oncogenic \[[@CR68], [@CR69]\]. Similarly, a role of the TM domain in receptor activation has been elegantly documented for PDGF receptors carrying artificially engineered dimerization motifs \[[@CR70]\]. These data clearly establish that the TM and the juxtamembrane domains are involved in regulating receptor kinase activity.

All three VEGF receptors contain tyrosine phosphorylation sites that have an either regulatory or signaling function. While phosphorylation of sites in the juxtamembrane domain and the lower lobe of the kinase domain presumably modulates receptor structure and kinase activity, others act as docking sites for SH2-domain-containing signaling molecules. Four experimental approaches have been used to study the biological function of these phosphorylation sites: (i) mutation analysis in which specific tyrosine residues were mutated and receptor-associated signaling molecules and biological output were determined, (ii) phosphopeptide mapping of receptors isolated from resting and ligand-stimulated cells labeled with inorganic phosphate *in vivo*, (iii) phosphopeptide mapping of *in-vitro*-phosphorylated receptors, (iv) determination of the phosphorylation state of specific residues with phosphotyrosine-specific antibodies. Each of these methods has its shortcomings and published data on receptor activity and binding of downstream signaling molecules are therefore often difficult to compare. Mutational analysis, for example, might change receptor structure and therefore biological activity, while results from *in vivo* and *in vitro* phosphorylation analysis are biased by the different turnover of tyrosine phosphates labeled under these conditions.

VEGFR-1 regulates blood vessel morphogenesis {#Sec6}
--------------------------------------------

VEGFR-1 is an 180-kDa glycoprotein expressed in many hematopoietic cells. The receptor is required for normal blood vessel development during embryogenesis, since homozygous deletion of VEGFR-1 is lethal in mice at embryonic day E8.5 due to severe malformation of the vasculature \[[@CR16]\]. A VEGFR-1 splice variant lacking the intracellular tyrosine kinase and the transmembrane domain, sVEGFR-1 or sFlt-1, has been shown to be deficient in signaling, yet is expressed in many tissues during normal embryonic development. This molecule apparently acts as a decoy for VEGF ligands \[[@CR71]--[@CR73]\] and is clinically associated with a placental insufficiency, called preeclampsia, observed in some patients late in pregnancy \[[@CR74]\]. The view that VEGFR-1 kinase activity is dispensable for vessel development at particular developmental stages is further supported by the finding that a kinase-inactive VEGFR-1 mutant rescues VEGFR-1 null mutant mice \[[@CR75]\]. More recent data indicate that the kinase activity of VEGFR-1 plays an essential role during pathological angiogenesis and in wound healing, by potentiating VEGFR-2 signaling \[[@CR76]--[@CR78]\], however, the molecular details for this receptor cross-talk have not yet been elucidated. Undisputed is the role of kinase-active VEGFR-1 in recruiting hematopoietic cells from bone marrow precursors \[[@CR79], [@CR80]\].

VEGFR-1 has poor kinase activity compared with VEGFR-2 due to the presence of a repressor motif in the juxtamembrane domain, making studies on receptor phosphorylation difficult \[[@CR81]\]. A wide variety of signaling molecules has been shown to be activated by VEGFR-1 upon recruitment to specific phosphorylation sites \[[@CR82]--[@CR86]\]. Tyr1213 and 1333 serve as binding sites for adaptor molecules such as Nck, Crk, Grb-2 \[[@CR84], [@CR87]\], Sck \[[@CR88]\], the regulatory p85 subunit of phosphatidylinositol (PI) 3-kinase \[[@CR85]\] and the phosphatase SHP-2 \[[@CR87]\]. Phospholipase C*γ*-1 (PLC*γ*-1) has been shown to associate with VEGFR-1 via Tyr794 and 1169 \[[@CR86], [@CR89]\]. Tyr1242 and 1327 are minor phosphorylation sites with no interacting intermediates described to date. The downstream signaling pathways activated by VEGFR-1 are not well characterized and only weak proliferative or migratory effects are mediated by this receptor in endothelial cells. Conflicting reports indicate that VEGFR-1 has mitogenic potential in a PLC*γ*-1- and phosphokinase C (PKC)-dependent or independent manner \[[@CR89]--[@CR91]\]. Taking into account more recent results from P. Carmeliet's group, these data may arise from receptor cross-talk with VEGFR-2 \[[@CR76]\]. A single study shows activation of Fyn and Yes, two members of Src family kinases \[[@CR92]\], but no biological function has been attributed to these interactions. The function of VEGFR-1 is best established in monocyte migration and differentiation \[[@CR79], [@CR93]\], in the recruitment of endothelial cell progenitors from bone marrow \[[@CR80], [@CR94], [@CR95]\], the migration and invasion of carcinoma cells \[[@CR96]\] , the production of growth factors by liver sinusoidal endothelial cells \[[@CR97]\] and the adhesion of natural killer cells to endothelial cells \[[@CR98]\].

VEGFR-2 is the predominant receptor in angiogenic signaling {#Sec7}
-----------------------------------------------------------

VEGFR-2 is a 200-kDa glycoprotein expressed in hematopoietic \[[@CR48], [@CR99], [@CR100]\], neural \[[@CR101]--[@CR103]\] and retinal cells \[[@CR104]\]. VEGFR-2 regulates endothelial cell migration, proliferation, differentiation and survival as well as vessel permeability and dilation. Among the 19 tyrosine residues present in the intracellular domain of VEGFR-2, seven putative phosphorylation sites have been described in some detail to date: Tyr801, 951, 996, 1054, 1059, 1175 and 1214. A comprehensive study of receptor activity based on *in vitro* phosphorylation of immunoprecipitated VEGFR-2, on receptor mutagenesis and on *in vivo* mapping with phosphorylation site-specific antibodies identified Tyr951, 1054, 1059, 1175 and 1214 as the most prominent phosphorylation sites and Tyr1305, 1309 and 1319 as minor sites, while Tyr801 and 996 phosphorylation was not detected in this study \[[@CR105]\]. Tyr1175 is clearly the most important site implicated in activation of many pathways via PLC*γ*-1 \[[@CR106]\]. A role for Tyr1175 in endothelial and hematopoietic cell signaling was also shown in knockin mice expressing a Tyr1175 mutant. In this study, animals expressing the mutant receptor died in utero from vascular defects similar to those observed in VEGFR-2 null mice \[[@CR107]\]. A mutagenesis study led to the identification of Tyr1008 as an additional site involved in activation of PLC*γ*-1, promoting endothelial cell tubulogenesis and differentiation but not cell proliferation \[[@CR108]\].

The mechanism of receptor activation is not understood in molecular detail. Tyr1054 and 1059 in the kinase domain, which are homologous to regulatory residues present in all protein kinases, were tentatively identified as autophosphorylation sites \[[@CR109], [@CR110]\]. Similar to other RTKs, the putative phosphorylation sites Tyr801 and 822 located in the juxtamembrane domain may be phosphorylated following dimerization of VEGFR-2 and maintain the receptor in an active conformation \[[@CR111]\]. Mutation analysis also showed that Tyr996 and 1214 in the kinase and the carboxy-terminal domain are essential for receptor activation; however, one should bear in mind that such results may derive from structural changes in these mutant receptors \[[@CR109], [@CR112]\].

VEGFR-2 is downregulated and dephosphorylated upon internalization into endocytic vesicles \[[@CR109], [@CR113]\]. Both inactivation by directly associated phosphatases as well as dephosphorylation upon association with other membrane receptors carrying associated phosphatases such as SHP-1 and -2 have been described \[[@CR114], [@CR115]\]. A particularly interesting case is the downregulation of VEGFR-2 by tumor necrosis factor *α* (TNF*α*), which recruits SHP-1 to the plasma membrane \[[@CR116], [@CR117]\]. Finally, a variety of less well characterized signaling molecules have been shown to associate with the activated receptor and to transmit downstream signals. These include the Ras GT-Pase-activating protein GAP \[[@CR118]\], the adaptor proteins Nck \[[@CR118]\], Grb-2, Grb-10 and Sck \[[@CR88], [@CR114], [@CR119], [@CR120]\] and the human cellular protein tyrosine phosphatase A (HCTPA) \[[@CR121]\].

VEGFR-2 promotes mitogenesis {#Sec8}
----------------------------

VEGF-mediated endothelial cell proliferation depends on the activation of multiple pathways downstream from VEGFR-2. Similar to other members of the RTK family, VEGFR-2 activates the classical Ras-dependent signaling cascade impinging on MAP kinases such as ERK1 and 2 \[[@CR122]\]. In this pathway, VEGFR-2 recruits Grb-2, either by a direct interaction involving the putative docking site at Tyr1214 \[[@CR123]\], or via association with the adaptor protein Shc \[[@CR114]\]. Phosphorylation of Grb-2 leads to activation of the nucleotide exchange factor Sos followed by activation of Ras and stimulation of the Raf1/MEK/ERK signaling cascade. Cross-talk between ERK1 and 2 and another MAP kinase family member, c-Jun N-terminal protein kinase (JNK), has also been described, rendering difficult the assignment of specific biological roles to the individual MAP kinase family members \[[@CR124]\]. Activated MAP kinases translocate to the nucleus where they phosphorylate transcription factors and regulate gene expression.

Most studies on VEGF signaling agree that receptor recruitment of PLC*γ*-1 upon phosphorylation of Tyr1175 is essential for stimulation of mitogenesis. Activation of PLC*γ*-1 promotes phosphatidylinositol 4,5-bisphosphate (PIP~2~) hydrolysis giving rise to 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP~3~). Production of DAG activates PKC resulting in Ras-independent Raf activation which leads to stimulation of ERK activity \[[@CR106], [@CR125]--[@CR128]\]. Treating cells with PKC inhibitors blocked mitogenic signaling by VEGF supporting a role for PKC in this pathway \[[@CR129]\]. Receptor association of PLC*γ*-1 via Tyr801 has been reported using receptor mutants \[[@CR86]\] and mutation analysis of VEGFR-2 showed that Tyr801 and 1175 are putative docking sites for the p85 subunit of PI 3-kinase \[[@CR130]\]. PI 3-kinase has also been implicated in mitogenic signaling by VEGFR-2, based on treatment of cells with specific inhibitors \[[@CR131]\]. This lipid kinase regulates the S6 kinase/Akt pathway which has been shown in many cell types to stimulate cell growth. Conflicting results suggest, however, that PI 3-kinase is not required for VEGFR-2-mediated mitogenesis \[[@CR122], [@CR126]\]. Taken together, these data, mostly based on mutagenesis studies, should be interpreted with some care since changes in receptor structure that alter association with signaling molecules may affect receptor readout.

Finally, c-Src and nitric oxide (NO) \[[@CR132]\] have been identified as intracellular mediators of VEGF signaling, and heparan sulfate and components of the ECM act as extracellular modulators in mitogenic signaling \[[@CR133], [@CR134]\], while the classical mechanism of 'contact inhibition of growth', described for epithelial cells or fibroblasts and mediated by VE-cadherin in endothelial cells, is reponsible for regulation of cell proliferation in a density-dependent manner \[[@CR135]\].

VEGFR-2 regulates cytoskeleton organization and cell migration {#Sec9}
--------------------------------------------------------------

The motogenic signals transduced by VEGFR-2 impinge on focal adhesion kinase (FAK), which regulates focal adhesion assembly and disassembly and actin organization. VEGF regulates FAK phosphorylation and activity and leads, together with paxillin and actin-anchoring proteins such as talin or vinculin, to recruitment of this kinase to focal adhesions \[[@CR136], [@CR137]\]. FAK activation has been shown to require signaling by PKC \[[@CR136]\]. In addition, Nck/PAK (p21-activated kinase) was shown to promote FAK phosphorylation and cell migration \[[@CR138]--[@CR141]\]. Src-dependent FAK phosphorylation was reported to regulate cell migration and survival \[[@CR142]\] and, consistent with these data, the Src kinase inhibitor M475271 blocked VEGF-induced endothelial cell migration \[[@CR143]\]. Besides its role in cellular proliferation, Tyr1175 phosphorylation has been shown to regulate stress fiber formation, focal adhesion assembly and cell migration upon recruitment of the adaptor protein Shb and subsequent activation of PI 3-kinase and FAK \[[@CR144]\]. More recently, Tyr951 located in the kinase insert domain and presumably phosphorylated by an associated kinase when VEGFR-2 is activated, was shown to recruit the adaptor molecule VRAP/TSAd (VEGF-receptor-associated protein/T-cell-specific adaptor molecule) in a subfraction of endothelial cells \[[@CR105]\]. This adapter molecule associates with Src, PI 3-kinase and PLC*γ*-1 and regulates actin organization and cell migration, but it also plays a crucial role in tumor angiogenesis \[[@CR105], [@CR145]\].

VEGF-induced endothelial cell migration is also mediated by stress activated protein kinase 2, SAPK/p38 \[[@CR139], [@CR146]\]. SAPK/p38 activity is regulated by Src and related focal adhesion protein kinase/proline-rich tyrosine kinase 2 (RAFTK/Pyk2) \[[@CR147]\]. Ca^2+^ mobilization and activation of Cdc42 have been shown to lead to SAPK/p38 phosphorylation \[[@CR147], [@CR148]\] and to induce phosphorylation of MAPKAP kinases 2 and 3 and the small heat shock protein HSP27 \[[@CR146], [@CR149]\]. This leads to the release of phosphorylated HSP27 from capped actin filaments, actin reorganization and the formation of stress fibers and lamellipodia which promote cell migration \[[@CR150]\]. Furthermore, a role for the small GTPases Rho and Rac, which modulate actin dynamics and cell contraction in endothelial cell migration and which are regulated by G proteins such as Gq/11 and Gbg and by PLC, is well documented \[[@CR151]\].

Signaling by VEGFR-2 is essential for cell survival {#Sec10}
---------------------------------------------------

VEGF also protects endothelial cells against apoptosis *in vitro* and *in vivo. In vivo*, the role of VEGF in preventing apoptosis is restricted to immature vessels that lack pericytes, as indicated by experiments showing that VEGF is essential for endothelial cell survival and blood vessel development in early postnatal life, but not in adult mice \[[@CR152]--[@CR154]\]. Activation of the PI 3-kinase/Akt pathway by VEGF protects cultured cells against apoptosis induced by serum starvation \[[@CR155], [@CR156]\]. Signal output from VEGFR-2 maintains adequate levels of active PI 3-kinase and thereby regulates the production of the second messenger phosphoinositide(3,4,5)trisphosphate (PIP~3~) which is required for activation of the serine/threonine kinase Akt \[[@CR157]\]. VEGF was also shown to induce the expression of anti-apoptotic molecules including the caspase inhibitors Bcl-2 and A1 \[[@CR158]\] and IAP (inhibitors of apoptosis) family proteins \[[@CR159]\]. Finally, survival signaling by VEGFR-2, PI 3-kinase and Akt depends on the integrity of adherens junctions that contain a transient tetrameric complex composed of VEGFR-2, PI 3-kinase, VE-cadherin and *β*-catenin \[[@CR160], [@CR161]\]. Disruption of this multimeric complex by loss or truncation of VE-cadherin induced endothelial cell apoptosis and blocked the transmission of PI 3-kinase-dependent survival signals.

VEGFR-2 regulates vessel permeability {#Sec11}
-------------------------------------

VEGF also regulates vascular permeability, and indeed was initially described as a vascular permeability factor \[[@CR3]\]. Increased vascular permeability is observed shortly after VEGF administration concomitant with the formation of so-called vesicular-vacuolar organelles, VVOs, \[[@CR162]\] and fenestrae \[[@CR163]--[@CR165]\]. These specialized regions in the plasma membrane of endothelial cells are highly permeable for macromolecules. How these membrane structures are formed is still unclear, but based on data from mutant mice lacking both c-Src and c-Yes, which showed reduced vascular permeability after VEGF administration, a role for Src family kinases has been suggested \[[@CR166]\]. Addition of VEGF to endothelial cells induces expression of the plasmalemmal vesicule-associated protein (PLVAP), a component of diaphragmed endothelial fenestrations. PLVAP expression is regulated by PI 3-kinase and SAPK/p38 \[[@CR167]\]. In mature vessels, VEGF also regulates vascular permeability by loosening the junctions between endothelial cells, giving rise to the formation of transcellular gaps. Phosphorylation of major components of tight, adherens and gap junctions, such as VE-cadherin \[[@CR168]--[@CR170]\], *β*-catenin \[[@CR168], [@CR171]\], occludin and zonula occluden 1 \[[@CR172], [@CR173]\] and of connexin 43 \[[@CR174]\] have been reported in response to VEGF. Vessel dilation and permeability are also regulated by NO which is upregulated by Akt upon induction of endothelial NO synthase (eNOS) expression \[[@CR175], [@CR176]\]. Blocking eNOS and cyclooxygenase with specific inhibitors prevents the release of NO and prostacyclin (PGI2) and blocks VEGF-induced vessel hyperpermeability \[[@CR177]\].

VEGFR-3 regulates lymphangiogenesis {#Sec12}
-----------------------------------

VEGFR-3 is synthesized as a 195-kDa precursor protein consisting of seven extracellular Ig-like domains, a TM and an intracellular kinase domain. The protein is proteolytically processed in the fifth Ig domain giving rise to a 125- and a 75-kDa chain held together by a disulfide bond \[[@CR178], [@CR179]\]. Expression of this receptor starts at E8.5 of mouse development in all embryonic endothelial cells. After E8.5, VEGFR-3 expression is only seen on developing veins and lymphatics but not on arteries \[[@CR59], [@CR180]\]. Later in development, the expression gradually becomes restricted to lymphatic vessels. In VEGFR-3 null mice, vascular remodeling and maturation are abnormal in larger vessels with a defective lumens causing fluid accumulation in the pericardial cavity and embryonic death at E9.5 \[[@CR181]\]. Recent reports also showed expression of the receptor on blood vessels in the vicinity of tumors as well as on several benign and malignant tumor cells \[[@CR182], [@CR183]\]. A role for VEGFR-3 in cell-to-cell signaling in adult blood vessel angiogenesis, where the expression of this receptor and its ligands seems to be induced by VEGF-A, has also been suggested \[[@CR184]\]. VEGFR-3 is activated by VEGF-C and -D, and proteolytic processing of these ligands gives rise to variants also interacting with VEGFR-2, although with lower affinity than with VEGFR-3. VEGFR-3 apparently also heterodimerizes with VEGFR-2 in lymphatic endothelial cells, expanding the repertoire of signaling pathways activated by this receptor \[[@CR185]\]. Paracrine expression of VEGF-C at sites of lymphatic sprouting further supports a role for VEGFR-3 in the development of lymphatic vessels \[[@CR186]\]. VEGFR-3 promotes cell migration and survival in lymphatic endothelia by inducing PKC-dependent MAP kinase activation and via a wortmannin-sensitive pathway requiring PI 3-kinase and Akt \[[@CR187], [@CR188]\]. In addition, a splice variant of this receptor has been shown to bind SHC \[[@CR189]\]. That signaling via VEGFR-3 is a prerequisite for lymphangiogenesis is further supported by data showing that a soluble, kinase-deficient receptor variant blocks lymphatic vessel formation \[[@CR190], [@CR191]\].

VEGF receptors form multiprotein complexes with various coreceptors {#Sec13}
===================================================================

VEGF signaling is complicated by the fact that the ligands and their receptors interact with additional cellular proteins such as neuropilins, heparan sulfate, integrins and cadherins. These interactions allow coordination of signal strength, timing and specificity with extracellular cues arising from soluble ligands, cell-cell and cell-substratum interactions.

Association with heparan sulfate and neuropilins {#Sec14}
------------------------------------------------

The short form of VEGF-A, VEGF-A121, encoded by exons 2--5 and 8, consists of a receptor-binding domain specific for VEGFR-1 and -2 and is a poor mitogen. The longer VEGF-A165 isoform, that also contains sequences encoded by exon 7, binds the receptors with similar affinity as VEGF-A121 but displays increased signaling potential \[[@CR192]--[@CR194]\]; the underlying mechanism remains unclear at present. All isoforms carrying exon 7, or 6 plus 7, such as VEGF-A165, VEGF-A183, VEGF-A189 and VEGF-A203, interact with proteins of the neuropilin family and with heparan sulfate \[[@CR193]--[@CR202]\]. Neuropilin-1 and -2 interact with VEGFR-2 and -1, respectively, stimulating signaling by these receptors. Interaction of VEGF-A165 with neuropilin-1 is particularly important for endothelial tip cell guidance where regulation of cell migration is the predominant signal output \[[@CR203], [@CR204]\]. VEGF-A165 has also been shown to interact with neuropilin-1 and VEGFR-2 expressed separately on adjacent cells. This may be particularly important during endothelial cell guidance when vessels are formed along tracks predetermined by neural cells \[[@CR205]--[@CR207]\].

Association with integrins {#Sec15}
--------------------------

Integrins play an important role in cell signaling linking intracellular signaling pathways activated by soluble factors to output elicited by cellular interactions with the ECM and with neighboring cells. Specific integrins bind to the extracellular domain of VEGFR-2 and augment receptor signaling \[[@CR208]\]. Integrins of the *β*~3~ subfamily specifically bind to the extracellular domain of VEGFR-2 resulting in increased receptor activation upon VEGF stimulation \[[@CR209]--[@CR211]\]. Direct interaction between *β*~3~ integrin and VEGFR-2 is restricted to *α*~v~*β*~3~ and was shown to be either ligand independent \[[@CR210]\] or dependent \[[@CR211]\]. VEGFR-2 signaling in the context of *α*~1~*β*~1~ and *α*~2~*β*~1~ integrins has been shown to regulate lymphangiogenesis during tissue repair, further demonstrating how output from VEGF receptors is modulated by cellular interactions with the ECM \[[@CR212]\]. VEGFR-2-mediated angiogenesis is also directly regulated by integrins as proposed by work performed in knockout mice lacking *β*~3~ or *β*~5~ integrin \[[@CR213], [@CR214]\]. Animals that did not express these integrins showed increased VEGFR-2 activity and tumor vascularization. Taken together these studies suggest that integrins act as 'gatekeepers', preventing aberrant stimulation of resting endothelial cells under non-pathological conditions, or as 'caretakers', which facilitate angiogenesis during vessel repair in disease \[[@CR215]\].

VEGF bound to the ECM promotes integrin-dependent cell spreading, migration and survival that do not require signaling by VEGF receptors \[[@CR216]\]. Similarly, sVEGFR-1 interacts with *α*~5~*β*~1~ integrin thus becoming part of the ECM and promoting cell migration and spreading \[[@CR217]\]. In addition, interaction of VEGF-A with fibronectin, an ECM component interacting predominantly with *α*~5~*β*~1~ and, to a minor extent, with the vitronectin receptor *α*~v~*β*~3~, has been reported recently \[[@CR218]\]. Finally, in cells grown on fibronectin instead of vitronectin or collagen, increased biological activity of VEGF and augmented endothelial cell migration and MAP kinase activity were reported \[[@CR219]--[@CR221]\].

Association with cadherins {#Sec16}
--------------------------

Cadherins are involved in the formation of adherens junctions in endothelial and epithelial cells and play an essential role in VEGF signaling \[[@CR160], [@CR222]--[@CR225]\]. Interaction of VEGFR-2 with VE-cadherin is regulated by *β*-catenin. At high cell density, the phosphatase PTP1/Dep1/CD148 associates with VE-cadherin and attenuates tyrosine phosphorylation of VEGFR-2 thereby suppressing signaling via PI 3-kinase, MAP kinases and PLC*γ*-1 \[[@CR135]\]. At low cell density, such as at the tip of developing blood vessels, VEGFR-2 associates with integrin *α*~v~*β*~3~ instead of cadherin and signal output is directed toward stimulation of cell migration and mitogenesis \[[@CR215]\]. Reduced turnover of VEGFR-2 at high cell density has also been demonstrated to depend on cadherin and to enhance VEGF-mediated activation of MAP kinases \[[@CR226]\]. To a large extent, vessel homeostasis is determined by cell-to-cell junctions that play an essential role during formation of a mechanosensory complex that regulates the response of endothelial cells to fluid shear stress \[[@CR227]\].

Structure of VEGF receptors and their ligands {#Sec17}
=============================================

The structures of VEGF-A \[[@CR228], [@CR229]\], PlGF \[[@CR230]\] and the snake venom components Vammin and VR-1 \[[@CR31]\] have been solved. These molecules form homodimers and fold into a cysteine-knot structure also described for other growth factors \[[@CR231]\]. The structure of Ig domain 2, which is part of the ligand-binding domain of VEGFR-1, has also been determined \[[@CR232]\] and there are structural data available for complexes formed between VEGF-A and PlGF with domain 2 of VEGFR-1 \[[@CR233]--[@CR235]\]. These structural studies were complemented with biochemical investigation of receptor-blocking peptides \[[@CR236], [@CR237]\]. Biochemical analysis also led to the identification of specific residues determining receptor selectivity \[[@CR56], [@CR229], [@CR238], [@CR239]\].

The extracellular Ig-like domain 2 is sufficient for high-affinity binding of VEGF-A to VEGFR-1, while domains 2 and 3 are required for binding to VEGFR-2 \[[@CR54]\]. VEGF mutants with altered loops L1 and L3 were used to determine the molecular basis for receptor selectivity of VEGF polypeptides \[[@CR239], [@CR240]\]. We created similar chimeric molecules based on our recently solved structure of the VEGFR-2-specific VEGF-E NZ2 variant. Loops L1 and L3 are tightly associated at their base and apparently function as a structural entity determining receptor selectivity, while L2 is required for interaction with both receptors \[unpublished data\].

The structure of the heparan-sulfate- and neuropilin-1-binding domain of VEGF-A, encoded by exons 7 and 8, has been solved by nuclear magnetic resonance spectroscopy \[[@CR241]\]. Analysis of the electrostatic surface potential shows an extended patch of positively charged residues on one side of the molecule that is a candidate binding site for negatively charged heparan sulfate side chains. All VEGFs carrying sequences encoded by exon 7 bind to heparan sulfate both *in vitro* and *in vivo* \[[@CR242]\]. However, high-affinity interaction with heparan sulfate, and particularly with neuropilin-1, also requires the short carboxy-terminal peptide encoded by exon 8 as shown by our laboratory \[unpublished data\].

Clearly, additional in-depth structural information is required for a comprehensive understanding of VEGF interaction with VEGFR-1 and -2, neuropilin-1 and heparan sulfate. Similarly challenging is the task to unravel the structural changes in the intracellular kinase domain following ligand binding to the extracellular domain. A first step in this direction is the resolution of a partial structure of the kinase domain of VEGFR-2 \[[@CR243]\]. Such information will be useful to dissect the activation mechanism of VEGF receptor kinases and to engineer more specific reagents interfering with receptor activation, with the goal to block or stimulate VEGF signaling in disease.

Conclusions {#Sec18}
===========

Endothelial cells integrate signals elicited by cell-cell contacts, cell-extracellular matrix interactions and angiogenic growth factors. The final signal output results from the formation of context-specific signaling modules in distinct membrane compartments where receptor activity is tuned to the specific needs of a particular cell and aberrant signaling is suppressed \[[@CR244]\]. Signal specificity of VEGF receptors arises from combinatorial activation of multiple cellular pathways. Each receptor subtype assembles a distinct set of signaling molecules in a spatially and temporally controlled manner giving rise to the formation of specific signal transduction modules or 'signalosomes' at the plasma membrane. *In vivo*, cells release VEGFs into their neighborhood in a temporally and spatially well-defined manner that allows gradual formation of receptor-ligand complexes. This significantly differs from tissue culture models where growth factors are usually administered as a single bolus, and great caution is required when extrapolating such *in vitro* data to the *in vivo* situation. Signal output is also determined by competition among the various VEGF receptors for VEGFs that interact with more than one receptor isoform and is influenced by the kinetics with which receptors are activated by different ligands. Finally, the exact three-dimensional structure of each ligand-receptor-coreceptor complex determines the efficacy with which intracellular tyrosine residues are phosphorylated and subsequently exposed to downstream signaling molecules. This has an impact on the strength and the kinetics with which individual signaling pathways are activated and execute their tasks.

This work was supported by grants from the Swiss National Foundation (3100A0-100204, 3100B0-10345/1 and 3100-054441), from Schweizerische Krebsliga (KLS-01220-02-2002), from the Hauptabteilung für die Sicherheit der Kernanlagen des Bundesamtes für Energiewirtschaft and by grants from the Paul Scherrer Institut. We are grateful to M. Pieren for critical reading the manuscript.

Received 15 September 2005; received after revision 11 November; accepted 24 November 2005


Patterns of outpatient use of mental health services for Medicaid beneficiaries under a prepaid mental health carve-out.
This study examines the impact of a mental health carve-out, the Utah Prepaid Mental Health Plan (UPMHP), on use of outpatient mental health services by Medicaid beneficiaries with schizophrenia. Data were collected through interviews with the same group of Medicaid schizophrenic beneficiaries. A pre/post comparison with a contemporaneous control group examined the impact of the program on type of outpatient services used by beneficiaries. The results indicate a greater reliance on medically-oriented outpatient mental health services in treatment of beneficiaries under the UPMHP. Medicaid beneficiaries with schizophrenia in the UPMHP group received relatively fewer day treatment visits, but relatively more medication visits and individual therapy visits over the first 3 1/2 years of the program.


Wednesday, March 28, 2012

Support for nuclear: Broad but shallow?

Coming just upon the heels of a recent post about public opinion on energy sources, I couldn't help but also notice this poll by Gallup which NEI points to, indicating a relatively constant support for nuclear following Fukushima. The takeaway? Despite a small uptick in opposition to nuclear following the events at Fukushima (from 38% to 40%), public support for nuclear is still high, sitting at 57% in favor or strongly in favor of nuclear energy. While Republicans (and Republican-leaners) more strongly favored nuclear energy (65% supporting vs. 34% opposed) compared to Democrats (and associated leaners - 50% support / 45% opposed), support across the Gallup poll appears to generally be broad across parties.

The starkest reported differences in opinion were between men and women - 74% of men vs. 42% for women. Tellingly, a nearly parallel trend occurs for the perceived safety of nuclear (with 72% of men believing nuclear is safe compared to 43% of women).

Given the somewhat less sunny projections from the recent Pew poll, how does one square the difference?

Digging into the Gallup data, one observes that strong support and opposition have both historically ranged around 23% and 21%, respectively; the bulk of support and opposition has been in the more moderated "somewhat support" (33%) and "somewhat oppose" (19%). How has this changed in the events following Fukushima? Overall, not much - overall support remains constant at 57%, although one observes some erosion in self-identified "strong" support. Meanwhile, strong opposition has hardened (growing from 18% to 24% in the last polling period).

One might square this against the Pew data in the sense that Pew specifically asked about the expansion of nuclear energy (particularly in comparison to other energy sources), while Gallup simply gauged overall public support. If one takes both trends as true, it paints a picture of public opinion over nuclear as stuck in neutral - a public which overall supports nuclear energy but is divided about its expansion in light of other perceived alternatives.

Meanwhile, going back to the Gallup results, overall public perception of nuclear safety is relatively unchanged (57% responding that U.S. nuclear plants are safe, while 40% respond that they feel U.S. nuclear plants are unsafe). While the continued (accurate!) perception of the safety of the U.S. nuclear industry is encouraging, it speaks to the need for further outreach efforts, as well as perhaps the unique perception of nuclear compared to other sources (i.e., where constant, elevated risk - such as hazards presented by sources such as coal - is tolerated much more readily than low-frequency, highly dramatic events, even if the ultimate public health consequences are minuscule).

Given the above, clearly nuclear is not in danger of a German-style phaseout. Yet the obvious challenge for public support of nuclear - beyond simply tolerating it, but in expanding it - is in making the case to the public that nuclear is both safe and essential - the latter of which comes down to arguments both over economics and the environment.


80% Chance Precip. • Wind 13 mph • Gusts at 20 mphSnow showers in the morning will give way to a mixture of rain and snow in the afternoon. High 39F. Winds S at 10 to 20 mph. Chance of precip 80%. Snow accumulations less than one inch.


admin General

Last week I introduced baby Sean to sweet potatoes and this week we will start on peas. This recipe will make about 6 servings (2 tablespoons each) Feel free to double up on this recipe and freeze the extras. This is a great way to be prepared for the following week.

2 cups frozen peas or fresh if they are in season

1/2 cup water

1/2 formula or breast milk


I've been contacted by jaynyy and we've discussed moving this update forward. Jay is going to start by replacing the ReplayGain 1.0 loudness measurement with BS 1770-2. There are pointers to some of the other improvements we've discussed on the wiki talk page.

Further discussions of other improvements can happen here or on the talk page.


Q:

absolute/relative paths for php includes

I have a function(orders) with many php includes in a file called extract.php
http://example.com/application/dir1/dir2/dir3/extract.php
function orders ($username)
{
 require ("functions.inc.php");
 define("DATAGRID_DIR", "./../datagrid/");
 define("PEAR_DIR", "./../datagrid/pear/");
 require_once (DATAGRID_DIR . 'datagrid.class.php');
 require_once (PEAR_DIR . 'PEAR.php');
 require_once (PEAR_DIR . 'DB.php');
 require_once ('./../datagrid/classes/database.class.php');

//function code here
}

I have another file(install.php) that calls this function inside the same directory and it works very well.
<?php
include ("extract.php");
orders ("643443876996");
?>

However, when i include it in another function in this path
http://example.com/orders/getorder.php, it gives me error that it cannot find my files 'datagrid.class.php';
    <?php
    include ("include('../application/dir1/dir2/dir3/extract.php');");
    orders ("643443876996");
    ?>

How can I get the path referencing correct?

A:

Reference from the server root, either with $_SERVER['DOCUMENT_ROOT'] or
require_once (DATAGRID_DIR . '/application/dir1/dir2/dir3/datagrid.class.php');
etc...


Q:

How to attach extra data to user (for password reset) with Kuzzle

I want to make a reset password feature on a Kuzzle stack but I don't know where to put extra data (like a token for reset password) in the user document.
Furthermore, the user should not be able to change the data itself.
Should I use security.updateUser endpoint and prevent user access with a hook or a pipe ?

A:

Yes you can use the security.updateUser endpoint to add extra data to your user.
What you can do to prevent the user itself to change his data is either create a role which forbid access to the controller security and action updateUser (see https://docs.kuzzle.io/guide/1/essentials/security) or make a pipe plugin on before:updateUser which will remove the extra data from the query if present (see https://docs.kuzzle.io/plugins/1/essentials/pipes)
Full disclosure: I work at Kuzzle


Silencer Swap?

timoyz1

On my last high speed get off of my 07 WR 450, one of the damaged items was my silencer. The tubing is kinked enough that it is beyond saving. Being broke, and needing to get back on my bike, I found a 06 YZ 450 silencer for pretty cheap. I know it won't have a spark arrestor (dont worry, I'll fix it), but will it bolt up to my bike? I didn't know if the aluminum frames were a copy from the YZ's or not. It obviously doesn't have the same part number, but anyone know if it will work, or do I keep shopping?

odlaw19

I'm running a YZ450 06 muffler on my WR450 07. Bolts up fine. The only thing is that the WR header is a smaller diameter. I had to use some exhaust tape to seal it properly.

I have since put a YZ450 08 titanium header on. It is even bigger diameter than the 06 YZ header. I had an exhaust shop expand the clamp section of the tube and i installed a new YZ450 08 gasket. Its all good now!!!


[Muscle reflex responses to paired electrical stimulation of peripheral nerves].
Double-shock bifocal peripheral nerve stimulation with varying interstimulus intervals (ISI) was applied to healthy human subjects. Short (SLR) and long latency reflexes (LLR) from the slightly active first dorsalis interosseous muscle of the hand were recorded after an electrical conditioning stimulation of the index finger and test stimulation of the median nerve. It was found with intervals from 1 to 5 ms the size of the SLR and LLRs was smaller and unstable, possibly due to transmitter depletion at these short ISIs. With intervals of 10 ms and longer, the test responses progressively increased and were far larger than the control responses, probably due to some temporal summation with double-shock stimulation and some release of the transmitter substance after the conditioning cutaneous stimulus. (Fig. 3, Ref. 18.).


15 Kid-Friendly Halloween Crafts

Halloween is quickly approaching, so the time to start picking your costume and making jack-o-lanterns is now! On top of those fun things, it’s also time to make your home a little spookier, both inside and outside.

Instead of heading to the store and picking up the items you’ll need for frights and screams, you can spend some quality time with your family and make your own Halloween decorations. Not only will you be able to unleash your children’s creativity, but you’ll be able to fill your house with ghosts, skeletons and witches for a fraction of the price.

Here are 15 crafts you can do together with your family so that Halloween is as festive and fun as it should be!

We’ve never seen something like this before, but we really want to try it out!

Once you’ve cleaned up the paper scraps, wiped up the glue and rinsed out the paint brushes, come get a bite to eat at Cheddar’s. We prepare our food just like you did your crafts: straight from scratch. We’d love to serve you some of our delicious scratch-made food that’s scary good!

About

At Cheddar’s Scratch Kitchen, we’ve been graciously serving made-from-scratch goodness since 1979: Quality ingredients, prepared fresh in our kitchen. It’s the right way, not the easy way. Because it tastes better that way.


Studies of female infertility include use of a GnRH agonist to treat endometriosis in monkeys, development of surrogate embryo transfer combined with exogenous steroid therapy and use of "pure" FSH to stimulate ovarian folliculogenesis. We have made antisera to a follicular fluid factor that blocks estrogen-induced LH surges and employed a mechanical pulse pump to treat secondary amenorrhea. An anti-progesterone compound was evaluated for its contraceptive potential by direct action on the endometrium for induction of menses. CT scans were employed to verify the conservation of the cortical mantel in hydrocephalic monkeys given shunts in utero. Thymic peptides were found to stimulate the hypothalamic-pituitary-adrenal axis.


Q:

PowerBI Service Account granting duplicate execute permissions on stored procedure almost constantly

I have PowerBi Server (Jan 2019) installed on my server. I can see from the default trace that that the PowerBi Service account NT SERVICE\PowerBIReportServer is granting execute permissions on a stored procedure ExtendEditSessionLifetime to the RSExecRole (within the ReportServer database):
GRANT EXECUTE ON [dbo].[ExtendEditSessionLifetime] TO RSExecRole

This is happening frequently many times during the hour, most hours (around 50 times an hour) there doesn't seem to a pattern that I can see and I can't find any documentation online about what the stored procedure does. I can't see anything obvious in the PowerBI logfiles or the event viewer either.
What could be causing this (or is it intended behaviour?)

A:

I have worked out what is happening here
If I run:
SELECT * FROM sys.sql_modules WHERE definition LIKE '%GRANT EXECUTE ON \[dbo\].\[ExtendEditSessionLifetime\] TO RSExecRole%' ESCAPE '\'

I can see stored procedures that have that grant statement within them.
This returned one record and the definition field begins
CREATE PROC [dbo].[ExtendEditSessionLifetime]      @EditSessionID varchar(32)......

If I navigate to this stored procedure in the object tree and right click and script it out, I can see the stored procedure is written in this format
CREATE PROC [dbo].[ExtendEditSessionLifetime]
    -- params
AS
BEGIN
    --code
END

GRANT EXECUTE ON [dbo].[ExtendEditSessionLifetime] TO RSExecRole
GO

I wasn't aware of this but it seems that when you create a stored procedure, you can place a GRANT after the END and it becomes part of the definition (I thought it had to be part of the BEGIN .... END construct (when there is one)
Therefore, everytime ExtendEditSessionLifetime is run, it is re-granting permissions to RSExecRole


---
author:
- |
    Sahil Bhatia\
    \
    \
    Rishabh Singh\
    \
    \
bibliography:
- 'references.bib'
title: Automated Correction for Syntax Errors in Programming Assignments using Recurrent Neural Networks
---

=1


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Desde la Star Wars Celebration 2019 en Chicago, Electronic Arts ha revelado los primeros detalles, imágenes y tráiler de Star Wars Jedi: Fallen Order, el ambicioso juego de aventuras y acción en tercera persona, de Respawn Entertainment, centrado en la narrativa del universo Star Wars.

Con su lanzamiento fijado para el 15 de noviembre de 2019 en PC (Origin), PlayStation 4 y Xbox One; y alejado de las tendencias actuales, Star Wars Jedi: Fallen Order ofrecerá una campaña para un único jugador, sin opciones multijugador y sin microtransacciones ni loot boxes.

La historia de Star Wars Jedi: Fallen Order

Ambientado en la saga ‘Tiempos Oscuros’ tras Star Wars Episodio III – La Venganza de los Sith y la caída de los Jedi, los jugadores encarnarán a Cal Kestis, interpretado por el actor Cameron Monaghan (Shameless, Gotham), un joven padawan que ha escapado de la Orden 66, el decreto del Emperador para eliminar a todos los Jedi de la galaxia.

Con la misión de completar su entrenamiento Jedi y dominar el sable de luz, la historia comenzará en el planeta Bracca, donde Cal se oculta del Imperio. Allí se verá forzado a huir cuando sus poderes de la Fuerza quedan expuestos y comience a ser perseguido por la Segunda Hermana, una Inquisidora de élite, y los Soldados de Purga, unas nuevas fuerzas imperiales especiales entrenadas para rastrear a los Jedi y ayudar a los Inquisidores en su vil tarea.

Sin embargo, en su peligrosa historia, Cal Kestis no estará solo, pues su aventura le llevará a aliarse con amigos como Cere o su droide BD-1, así como algunas caras conocidas. Los fans de Star Wars reconocerán localizaciones, armas, equipamiento y enemigos, así como descubrirán nuevos personajes, lugares, criaturas, droides y adversarios.

“Cuando Respawn se acercó a nosotros con esta historia, les apoyamos de inmediato. Un videojuego de un solo jugador centrado en la narrativa del universo Star Wars es exactamente lo que estábamos buscando y también sabemos que los fans han estado esperándolo. Centrarse en la vuelta de Cal como Jedi tras la Orden 66 abre un gran abanico de oportunidades en la jugabilidad y de ricas tramas de la historia para desarrollar este nuevo personaje y su trasfondo.”, declaró Steve Blank, Director de Contenido y Estrategia de Franquicia de LucasFilm.

La jugabilidad de Star Wars Jedi: Fallen Order

Desarrollado en Unreal Engine 4, Star Wars Jedi: Fallen Order se configura como un juego de combate cuerpo a cuerpo intenso y meditado donde se combinan las técnicas melee con un innovador sistema de combate con sables luz y habilidades de la Fuerza.

En el desplazamiento, y heredado de otros juegos del estudio -como Titanfall-, se incluyen habilidades de desplazamiento al estilo parkour con las que “acabar con oponentes de forma estratégica y solucionar puzles en su camino a través de esta aventura por la galaxia”.

Todo este sistema de juego, y a falta de verlo en movimiento con un gameplay del título, puntualizan desde Electronic Arts que ha sido diseñado con la intención de replicar las intensas batallas con sable de luz que se ven en las películas.

“Muchos fans de Star Wars, entre los que nos incluimos, estamos muy interesados en la saga ‘Tiempos Oscuros’ y queríamos explorar esa misteriosa y peligrosa época. El equipo de LucasFilm han sido unos colaboradores increíbles, ayudándonos a explorar esta parte de la línea de tiempo y crear una narrativa en la que el Imperio está en la cumbre del poder y los Jedi se esconden. En este contexto, hemos creado un videojuego de combate cuerpo a cuerpo intenso, meditado y divertido haciendo uso del sable de luz y los poderes de la Fuerza y permitiendo a todo el mundo cumplir el sueño de convertirse en Jedi.”, declaró Stig Asmussen, director del juego y reputado desarrollador tras aclamados títulos como la trilogía God of War.

A continuación puedes ver el primer tráiler de Star Wars Jedi: Fallen Order.


OIG Says Lab Can’t Provide Free Services

In a recent advisory opinion, the Office of Inspector General (OIG) nixed a proposed arrangement whereby a multi-regional laboratory (Lab) sought to provide free services to out-of-network patients in exchange for exclusivity in referrals from the physicians.

Under the proposed arrangement the Lab envisioned entering into agreements with physician practices to provide all lab services required by the practice regardless of patients’ insurance coverage. In exchange, explained the Lab, the physicians obtained the “ease of communication and consistency in the reporting of test results,” that is often associated with working with a single laboratory.

Approximately 70% of the Lab’s physician-practices, however, have patients whose insurances limit the provision of lab services to specific labs (this amounted to about 10-40% of patients). To remove this roadblock the Lab proposed not to bill the patient, the practice or the health plan if it performed out-network services as long as the Lab had the exclusive agreements with the physician practice.

Under the agreement each party would represent that neither is getting a financial benefit from such an arrangement and the only item the Lab would offer to the physician would be a limited-use interface for electronic submissions.

Not so fast.

The OIG took a negative view of the proposed arrangement. It stated that the proposed arrangement potentially implicates the anti-kickback statute and the prohibition on charging Medicare or State health care programs substantially in excess of the provider’s usual charges.

The OIG took issue with the Lab’s locking down the physician referral stream. Since the OIG saw the “main purpose” of the arrangement as securing all of the referrals from a physician practice, including services paid for by federal funds, it analyzed whether remuneration would flow to the referral source.

The OIG’s position on the provision of free or below-market goods or services to actual or potential referral sources is longstanding and clear: such arrangements are suspect and may violate the anti-kickback statue, depending on the circumstances. If the physicians or physician practices would receive remuneration from the [Lab], the anti-kickback would be implicated.

In this case, while there would be no direct payments from the Lab to the physician/physician practice, the proposed arrangement would still be problematic, said the OIG. The proposed arrangement would offer the physician practices a reduction in administrative burden (the convenience of consistent lab references) and possibly financial costs associated with using multiple labs (avoiding fee that some electronic health record vendors charge to connect to different labs). Furthermore, the arrangement had the potential to lead to “inappropriate steering of patients,” observed the agency.

The OIG also found problematic the elimination of fees for out-of-network patients while Medicare and Medicaid would continue to be billed at regular fees for services rendered to beneficiaries. “Thus, the proposed arrangement would essentially result in a two-tiered pricing structure,” said the OIG.

As proposed, the arrangement would generate prohibited remuneration, said the agency, and the parties stood to face potential sanctions such as exclusion and the imposition of civil money penalties.

If you have questions about this advisory opinion, the anti-kickback statute or have other health law questions, please contact our office.

Deniza Gertsberg, Esq. is an attorney representing healthcare providers in New York and New Jersey. This article is published for informational purposes only; it contains no legal advice whatsoever. Publication of this article does not create an attorney-client relationship. Please see our full disclaimer.

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Physicochemical, functional, and macromolecular properties of waxy yam starches discovered from "Mapuey" (Dioscorea trifida) genotypes in the Venezuelan Amazon.
"Mapuey" tubers in Venezuela are staple food for indigenous peoples from the Caribbean coast and Amazon regions. Noticeable differences between genotypes of yam starches were observed. Granules were large, triangular, or shell-shaped with monomodal particle size distribution between 24.5 and 35.5 μm. Differential scanning calorimetry (DSC) analyses revealed onset gelatinization temperatures from 69.1 to 73.4 °C with high gelatinization enthalpy changes from 22.4 to 25.3 J g(-1). All X-ray diffractograms of starches exhibit B-type crystallinity. Crystallinity degrees varied from 24% to 40%. The highest crystallinity was found for the genotype having the highest amylose content. Iodo-colorimetric, amperometric, and DSC amylose determinations varied from 1.4 to 8.7%, 2.2 to 5.9%, and 1.4 to 3.5% for Amazonian genotypes, in comparison with commercial Mapuey starches: 12.0, 9.5, and 8.7%, respectively. Solubility and swelling power at 90 °C varied from 2.1 to 4.4% and 20.5 to 37.0%, respectively. Gel clarity fluctuated from 22.4 to 79.2%, and high rapid visco analyzer (RVA) viscosity was developed at 5% starch suspension (between 1430 and 2250 cP). Amylopectin weight average molar mass M(w), radius of gyration R(G), hydrodynamic coefficient ν(G), and apparent molecular density d(Gapp) were determined using high-performance size exclusion chromatography (HPSEC) and asymmetrical flow field flow fractionation (A4F) techniques coupled with multiangle laser light scattering (MALLS) on the Dioscorea trifida genotypes exhibiting the lowest and highest amylose contents. Amylopectins showed very similar molecular conformations. M(w) values were 1.15 × 10(8) and 9.06 × 10(7) g mol(-1) using HPSEC and A4F, respectively, thus, 3-5 times lower than those reported with the same techniques for other yam species, and very close to those of potato and cassava amylopectins. This discovery of a new natural amylose-free starch in the neglected yam "Mapuey" could present some potential for the food industry.


Posted by Archie Magoulas, Technical Information Specialist, Food Safety and Inspection Service, USDA in Health and Safety

Summer object and food icons. Text: USDA’s summer food safety tips.

Whether it’s a family BBQ, camping, hiking or going to the beach, summer activities can get hot! When food is a part of those activities, keep in mind the old saying: Safety first!

USDA’s Meat and Poultry Hotline is here to help you with food safety for all your summer plans. It has been assisting Americans with all types of food safety questions and concerns since 1985. Here are just a few:

Question: I fell asleep and left hot dogs and hamburgers sitting out on the counter all night. If I reheat them, they will still be safe to eat, because I’ll kill all of the bacteria, right?

Answer: No. Cooking these items may kill the bacteria and other organisms, but it does not get rid of the dangerous toxins formed in the “danger zone” between 40°F and 140°F. Room temperature, of course, is part of that ‘zone.’ Never leave perishable foods out any longer than two hours at room temperature, no longer than one hour when the temperature is 90°F or higher. Those hot dogs and hamburgers left out should be tossed.

Question: We grilled our beef burgers to 160°F, as measured by a food thermometer, but they are still pink inside. Are they still safe to consume?

Answer: Yes. Ground beef can be pink inside, even after it is safely cooked. The pink color can be due to a reaction between the heat and myoglobin, which causes a red or pink color. It can also occur when vegetables containing nitrites are cooked along with the meat. Because doneness and safety cannot be judged by color, it is very important to use a food thermometer when cooking ground beef. To be sure all harmful bacteria are destroyed, cook ground beef to an internal temperature of 160°F as measured with a food thermometer. For burgers, be sure to insert the thermometer probe into the side of the burger and get close to the center for an accurate read.

Question: What’s the best way to travel with perishable food items that will later be grilled at the park?

Answer: Pack perishable foods directly from the refrigerator or freezer into the cooler. A full cooler will maintain its cold temperatures longer than one that is partially filled. Meat and poultry items may be packed while still frozen, keeping them colder longer. Be sure to keep raw meat and poultry at the very bottom of the cooler, wrapped separately from cooked foods and foods meant to be eaten raw, such as fruits. If the cooler is only partially filled, pack the remaining space with more ice or cooling packs. For long trips, take along two coolers — one for immediate food needs, such as lunch, drinks or snacks for the car trip, and the other for perishable foods to be used later. Limit the number of times the cooler is opened. Open and close the lid quickly.

Question: I’m planning ahead for my family’s trip to the beach, are there any food safety tips I should consider?

Answer: Good thinking in planning ahead! Take along only the amount of food that can be eaten to avoid having leftovers. To keep your cooler cool, partially bury it in the sand, cover it with blankets and shade it with a beach umbrella. Bring along disposable moist towelettes for cleaning hands. If dining along the boardwalk. The “danger zone” is between 40 and 140°F, so don't eat anything that has been sitting out in the hot sun for more than two hours (one hour when the temperature is above 90°F) — that would be an invitation for foodborne illness and a spoiled vacation.

The hotline helps prevent foodborne illness by answering your questions about the safe food storage, handling and preparation. Live food safety experts with backgrounds in food science, nutrition and health science are available to answer your questions.

If you have a question about food safety for your summer activities, call 1-888-MPHotline (1-888-674-6854). Hours of operation are 10 a.m. to 6 p.m., Monday through Friday, ET. Callers may also listen to food safety messages on various topics, recorded in English and Spanish, 24 hours a day. You can also email or chat via Ask Karen and Pregúntele a Karen.


Combine this discounted with this latest promotion at HS Sweden! Socks, briefs, and boxers are on sale for up to 50% off in the summer clearance sale. In addition, you will receive free shipping: no minimum order is needed. Then, you can use our Exclusive coupon codeBargainMoose_HS_Sweden to receive an extra 15% off your order.

Summertime is when I become addicted to ankle socks. While I appreciate the added warmth of a mid-calf sock during the winter; the summer is just too hot for that much fabric. In the sale, these Leopard Low Socks are on sale from $12 down to $5.88 along with the Zebra low socks. Each pair of ankle socks comes down to $4.99 after our exclusive coupon code. You will also receive free shipping.

If you prefer the higher socks, then there are plenty of options as well. I have pulled out some of the socks that are steeply discounted as examples. The Striped sock (Black, Blue, Green, Grey, Orange, Red, and Yellow) was $13, and is now $6.37. After coupon code, pay just $5.41. In fact, you can grab a pair of blue/red Argyle Socks for the same price and as well as many other socks including the Five-Colour Sock (Black, Green, Grey, and Yellow) and the Stripe Toe Sock in pink/red.

If your sock box is already full (I know that mine is), then you may want to refresh your underwear drawer. There are plenty of underwear choices for women under $10 per pair. I rather like the pattern on the Bark Women's Brief. Originally $13, these underwear are on sale for $8.40. Use the promo code above to score a pair for just $7.14. I like HS Sweden underwear because they are made of 95% cotton and 5% Elastane. Essentially, they are quite breathable and gentle on the skin. They are perfect for hot weather.

For men, there are a couple different styles available. I prefer the Dot Boxer Brief as a basic but not too basic pair of men's briefs. I like the more formfitting cut and the mini white dots on black are nice too. Originally $20, these briefs are on sale for $14. Use our exclusive coupon code to pick up each pair for just $11.90.

With free shipping on any order, you can buy as many or as few pairs as you want. Usually, HS Sweden requires four 'pairs' (underwear or socks) in order to receive free shipping.