text:
Effects of the dipolar form of phloretin on potassium conductance in squid giant axons
G. R. Strichartz, G. S. Oxford, F. Ramon
The effects of phloretin on membrane ionic conductances have been studied in the giant axon of the squid, Loligo pealei. Phloretin reversibly suppresses the potassium and sodium conductances and modifies their dependence on membrane potential (Em). Its effects on the potassium conductance (GK) are much greater than on the sodium conductance; no effects on sodium inactivation are observed. Internal perfusion of phloretin produces both greater shifts in GK (Em) and greater reductions in maximum GK than does external perfusion; the effect of simultaneous internal and external perfusion is little greater than that of internal perfusion alone. Lowering the internal pH, which favors the presence of the neutral species of weakly acidic phloretin (pKa 7.4), potentiates the actions of internally perfused phloretin. Other organic cations with dipole moments similar to phloretin's have little effect on either potassium or sodium conductances in squid axons. These results can be explained by either of two mechanisms; one postulates a phloretin 'receptor' near the voltage sensor component of the potassium channel which is accessible to drug molecules applied at either the outer or inner membrane surface and is much more sensitive to the neutral than the negatively charged form of the drug. The other mechanism proposes that neutral phloretin molecules are dispersed in an ordered array in the membrane interior, producing a diffuse dipole field which modifies potassium channel gating. Different experimental results support these two mechanisms, and neither hypothesis can be disproven.
Biophysical Journal
Decapodiformes
Potassium Channels
Loligo
Membrane Potentials
Cations
Strichartz, G. R., Oxford, G. S., & Ramon, F. (1980). Effects of the dipolar form of phloretin on potassium conductance in squid giant axons. Biophysical Journal, 31(2), 229-246.
Effects of the dipolar form of phloretin on potassium conductance in squid giant axons. / Strichartz, G. R.; Oxford, G. S.; Ramon, F.
In: Biophysical Journal, Vol. 31, No. 2, 1980, p. 229-246.
Strichartz, GR, Oxford, GS & Ramon, F 1980, 'Effects of the dipolar form of phloretin on potassium conductance in squid giant axons', Biophysical Journal, vol. 31, no. 2, pp. 229-246.
Strichartz GR, Oxford GS, Ramon F. Effects of the dipolar form of phloretin on potassium conductance in squid giant axons. Biophysical Journal. 1980;31(2):229-246.
Strichartz, G. R. ; Oxford, G. S. ; Ramon, F. / Effects of the dipolar form of phloretin on potassium conductance in squid giant axons. In: Biophysical Journal. 1980 ; Vol. 31, No. 2. pp. 229-246.
@article{5c19cea1806b40a894f5654195c331e9,
title = "Effects of the dipolar form of phloretin on potassium conductance in squid giant axons",
abstract = "The effects of phloretin on membrane ionic conductances have been studied in the giant axon of the squid, Loligo pealei. Phloretin reversibly suppresses the potassium and sodium conductances and modifies their dependence on membrane potential (Em). Its effects on the potassium conductance (GK) are much greater than on the sodium conductance; no effects on sodium inactivation are observed. Internal perfusion of phloretin produces both greater shifts in GK (Em) and greater reductions in maximum GK than does external perfusion; the effect of simultaneous internal and external perfusion is little greater than that of internal perfusion alone. Lowering the internal pH, which favors the presence of the neutral species of weakly acidic phloretin (pKa 7.4), potentiates the actions of internally perfused phloretin. Other organic cations with dipole moments similar to phloretin's have little effect on either potassium or sodium conductances in squid axons. These results can be explained by either of two mechanisms; one postulates a phloretin 'receptor' near the voltage sensor component of the potassium channel which is accessible to drug molecules applied at either the outer or inner membrane surface and is much more sensitive to the neutral than the negatively charged form of the drug. The other mechanism proposes that neutral phloretin molecules are dispersed in an ordered array in the membrane interior, producing a diffuse dipole field which modifies potassium channel gating. Different experimental results support these two mechanisms, and neither hypothesis can be disproven.",
author = "Strichartz, {G. R.} and Oxford, {G. S.} and F. Ramon",
journal = "Biophysical Journal",
publisher = "Biophysical Society",
T1 - Effects of the dipolar form of phloretin on potassium conductance in squid giant axons
AU - Strichartz, G. R.
AU - Oxford, G. S.
AU - Ramon, F.
N2 - The effects of phloretin on membrane ionic conductances have been studied in the giant axon of the squid, Loligo pealei. Phloretin reversibly suppresses the potassium and sodium conductances and modifies their dependence on membrane potential (Em). Its effects on the potassium conductance (GK) are much greater than on the sodium conductance; no effects on sodium inactivation are observed. Internal perfusion of phloretin produces both greater shifts in GK (Em) and greater reductions in maximum GK than does external perfusion; the effect of simultaneous internal and external perfusion is little greater than that of internal perfusion alone. Lowering the internal pH, which favors the presence of the neutral species of weakly acidic phloretin (pKa 7.4), potentiates the actions of internally perfused phloretin. Other organic cations with dipole moments similar to phloretin's have little effect on either potassium or sodium conductances in squid axons. These results can be explained by either of two mechanisms; one postulates a phloretin 'receptor' near the voltage sensor component of the potassium channel which is accessible to drug molecules applied at either the outer or inner membrane surface and is much more sensitive to the neutral than the negatively charged form of the drug. The other mechanism proposes that neutral phloretin molecules are dispersed in an ordered array in the membrane interior, producing a diffuse dipole field which modifies potassium channel gating. Different experimental results support these two mechanisms, and neither hypothesis can be disproven.
AB - The effects of phloretin on membrane ionic conductances have been studied in the giant axon of the squid, Loligo pealei. Phloretin reversibly suppresses the potassium and sodium conductances and modifies their dependence on membrane potential (Em). Its effects on the potassium conductance (GK) are much greater than on the sodium conductance; no effects on sodium inactivation are observed. Internal perfusion of phloretin produces both greater shifts in GK (Em) and greater reductions in maximum GK than does external perfusion; the effect of simultaneous internal and external perfusion is little greater than that of internal perfusion alone. Lowering the internal pH, which favors the presence of the neutral species of weakly acidic phloretin (pKa 7.4), potentiates the actions of internally perfused phloretin. Other organic cations with dipole moments similar to phloretin's have little effect on either potassium or sodium conductances in squid axons. These results can be explained by either of two mechanisms; one postulates a phloretin 'receptor' near the voltage sensor component of the potassium channel which is accessible to drug molecules applied at either the outer or inner membrane surface and is much more sensitive to the neutral than the negatively charged form of the drug. The other mechanism proposes that neutral phloretin molecules are dispersed in an ordered array in the membrane interior, producing a diffuse dipole field which modifies potassium channel gating. Different experimental results support these two mechanisms, and neither hypothesis can be disproven.
JO - Biophysical Journal
JF - Biophysical Journal


text:
Q: Unavailable Shared Library - INSTALL_FAILED_MISSING_SHARED_LIBRARY I am taking over a project, and am trying to launch it in the Android Emulator. I get the following error in Logcat:
11-11 06:21:42.215: ERROR/PackageManager(58): Package com.android.xxx requires unavailable shared library com.google.android.maps; failing!
Console displays:
Installation error: INSTALL_FAILED_MISSING_SHARED_LIBRARY

How can I make sure com.google.android.maps is included in my project?
UPDATE:
I am using a min sdk level of 3 in my androidmanifest.xml:
</application>
    <uses-sdk android:minSdkVersion="3"/>

A: When you crate your emulated device, make sure you choose "Google APIs..." as the target. If you choose just "Android..." you won't have advanced APIs such as Maps available on the emulated device.

redpajama_set_name: RedPajamaStackExchange


The stock Azumi Speed 5 (Aka Speed 5B) firmware, ROM or flash file can be used to refresh the device model's firmware. It can also help with clearing malware and viruses and well as correct general firmware crashes resulting from corrupted files.
For those familiar with MediTek based devices, the Azumi Speed 5B should not be an issue. Anyone requiring a step by step guide or just assistance on this particular device model can post here.


text:
Chiral Modification of the Pd(111) Surface By Small Organic Molecules
Mausumi Mahapatra, University of Wisconsin-MilwaukeeFollow
Wilfred T. Tysoe
Dennis Bennett, Peter Geissenger, Alan Schwabacher, Jorg Woehl
Chiral modification of a metal surface is one of the most successful approaches of achieving enantioselective catalysis in heterogeneous phase. In this approach, an active metal surface is directly modified by adsorption of a chiral molecule (chiral modifier), the metal is responsible for catalytic activity while the adsorbed modifier controls the stereochemistry of adsorption and subsequent reactions through substrate interactions. So far, there are three types of widely recognized metal/modifier catalytic systems: tartaric acid modified Ni catalysts for the hydrogenation of β-ketoesters, cinchona modified Pt catalysts for hydrogenation of α-ketoesters and Pd catalysts modified with cinchona for selective activated alkene where high activity and enantiomeric excess is gained in comparison to the unmodified surfaces. However, the exact manner in which chirality is bestowed to a metal surface and how that affects a chiral reaction is not well understood and warrants the development of model studies (surface science analysis on well-defined single crystal surfaces under ultrahigh vacuum conditions). These model studies and surface science analysis are necessary to promote the fundamental understanding and to facilitate the rational design of a suitable metal/modifier system which is the principal focus of this dissertation.
This dissertation is primarily focused on two aspects. First, a number of complementary surface science studies have been performed to characterize four different chiral modifiers: D-alanine, (S,S) tartaric acid, L-aspartic acid and α-(1-naphthyl) ethylamine on a Pd(111) surface to gain insight into the way in which they impart chirality to the surface. Second, the enantioselectivity of the chirally modified surfaces has been measured in ultrahigh vacuum. This has been achieved by exposing the modified surface to both enantiomers of another chiral molecule (the probe), to see if there is any enantiospecific interaction between the modifier and the probe. The enantioselectivity is measured from the enantioselectivity ratio which is the ratio of relative coverages of two enantiomers of the probe on a surface modified with a single enantiomer of the modifier.
Combined experimental results and theoretical density functional theory calculations suggest that the amino acid modifiers impart chirality to the Pd(111) surface by an ensemble mechanism where they work collectively to form discrete chiral templates which interact with the chiral probe propylene oxide and glycidol enantiospecifically whereas, tartaric acid and naphthylethylamine provide individual chiral motifs which interact with the probes in a one to one fashion.
Mahapatra, Mausumi, "Chiral Modification of the Pd(111) Surface By Small Organic Molecules" (2015). Theses and Dissertations. 893.
Physical Chemistry Commons

redpajama_set_name: RedPajamaCommonCrawl


The Practice of the Presence of God: The Best Rule of Holy Life Brother Lawrence PREFACE "I believe in the communion of saints." SURELY if additional proof of its reality were needed, it might be found in the universal oneness of experimental Christianity in all ages and in all lands.... The Peace and the Presence of God. In this series, you'll find God's Word provides the encouragement and wisdom you need to stay on course, even in the toughest battles of the Christian life. In this series, you'll find God's Word provides the encouragement and wisdom you need to stay on course, even in the toughest battles of the Christian life.
THE PRACTICE OF THE PRESENCE OF GOD SERIES SPIRIT OF THANKSGIVING We are on a series of the Practice of the Presence of God. Last Sunday we spoke... The presence of God finds its greatest expression in Immanuel, God with us. God himself comes to save. Jesus Christ, the Son of God, entered human history to give his life as a ransom for many (Matt 20:28; Mark 10:45).
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The Practice of the Presence of God is a book of collected teachings of Brother Lawrence (born Nicholas Herman), a 17th-century Carmelite friar, compiled by Father Joseph de Beaufort.


text:
Taraz, April 2021
1 April 18 Shaʿban Thursday 05:37 06:54 13:21 17:50 19:44 21:01
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26 April 14 Ramadan Monday 04:49 06:13 13:15 18:09 20:12 21:37
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Lots of business will charge a shipping fee in addition to the rate they quote you, so make sure to ask. They use different bases for their fees. They stand by your side and help you in getting rid of trash in the easiest way at very affordable price. A couple of Chatham businesses operate in the majority of states, so take a look at a little selection and see where your nearest rental location is.
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text:
Research Article Free access | 10.1172/JCI40283
Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice
Mitomu Kioi,1 Hannes Vogel,2 Geoffrey Schultz,3 Robert M. Hoffman,4,5 Griffith R. Harsh,6 and J. Martin Brown1
1Division of Radiation and Cancer Biology, Department of Radiation Oncology, 2Department of Pathology, and 3Department of Surgery, Stanford University School of Medicine, California. 4AntiCancer Inc., San Diego, California. 5Department of Surgery, University of California, San Diego. 6Department of Neurosurgery, Stanford University School of Medicine.
Address correspondence to: J. Martin Brown, Department of Radiation Oncology, Stanford University School of Medicine, CCSR 1255, 269 Campus Drive West, Stanford, CA 94305-5152. Phone: 650.723.5881; Fax: 650.723.7382; E-mail: mbrown@stanford.edu.
Find articles by Kioi, M. in: JCI | PubMed | Google Scholar
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First published February 22, 2010 - More info
Published in Volume 120, Issue 3 on March 1, 2010
J Clin Invest. 2010;120(3):694–705. https://doi.org/10.1172/JCI40283.
First published February 22, 2010 - Version history
Received: June 23, 2009; Accepted: January 13, 2010
Resisting arrest: a switch from angiogenesis to vasculogenesis in recurrent malignant gliomas
Jeffrey P. Greenfield, … , William S. Cobb, David Lyden
The cellular and molecular events that initiate and promote malignant glioma development are not completely understood. The treatment modalities designed to promote its demise are all ultimately ineffective, leading to disease progression. In this issue of the JCI, Kioi et al. demonstrate that vasculogenesis and angiogenesis potentially play distinct roles in the etiology of primary and recurrent malignant gliomas, suggesting that patient therapy should perhaps be tailored specifically against the predominant vasculature pathway at a given specific stage of gliomagenesis.
Jeffrey P. Greenfield, William S. Cobb, David Lyden
Despite the high doses of radiation delivered in the treatment of patients with glioblastoma multiforme (GBM), the tumors invariably recur within the irradiation field, resulting in a low cure rate. Understanding the mechanism of such recurrence is therefore important. Here we have shown in an intracranial GBM xenograft model that irradiation induces recruitment of bone marrow–derived cells (BMDCs) into the tumors, restoring the radiation-damaged vasculature by vasculogenesis and thereby allowing the growth of surviving tumor cells. BMDC influx was initiated by induction of HIF-1 in the irradiated tumors, and blocking this influx prevented tumor recurrence. Previous studies have indicated that BMDCs are recruited to tumors in part through the interaction between the HIF-1–dependent stromal cell–derived factor–1 (SDF-1) and its receptor, CXCR4. Pharmacologic inhibition of HIF-1 or of the SDF-1/CXCR4 interaction prevented the influx of BMDCs, primarily CD11b+ myelomonocytes, and the postirradiation development of functional tumor vasculature, resulting in abrogation of tumor regrowth. Similar results were found using neutralizing antibodies against CXCR4. Our data therefore suggest a novel approach for the treatment of GBM: in addition to radiotherapy, the vasculogenesis pathway needs to be blocked, and this can be accomplished using the clinically approved drug AMD3100, a small molecule inhibitor of SDF-1/CXCR4 interactions.
Radiotherapy plays a major role in the treatment of glioblastoma multiforme (GBM). However, despite the use of radiosensitizers, radioactive implants, the combination of radiation with anticancer drugs, and most recently, high-dose boost stereotactic radiosurgery (1), GBMs invariably recur, leading to the death of more that 75% of the patients by 2 years. Importantly, most of the recurrences occur within the radiation field (2–4). Thus, any method of increasing the local control of the primary tumor by radiotherapy would produce a major improvement in the curability of GBM. At first sight, it is difficult to understand how the large radiation doses given locally to brain tumors do not result in cures, because even though all of the tumor cells may not be sterilized by radiation, it is unlikely that any of the endothelial cells in the tumor could survive the large doses given. Indeed, recent data demonstrate that the response of tumors to irradiation can be affected by the sensitivity of tumor endothelial cells (5, 6).
The tumor vasculature can arise from sprouting and proliferation of endothelial cells from local vessels (angiogenesis), co-option of preexisting vessels, or by colonization of circulating endothelial or other cells primarily from the BM (a phenomenon known as vasculogenesis). The paradox of the sensitivity of the vasculature to irradiation and the resistance of GBMs to radiotherapy could be resolved if, as postulated, circulating cells outside of the radiation field can recolonize and/or stabilize the tumor vasculature after irradiation, thereby supporting any remaining viable tumor cells. This restoration of the vasculature facilitated by bone marrow–derived cells (BMDCs) is observed in ischemic normal tissues and in malignant tissues (7–10). These BMDCs are also thought to create an environment for tumor growth and invasion, and metastasis (11–13).
Among the variety of BMDCs contributing to vasculogenesis, the population most investigated is circulating endothelial progenitor cells (EPCs) (8, 10, 14, 15), which are thought to incorporate into the vascular endothelium. However, the extent of BM-derived EPCs varies greatly with tumor type from 50% (5) to very few (16). CD45+ monocytic lineage, including CD11b+ myeloid cells, Tie2+ monocytes, VEGFR1+ hemangiocytes, and tumor-associated macrophages, has also been observed to incorporate into tumors. BM-derived myelomonocytic cells are often observed in the perivascular regions of the endothelium (17, 18) or colocalized with endothelial cells (19–21) and have been shown to stabilize the tumor vasculature (15).
In our previous study, we showed that mouse MT1A2 tumors subcutaneously implanted in preirradiated normal tissues produce a massive influx of BM-derived, MMP-9–expressing CD11b+ myelomonocytic cells into the tumors (22). Moreover, tumors cannot grow in preirradiated sites if the function of these monocytes is abrogated by growing the tumors in MMP-9–knockout mice. We postulated that this is a result of the fact that the local irradiation abrogates the sprouting and proliferation of endothelial cells from adjacent normal vessels, thereby forcing the tumor to rely on the "backup" pathway of vasculogenesis from circulating endothelial and other proangiogenic cells. In such a situation, blocking the vasculogenesis pathway (by deleting MMP-9) prevented tumor growth.
In the present study, we have tested the hypothesis that blocking the vasculogenesis pathway could improve tumor control by irradiation using an orthotopic model of GBM. We have identified 3 key components of the vasculogenesis pathway involving the mobilization of proangiogenic BMDCs and their recruitment to tumors. First, solid tumors develop regions of hypoxia, leading to an induction of the transcription factor HIF-1 (23), which has been shown to be a major player in the recruitment of BMDCs to tumors including GBM (24) and to ischemic regions of normal tissues (7). Importantly, tumors regrowing after irradiation or transplanted into an irradiated tissue are more hypoxic than control tumors (25–27) and would therefore be expected to have higher levels of HIF-1. In addition, there is evidence that local tumor irradiation and reoxygenation can induce HIF-1 in tumors (28). Thus, we postulated that inhibition of HIF-1 after tumor irradiation would reduce or abrogate the accumulation of proangiogenic BMDCs into tumors. Second, several investigators have shown that BMDCs are recruited to and retained in hypoxic normal tissues or in tumors by the hypoxia-dependent secretion of stromal cell derived factor–1 (SDF-1, CXCL12) which binds its receptor, CXCR4, on the BMDCs (7, 29–32). Thus, we tested AMD3100, an inhibitor of the SDF-1/CXCR4 interaction (33), and neutralizing antibodies to CXCR4 in combination with local tumor irradiation. Finally, we tested the effect of monocyte/macrophage depletion on the ability of the GBM model tumor to regrow after irradiation. With all 3 of the above strategies to interfere with the vasculogenesis pathway, we show that the radiation-induced uptake of BMDCs, specifically CD11b+ myelomonocytes, into tumors can be prevented and that tumors can be controlled by doses of radiation that, in the absence of these strategies, are ineffective in sterilizing the tumors. Our data therefore suggest that inhibiting the vasculogenesis pathway after local tumor irradiation represents a new strategy that has the potential to increase the cure rate of human tumors by radiotherapy.
Irradiation promotes recruitment of BMDCs into GBM. To investigate our hypothesis, we established an orthotopic brain tumor in a mouse model using human U251 GBM cells that were retrovirally transduced with the firefly luciferase gene in order to monitor tumor growth in real time. As shown in Figure 1, A and B, intracranially (i.c.) implanted U251 tumors grew progressively, and 5 daily doses of 2 or 4 Gy delivered locally to the tumors by irradiating the whole brain caused the tumors to regress and then regrow in a dose-dependent manner. We confirmed the tumor growth, infiltration into the surrounding normal brain, and regrowth after irradiation by histology and by MRI (Supplemental Figure 1, A and B; supplemental material available online with this article; doi: 10.1172/JCI40283DS1).
Irradiation promotes homing of BM-derived CD11b+ myeloid monocytic cells into GBM. (A) Representative bioluminescent images of mouse heads with or without fractionated irradiation. Firefly luciferase–transduced U251 (U251/pFB-Luc) tumors were implanted i.c. and were irradiated from day 11 to day 15. 5 × 2 Gy, 2 Gy irradiation 5 times daily. (B) Tumor radioresponse was measured by BLI (n = 5 per group). Error bars indicate SD. (C) Radiation induced the influx of BMDCs into the U251 i.c. tumor. GFP-BM–transplanted nude mice received whole brain irradiation at 0, 8, or 15 Gy on day 22. Scale bar: 50 μm. (D) Quantification of GFP-BM influx into the tumor. Error bars indicate SEM. ***P < 0.001 versus control. (E) Representative images of IHC for GFP-BM cells and CD11b+ myeloid monocytic cells in U251 i.c. tumor before and after irradiation. Arrowheads indicate CD11b+ GFP-BM cells. Scale bar: 50 μm. (F) Quantification of CD11b and GFP-BM influx into the tumor. Error bars indicate SEM. ***P < 0.001. (G) Characterization of CD11b+ infiltrating cells into tumor after irradiation. Representative images of IHC staining for DAPI (blue), CD11b (red), and other markers (VEGFR1, VEGFR2, Tie-2, or Gr-1; green) in i.c. tumors after irradiation. Scale bar: 50 μm. IR, irradiation.
We next examined whether local irradiation induced the recruitment of BMDCs into the tumors. Tumor-bearing nude mice that had been transplanted earlier with GFP-expressing BM (34) were sacrificed when the tumors grew back to their preirradiation size after irradiation at either 8 or 15 Gy. We found by both immunohistochemistry (IHC) and quantitative real-time PCR (qPCR) that irradiation induced BMDC influx in a dose-dependent manner (Figure 1, C and D, and Supplemental Figure 2A). If the cells that colonized the irradiated tumor were circulating EPCs derived from the BM, we would expect that the proportion of endothelial cells arising from the BM would be increased in tumors regrowing after radiation. However, we found that although the GFP BMDCs in the tumors were often associated with CD31-staining endothelial cells, they were not colocalized (<1%, Supplemental Figure 2B). This is in agreement with our previous study (22) that showed no increase in EPCs in tumors growing in preirradiated sites and is also consistent with several recent reports (16, 35–37). Significantly, we found that most of the induced influx of BMDCs into the tumors were CD11b+ myelomonocytes (Figure 1, E and F). However, non-GFP CD11b+ cells were modestly increased after irradiation (from 3 to 10 GFP–CD11b+ cells compared with 6 to 72 GFP+CD11b+ cells per high-power field [HPF]). CD11b+ cells levels in normal brain were very low compared with those in the irradiated tumor (Supplemental Figure 2C). Further characterization of the CD11b+ cells in the irradiated tumors revealed that most of them did not express VEGFR1 and VEGFR2. Approximately 20% of the CD11b+ cells expressed Gr-1. Interestingly, approximately 50% of the CD11b+ cells were Tie2 positive, although they were not found in nonirradiated tumors (Figure 1G and Supplemental Figure 2D).
Irradiation causes vascular damage and increased tumor hypoxia. We postulated that the CD11b+ BMDC recruitment is a result of the fact that local tumor irradiation sterilizes sufficient numbers of endothelial cells in and adjacent to the tumor to abrogate the sprouting and proliferation of endothelial cells from adjacent normal tissues and in tumor vessels, thereby forcing the tumor to rely on the vasculogenesis pathway stimulated by increased tumor hypoxia (7, 22, 24). To test this, we determined the level of tumor endothelial cells, blood flow, and hypoxia by measuring CD31 expression, lectin perfusion, and pimonidazole staining after local tumor irradiation. We found that CD31-stained cells were reduced to some 25% of control levels by 2 weeks after 15 Gy of irradiation (Supplemental Figure 2E) and that this was accompanied by a large decrease in tumor perfusion and a large increase in tumor hypoxia (Figure 2, A and B). These data suggest that the increased tumor hypoxia caused by the severe vascular damage and decreased vessel perfusion after tumor irradiation could be the stimulus leading to the recruitment of BMDCs.
HIF-1 activity is increased in irradiated tumors and is necessary for influx of BMDCs and tumor recurrence after irradiation. (A) Representative images of IHC staining for hypoxia (pimonidazole [pimo]) and functional vessels (lectin perfusion [perf]) in i.c. tumors after irradiation. Scale bar: 50 μm. (B) Quantification of hypoxic areas and vessels in the tumors of A. Error bars indicate SEM. *P < 0.05, **P < 0.01 versus control. Note the log scale on the ordinate. (C) BLI of 5HRE-Luc or pFB-Luc U251 i.c. tumors after a single dose (arrowhead) of irradiation on day 32. The divergence of the 2 curves starting at about 2 weeks after irradiation indicates increased HIF-1 activity. Error bars indicate SD. (D) Effect of NSC-134754 on inhibition of HIF-1 activity in vivo. 5HRE-Luc or pFB-Luc U251 tumor–bearing mice were treated with 15 Gy of irradiation, followed by daily injection of NSC-134754 for 3 weeks. Images were taken on the day of irradiation and 3 weeks later. (E) IHC staining for leukocyte (CD45) and monocyte (CD11b, top row) or macrophage (F4/80, bottom row) infiltration into i.c. tumors. Tumors were harvested on the day of irradiation for controls and 17 days after irradiation in treatment groups. Scale bar: 50 μm. (F) Quantification of CD11b+ and F4/80+ cell influx in tumors of E. Error bars indicate SEM. **P < 0.01, ***P < 0.001 versus control. (G) Growth curves of i.c. tumors treated with irradiation, NSC-134754, irradiation+NSC-134754, and control (n = 5 per group). Error bars indicate SD. *P < 0.05. (H) Growth curves of orthotopic U251/pSR (vector) and U251 HIF1KD1+4 tumors using BLI (n = 7). A whole brain irradiation was given on day 22. Error bars indicate SD. *P < 0.05.
HIF-1 is a key factor responsible for BMDC influx by radiation and tumor recurrence. Consistent with our results (Figure 2, A and B), other investigators have shown that tumors recurring following irradiation or growing in an irradiated normal tissue are more hypoxic (25, 26, 38) and would therefore be expected to have increased HIF-1 levels. To determine HIF-1 activity in real time in our brain-implanted U251 GBM, we stably expressed the HIF-1 reporter construct 5HRE-Luc (39) in U251 cells and monitored luciferase activity in control and irradiated tumors. By comparing the bioluminescence from the tumors expressing the constitutive pFB-Luc (proportional to total cells) with that of those expressing 5HRE-Luc (proportional to total HIF-1 activity), we could monitor tumor HIF-1 activity as a function of tumor size. HIF-1 levels were relatively high immediately after inoculation and then decreased, most likely as tumor vessels formed (data not shown). After that, HIF-1 levels increased as the tumor grew. As shown in Figure 2C, HIF-1 activity paralleled tumor growth in nonirradiated tumors but increased more rapidly than tumor growth starting at about 2 weeks after 15 Gy irradiation, indicating increased HIF-1 levels coincident with the increase in tumor hypoxia at this time.
To test our hypothesis that the increased HIF-1 levels were responsible for the increased influx of CD11b+ cells into the tumors, we used the HIF-1 inhibitor NSC-134754 (40). This compound prevented the increased HIF-1 activity produced by hypoxia in vitro (data not shown) and by tumor irradiation in vivo (Figure 2D). When this inhibitor was given daily for 2 weeks, starting immediately after irradiation, the increased tumor levels of CD11b+ monocytes (and also the more mature macrophages, staining for F4/80) observed at 17 days after 15 Gy were abrogated (Figure 2, E and F). As shown in Supplemental Figure 3, A and B, approximately 75% of infiltrating CD11b+ cells also expressed F4/80. These data indicate that HIF-1 plays a major role in the recruitment of BMDCs into tumors after radiotherapy.
To test our hypothesis that the recruitment into the tumor of CD11b+ and other proangiogenic cells promoted tumor growth after irradiation, we treated the mice with NSC-134754 in combination with local tumor irradiation. We started the drug delivery after irradiation to avoid any drug-induced changes in oxygen levels that would affect tumor radiosensitivity. Although the growth of the unirradiated tumor was not affected by NSC-134754, the response of the irradiated tumor was profoundly changed, with constant shrinkage and no regrowth over the time of the experiment (Figure 2G), even after the end of the treatment with NSC-134754.
We also determined the effect of HIF-1 on the radiosensitivity of GBM by genetic means, using both brain-implanted HIF-1α–knockdown (KD) U251 cells and Hif1a-KO mouse GBM cells. Increased tumor radiosensitivity was found in the HIF-1–KD tumors that depended on the KD levels of HIF-1 in i.c. U251 (Figure 2H and Supplemental Figure 4, A and B). Hif-1–null murine GBM tumors were also more sensitive to irradiation (Supplemental Figure 4, C and D), resulting in longer survival of the Hif-1–KO tumor-bearing mice compared with the control tumor-bearing mice (Supplemental Figure 4E). This increase in tumor radiosensitivity of the HIF-1α–KD tumors was not the result of any change in radiosensitivity or growth of the cells in vitro (data not shown). These data suggest that blocking the HIF-1 signal caused by local irradiation attenuates recruitment of CD11b+ BMDCs, thereby preventing tumor vasculogenesis and tumor regrowth.
HIF-1–induced SDF-1 activates its receptor and sustains tumor blood flow. Several investigators have shown that BMDCs are recruited to and retained in hypoxic normal tissues and in tumors by the hypoxia-dependent secretion of SDF-1, which binds to its receptor, CXCR4, on BMDCs (7, 29–32). Indeed, we found that SDF-1 secretion was upregulated under hypoxic conditions in vitro, and this upregulation was abolished by NSC-134754 (data not shown). In our brain-implanted GBM, SDF-1 levels in the tumors rose to a maximum level by 2 weeks after irradiation and then returned to control levels at 4 weeks (Figure 3, A and B). In addition, we found that local irradiation increased phosphorylation of CXCR4 in the tumor, indicating interaction of SDF-1 with CXCR4 (Figure 3, C and D). Approximately 20% of the BMDCs recruited into the tumor after irradiation exhibited phospho-CXCR4, whereas very low levels of phosphorylation of BMDCs were found prior to irradiation (Supplemental Figure 5A).
The interaction of SDF-1 and CXCR4 promotes tumor influx of BMDCs and restoration of tumor vasculature. (A) Irradiation promotes the expression of SDF-1 in U251 i.c. tumor. Representative image of IHC staining for SDF-1. Scale bar: 50 μm. (B) Quantification of SDF-1 in the irradiated tumors. Error bars indicate SEM. ***P < 0.001 versus control. (C) Phosphorylation of CXCR4 on BMDCs in tumors was induced after irradiation. IHC staining for GFP-BM and pCXCR4 in U251 i.c. tumors 3 weeks after 15 Gy whole brain irradiation. Arrowheads indicate phospho-CXCR4 GFP-BM cells. Scale bar: 50 μm. (D) Quantification of pCXCR4 GFP-BM cells in U251 i.c. tumor after irradiation. Error bars indicate SEM. ***P < 0.001. (E) AMD3100 prevents the restoration of tumor blood flow (green) after irradiation. Representative ultrasound images from U251 s.c. tumors treated with 15 Gy irradiation, AMD3100, irradiation+AMD3100, and control. Scale bar: 1 mm. (F) Quantification of blood flow in tumor of E. Blood flow was reduced by irradiation but recovered by 3 weeks. AMD3100 plus IR prevents the recovery of tumor blood flow. Error bars indicate SD. *P < 0.05.
To determine the effect of inhibiting the interaction of SDF-1 with CXCR4 on tumor blood flow in the irradiated tumors, we used the clinically approved drug AMD3100, an inhibitor of the interaction of SDF-1 with CXCR4, utilizing the technique of ultrasound analysis with microbubbles (41) in the U251 s.c. tumor. As shown (Figure 3, E and F, and Supplemental Videos 1 and 2), AMD3100 had no effect on blood flow compared with control tumor but prevented the return of blood flow in the irradiated tumors. These data suggest that HIF-1–upregulated SDF-1 is an important factor for retention of BMDCs in the irradiated tumor and can promote the recovery of the tumor blood supply after its reduction by irradiation.
Blocking SDF-1 prevents tumor recurrence following irradiation and is significantly more effective than VEGF blockage. We next investigated the therapeutic effect of AMD3100 in blocking the interaction of SDF-1/CXCR4 stimulated by irradiation. Tumor-bearing mice were infused with AMD3100 starting immediately after irradiation. This had no significant effect on the growth of unirradiated tumors in the brain but completely inhibited the recurrence of the irradiated tumors after either a single dose of 15 Gy (Figure 4C) or the more clinically relevant scheme of 5 daily doses of 2 Gy (Figure 4, A and B). Of note is the fact that the tumors did not regrow after the end of the infusion period, and tumor recurrence was not observed at the end of the experimental period (more than 100 days; Supplemental Figure 5, C and D). We also confirmed the eradication of U251 i.c. tumor by histological and MRI analysis (Supplemental Figure 1C). Importantly, AMD3100 had no effect on the growth of the U251 cells treated in vitro with daily doses of 2 Gy (data not shown). As a check of the specificity of the effect of AMD3100 in blocking the SDF-1/CXCR4 interaction, we also performed the therapy study with 15 Gy using a neutralizing antibody against CXCR4 instead of the AMD3100 infusion. We found a similar abrogation of the recurrence of the irradiated tumors (Figure 4D).
Therapeutic effect of blocking the interaction of SDF-1 with CXCR4 after whole brain irradiation. (A) Growth curves of i.c. U251 early tumor model after fractionated irradiation (5 daily doses of 2 Gy starting on day 11 after transplantation). *P < 0.05. (B) BLI images after fractionated irradiation treated with or without AMD3100. (C and D) Growth curves of i.c. U251 advanced tumor model after a single dose of irradiation (15 Gy on day 22 after transplantation), treated with AMD3100 (21 day infusion; C, *P < 0.05) or with neutralizing anti-CXCR4 Ab (D, *P < 0.05), starting immediately after irradiation. (E) Growth curves of U251 i.c. tumor after 15 Gy irradiation, treated with DC101. Arrowheads indicate the treatment of DC101 (started immediately after irradiation and maintained for 21 days). (F) AMD3100 is more effective than DC101 in reducing tumor blood flow. Quantification of endothelial cells and functional vessels in U251 s.c. tumors after 15 Gy irradiation and combined with AMD3100 or DC101. Samples were taken 17 days after irradiation. Error bars indicate SEM. **P < 0.01, ***P < 0.001 versus IR.
To test this strategy with another tumor, we used the U87 GBM cell line, which we retrovirally transduced with luciferase. Again, AMD3100 enhanced the radiosensitivity of the tumor, as demonstrated by tumor growth and by mouse survival time (Supplemental Figure 6, A and B).
It has been reported that tumor-produced VEGF not only promotes local angiogenesis but also mobilizes endothelial and hematopoietic progenitors from the BM (42, 43). As VEGF is also an HIF-1 target gene, we tested the inhibition of VEGF using the mouse VEGFR2-neutralizing monoclonal antibody DC101. DC101 treatment alone slightly reduced the tumor growth rate (Supplemental Figure 5B). As shown in Figure 4E, this treatment given for 3 weeks after irradiation delayed the regrowth of the tumors but was not as effective as AMD3100 (or the HIF-1 inhibitor NSC-134754) in preventing recurrence of the irradiated tumors. We also compared the effect of AMD3100 and DC101 on tumor vascularity and blood perfusion after irradiation. Whereas both agents decreased the number of endothelial cells in the irradiated tumors, only AMD3100 produced an essentially total blockage of tumor perfusion when combined with irradiation (Figure 4F).
We further tested whether AMD3100 prevented the vasculogenesis pathway by using the model system of transplanting tumors into an irradiated normal tissue to eliminate local angiogenesis. We found that HIF-1 KD or treatment of tumor-bearing mice with AMD3100 totally abrogated tumor growth in the preirradiated normal tissue (Supplemental Figure 6, A and B). These data show that tumor regrowth is absolutely dependent on the vasculogenesis pathway when angiogenesis is impaired by local irradiation and that blocking the HIF-1 or SDF-1/CXCR4 signal prevents tumor recurrence.
Myelomonocytic cells are critical for tumor regrowth after irradiation. We next tested the importance of myelomonocytic cells for tumor regrowth following irradiation. To do this, we depleted the monocyte/macrophage lineage using carrageenan (44). This treatment reduced the number of CD11b+ BMDCs in the U251 i.c. tumor (Figure 5B). We also found that this prevented tumor recurrence after irradiation, although depletion of myeloid cells did not reduce the growth rate of unirradiated i.c. tumor (Figure 5A and Supplemental Figure 8A). These data are consistent with previous reports suggesting involvement of myeloid lineage cells in tumor progression and vasculogenesis (20, 22, 35).
CD11b+ cells are associated with GBM tumor recurrences in U251 i.c. tumors in mice and in patients. (A) Growth of U251 i.c. tumor assessed by BLI. Tumor-bearing mice were treated with 15 Gy whole brain irradiation, carrageenan (CAR), or the combination of irradiation and CAR. The arrow indicates irradiation, and the bar indicates the period of CAR treatment. Error bars indicate SD. *P < 0.05. (B) IHC of U251 i.c. tumor of A for CD11b. The average of CD11b cells per HPF was 6.3 (control), 72.6 (IR), 2.6 (CAR), and 2.0 (CAR+IR). (C) IHC of GBM clinical samples staining for CD11b. (D) Significantly increased levels of CD11b+ cells in the recurrent human GBMs compared with the untreated tumors. Quantification of CD11b based on IHC with CD11b staining. Eight of twelve samples showed significant increases in CD11b+ cells in recurrent GBMs (P < 0.05). Error bars indicate SEM. Scale bars: 50 μm.
CD11b+ myelomonocytic cells are increased in recurrences of human GBM. Finally, we determined the level of CD11b+ cells in human GBM tumors both prior to therapy and when the tumors had recurred in the same patients. We observed higher levels of CD11b+ cells in the recurrent tumors compared with those in the untreated tumors in 10 of the 12 paired samples, which was statistically significant (P < 0.05) in 8 of the 12 pairs (Figure 5, C and D). CD11b+ cells were not found or were at undetectable levels in normal brain tissues, indicating that the presence of large number of infiltrating CD11b+ cells in recurrent tumors is stimulated by tumor-derived cytokines.
The present series of experiments tested our hypothesis that the growth of a tumor, after radiation doses similar to those given in radiotherapy, has to rely on the vasculogenesis pathway, which is dependent on circulating cells, primarily of BM origin (Figure 6), and that inhibition of this pathway will prevent, or delay, tumor recurrence. Our results in the i.c. implanted GBM in mice support this hypothesis. We demonstrate that there is a large influx of BMDCs in the irradiated tumors as they begin to regrow after irradiation, and that 3 different ways of abrogating this influx (inhibition of HIF-1 or SDF-1/CXCR4 or depletion of monocytes/macrophages) prevent, or substantially delay, tumor recurrence. Our data suggest that the stimulus for the influx of BMDCs is increased tumor hypoxia caused by loss of functional vasculature after irradiation. Notably, we demonstrate that hypoxia is dramatically increased in the well-vascularized U251 brain tumor as it initiates regrowth after irradiation and this coincides with increased tumor HIF-1 levels. Further, inhibition of HIF-1 or inhibition of the interaction of the HIF-1–upregulated SDF-1 with its receptor, CXCR4, after irradiation prevents both the recruitment of BMDCs into the tumors and their regrowth after irradiation.
Model of the main contributions of BMDCs; and cytokines that promote restoration of tumor vasculature after irradiation. (A) Prior to irradiation, tumor growth is governed largely by local angiogenesis. When local angiogenesis is inhibited by irradiation, growth of the tumor vasculature (essential for recurrence of the tumor) can only occur from circulating cells, of which BMDCs are an essential component. (B) After irradiation, the tumor becomes more hypoxic, and HIF-1 is increased as the tumor attempts to regrow. This induces SDF-1 and promotes the mobilization of CD11b+ monocytes from the BM and retention of these BMDCs into the tumor. SDF-1 is the key factor for the influx of BMDCs, as AMD3100, an inhibitor of CXCR4/SDF-1, and antibodies against CXCR4 block the recruitment and/or tumor retention of the BMDCs, inhibit restoration of the tumor vasculature, and prevent tumor recurrence. The various inhibitors and the points in the cycle at which they act are shown in boxes. Mϕ, macrophages.
As discussed earlier, the 2 principal ways in which a tumor can expand its vasculature as it grows is by angiogenesis involving sprouting of endothelial cells from nearby normal vessels or by vasculogenesis, which occurs by the recruitment into the tumor of circulating endothelial and other cells. Our data suggest that for the intracranial growth of the U251 tumor, the angiogenesis pathway is dominant, as none of the 3 methods we used to inhibit vasculogenesis affected the growth of the unirradiated control tumors (Figures 2, 4, and 5). However, once the tumor received irradiation, most of the CD31+ endothelial cells in the tumor were eradicated (Supplemental Figure 2E). Also, those in the surrounding normal vessels would be expected to be sterilized, thereby inhibiting the angiogenesis pathway. Indeed, we and others have shown that irradiation abrogates tumor angiogenesis (45). This loss of functional vasculature in the tumor combined with an inability of the normal vasculature to provide vessels is likely the reason for the large increase in tumor hypoxia we observed 2 weeks after irradiation (Figure 2, A and B) and the resulting increase in tumor HIF-1 levels (Figure 2C), thereby providing the stimulus for vasculogenesis. It has been reported that local irradiation promotes HIF-1 activation due to tumor reoxygenation and increased free radicals (28). In our i.c. tumor model, HIF-1 was activated by local irradiation, but not by the same mechanism as reported, because it occurred much later than that caused by free radical production and was more consistent with the loss of tumor vasculature. Consistent with HIF-1 being the signal for vasculogenesis, we show that both pharmacological and genetic inhibition of HIF-1 attenuated BMDC recruitment and inhibited tumor recurrence. Of note is the fact that the effect of the HIF-1 inhibitor NSC-134754 is greater than that of HIF-1 KD or KO in the tumor cells (Figure 2 and Supplemental Figure 3). The likely reason for this is that in the HIF-1–KO tumors there are also stromal cells that can express HIF-1 (24).
AMD3100, an FDA-approved orphan drug, promotes the mobilization of BMDCs from the BM niche to peripheral blood by inhibiting the interaction of SDF-1 with CXCR4 (46). As SDF-1 is also known to have an important role in retention of CXCR4+ BMDCs in perivascular hypoxic areas of ischemic tissue (7, 29–32), we used AMD3100 to inhibit this part of the vasculogenesis pathway. Although it has been reported that SDF-1 inhibition by AMD3100 increases the therapy-induced growth delay of GBM in a mouse model by inhibiting the Erk and Akt pathways (47), this is not the mechanism of the effect we observed for 2 reasons. First, we only applied the drug after tumor irradiation, thus making any effect on tumor cell radiosensitivity unlikely. Second, we saw no effect of AMD3100 on the growth of the cells in vitro with or without irradiation. However, the effect of this drug in blocking BMDC influx and tumor regrowth was dramatic after either single or fractionated doses of irradiation. An important feature of our data is that although we completed the drug infusion before the tumors were eradicated, the tumors continued to shrink and did not recur even long after the end of the infusion.
The BMDC accumulation in the U251 tumors following irradiation was composed largely of CD11b+ monocytes. These cells have been shown to be increased in the BM, spleens, and peripheral blood of tumor-bearing mice and in the peripheral blood of cancer patients (48, 49). There is strong clinical evidence that infiltration levels of macrophages that are of myeloid cell lineage correlate with poor prognosis in breast, prostate, ovarian, and cervical cancers (50). In our tumor model, irradiation significantly increased the number of CD11b+ myeloid cells in the tumor. These cells are highly proangiogenic, as we and others have reported, suggesting that they are attractive targets for enhancing tumor response to irradiation (20, 22). In addition, it has been reported that CD11b+Gr-1+ myeloid cells mediate tumor refractoriness to anti-VEGF treatment (51). A significant finding in the irradiated tumors was that approximately 50% of the CD11b+ infiltrating monocytes also expressed the angiopoietin receptor Tie2, defining them as Tie2-expressing monocytes (TEMs). These have been shown to be highly related to tumor-associated macrophages (TAMs) and to express a gene signature associated with active remodeling and angiogenesis (52). Significantly, these cells were undetectable in the unirradiated U251 tumors. Although it is possible that CD11b– BMDCs contribute to the vasculogenesis after irradiation, we found no evidence for the involvement of EPCs. However, in other studies, we have found that antibody neutralization of CD11b had a significant radiosensitization effect on transplanted tumors, whereas depletion of Gr-1+ cells did not show the effect (G.O. Ahn et al., unpublished observations). This implicates CD11b+Gr-1– cells as the important proangiogenic population, a conclusion similar to that of F. Pucci et al. (52).
Anti-VEGF treatment combined with irradiation is an emerging strategy currently being tested in the treatment of GBM. However, recent reports of preclinical data indicate that the benefits are transitory, followed by rapid regrowth and increased tumor aggressiveness (53, 54). Our data also show that inhibiting the VEGF pathway through blockage of VEGFR2 by DC101 produces an increase in growth delay by irradiation. However, the effect was transitory, and the tumors regrew following the cessation of the treatment (Figure 4E). In addition, VEGF blockade was less effective than AMD3100 treatment in inhibiting tumor perfusion after irradiation (Figure 4F). Thus, blocking angiogenesis is inferior to inhibiting the vasculogenesis pathway in preventing both the postirradiation return of tumor perfusion and tumor recurrences.
In summary, our data suggest that the novel strategy of inhibiting the vasculogenesis pathway after local tumor irradiation has the potential to improve the control of human GBM by radiotherapy. In essence, this strategy abolishes the need to kill all the tumor cells, replacing it with the need only to kill the endothelial cells in and around the tumor, which will require significantly lower radiation doses than those needed to sterilize all the tumor cells.
Cell culture and retroviral transduction. U251 and U87MG cells were obtained from R.K. Puri (FDA, Bethesda, Maryland) and maintained in DMEM with 10% FBS (Gibco, Invitrogen). Murine GBM Hif1a wild-type and KO cell lines were obtained from G. Bergers (UCSF, San Francisco, California) and maintained in DMEM with 10% FBS.
The retrovirus-mediated RNA interference study was performed with the pSuper RNA interference system (Oligoengine). HIF-1α target sequences were as follows: HIF1KD1, 5′-GGAAATGAGAGAAATGCTTAC-3′; HIF1KD2, 5′-GCTGGAGACACAATCATATCT-3′; HIF1KD3, 5′-GCTTTGAGTCAAAGAACTACA-3′; HIF1KD4, 5′-GAGGAAGTACCATTATAT-3′. HIF1KD4 was obtained from D. Chan (Stanford University, Palo Alto, California). For retroviral packaging, 293T cells were cotransfected with pVPack-VSV-G, pVPack-GP (Stratagene), and the HIF-1α–KD or pSUPERretro (pSR) control constructs, and viral supernatant was harvested after 48 hours. The transduction of the firefly luciferase gene was performed with the pFB-Luciferase retrovirus system (Stratagene). For infections, cells were incubated with viral supernatant in the presence of 1 μg/ml DEAE-dextran, and after 48 hours, infected cells were selected with puromycin (1 μg/ml) for 72 hours.
BM transplantation. All animal procedures were approved by Stanford University's Administrative Panel on Laboratory Animal Care (APLAC). Athymic nu/nu nude mice were purchased from Charles River Laboratories. GFP nude mice were obtained from AntiCancer Inc. Mice were maintained in a germ-free environment and had access to food and water ad libitum. BM was harvested from 6- to 12-week-old GFP nude mice by flushing the femurs and tibias with 2% FBS in Hank's balanced salt solution (Invitrogen). Cells (>2 × 106) were injected i.v. through the tail vein of lethally irradiated (8.5 Gy) recipient mice. Tumor cell implantation was performed i.c. or s.c. 4 weeks later.
Orthotopic i.c. tumor implantation and irradiation. For i.c. implantation of U251, U87, or murine GBM cells, 6- to 12-week-old nude mice were implanted i.c. as described previously (55) with 5 μl of 1 × 106 U251/pFB-Luc, U251/pFB-LucHIF1KD, U251/5HRE-Luc, U87/pFB-Luc or 1 × 105 murine pFB-Luc GBM HIF1 WT or KO cells. Anesthetized mice were placed in individual lead boxes with a cut-out through which the whole brain protruded for irradiation but that shielded the mouth and throat cavity. All treatment experiments were repeated 2–3 times.
For the s.c. tumor inoculation, U251 cells were inoculated at 1 × 107 cells/mouse on the back of the mouse approximately 1 cm proximal to the base of the tail. Unanesthetized mice were placed in lead jigs through which the established tumors (approximately 200 mm3 in volume) protruded for irradiation.
Irradiation was performed with a Phillips X-ray unit operated at 200 kVp with a dose rate of 1.21 Gy/min (20 mÅ with added filtration of 0.5 mm copper, distance from X-ray source to the target of 31 cm, and a half value layer of 1.3 mm copper). Tumor volume (V) was calculated using the formula for a spheroid: V = 0.5 × width × length × depth.
In vivo real-time optical imaging. Mice were anesthetized and injected i.p. with 150 mg/kg D-luciferin (Caliper). Imaging was completed between 5 and 30 minutes after injection with the IVIS system (Caliper) coupled to Living Image acquisition and analysis software. For bioluminescence index (BLI) plots, photon flux was calculated for each mouse by using a rectangular region of interest encompassing the head of the mouse in a prone position. This value was scaled to a comparable background value (from a luciferin-injected mouse with no tumor cells) and then normalized to the value obtained immediately after xenografting (day 0), so that all mice had an arbitrary starting BLI signal of 100. Error bars shown in figures indicate SD.
Ultrasound analysis. Ultrasound imaging was performed using a dedicated small-animal high-resolution imaging unit (Vevo 770; VisualSonics). Mice were anesthetized with isoflurane and maintained in a supine position. The animals' body temperature was maintained with a heated platform and an auxiliary heat lamp. A tail vein cannulation was performed using a 27-gauge butterfly needle apparatus (VisualSonics), through which all contrast agents were injected. To measure microvascular perfusion, a 50-μl bolus (1 × 108 microbubbles) of Non-Targeted MicroMarker Contrast Agent was injected through the tail vein cannula. The first-pass bolus wash-in was viewed and used to quantify blood flow. The change in intensity from baseline to plateau gave an indication of relative blood volume, and the slope of the curve represented the relative blood flow.
Drug treatment. We obtained DC101 hybridoma from C.J. Kuo (Stanford University) and NSC-134754 from Developmental Therapeutics Program (NCI/NIH, Bethesda, Maryland). Nude mice injected i.c. with U251 cells were randomized into 4 cohorts (n = 5–7) for treatment with inhibitors alone (NSC-134754, AMD3100, or DC101), irradiation alone, inhibitors combined with irradiation, or control. Treatment with all the inhibitors was started immediately after irradiation and maintained for 3 weeks. The doses, sources, and routes of drug administration were as follows: NSC-134754 (Developmental Therapeutics Program, NCI/NIH): 5 mg/kg dose, injected i.p. daily; AMD3100 (Sigma-Aldrich): 5 mg/kg, continuous infusion by osmotic pump (Durect); neutralizing anti-CXCR4 antibody (Torrey Pines Biolabs): 0.4 mg/kg, injected i.p. daily; DC101: 40 mg/kg, purified from hybridoma supernatant as described previously (56), injected i.p. every 3 days. To deplete the monocyte/macrophage lineage, carrageenan (2 mg/mouse) was i.p. injected 7, 3, and 1 day prior to tumor inoculation and continued twice per week for the period indicated in Figure 4.
Body weight of the mice was monitored during all the treatments. No significant losses in body weight (less than 10%) were observed.
TaqMan qPCR. Total RNA was extracted from mouse brain including U251 i.c. tumor by TRIzol (Invitrogen). After reverse transcription, cDNA was then subjected to qPCR with gfp-specific TaqMan probe and primer set (probe: 5′-6FAM-CCAGTCCGCCCTGAGCAAAGACC-MGB-3′; forward primer: 5′-CTGCTGCCCGACAACCAC-3′; reverse primer: 5′-TCACGAACTCCAGCAGGAC-3′).
MRI. High-resolution MR images were acquired on a 7.0-T/31-cm animal scanner system (Varian). The imaging protocol consisted of scout imaging in 2 planes, followed by a spin-echo sequence (TR = 2,500 ms, TE = 45 ms, NEX = 4, matrix = 256 × 256, FOV = 30 mm, slice thickness = 1 mm, gap = 0 mm). During imaging, animals were under isoflurane anesthesia and monitored for respiration and heart rate; the temperature was kept at 37°C. Images were analyzed by OsiriX DICOM viewer.
Tissue preparation. When the luciferase signal reached approximately the same as that at irradiation, we performed cardiac perfusion in asphyxiated tumor-bearing mice with PBS, followed by 4% paraformaldehyde in PBS. To determine the hypoxia level, we injected pimonidazole hydrochloride 90 minutes prior to tissue harvest. For visualization of the functional tumor vessels, biotinylated tomato lectin was administered via tail vein injection before performing cardiac perfusion. Tumors were removed, embedded in optimal cutting temperature (OCT) compound (Sakura Finetek), and frozen at –80°C until cryosectioning and immunostaining.
IHC. Frozen sections were used for immunohistochemical analysis. Tumor endothelial cells were visualized by staining with a 1:500 dilution of rat anti-mouse CD31 antibody (BD Biosciences). BMDC populations were analyzed by staining with 1:500 anti-mouse CD11b, F4/80, CD45, Gr-1 (BD Biosciences), VEGFR1 (Abcam), VEGFR2, Tie-2 (eBioscience) antibodies or by detecting GFP in GFP-BM–transplanted mice. Phosphorylation of CXCR4 was visualized with anti–phosphorylated CXCR4 antibody obtained from J.B. Rubin (Washington University in St. Louis, Missouri). SDF-1 was detected with anti–SDF-1 antibody (Novus Biologicals). Hypoxic areas were detected with FITC-conjugated anti–Hypoxyprobe-1 antibody (Natural Pharmacia International). CD11b staining was performed on paraffin-embedded human GBM specimens with anti-human CD11b antibody (Abcam). For fluorescence visualization of antibody reactions, primary antibodies were detected using secondary antibodies labeled with the fluorochromes Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 594 or by streptavidin labeled with Alexa Fluor 488 or Alexa Fluor 555 (1:1,000; Molecular Probes), while nuclei were detected with DAPI. Photomicrographs were taken with a Leica DM6000B microscope using Plan 10×/0.30, 20×/0.50, and 40×/0.85 objective lenses with 10×/25 eyepieces.
Quantification of expression and colocalization. Fluorescence images of tumor sections were acquired with a QImaging digital camera and Imaging Pro Plus software (MediaCybernetics). The average of signal intensity for CD31, lectin, or pimonidazole staining was measured by the software from 4 or 5 random fields using ×20 objective with ×10 eyepieces of the fluorescence microscope. Quantitative analysis for GFP-BM, CD11b, and phospho-CXCR4–positive cells was performed by determining the number of cells in 5 randomly photographed fields using a ×40 objective with ×10 eyepieces of the fluorescence microscope. Quantification was performed in 4–6 animals per group. Error bars shown in the figures indicate SEM.
Histological assessment. Human GBM specimens of primary tumor and recurrences after radiation therapy were obtained from biopsy or surgical resection by the Department of Neurosurgery at Stanford Hospital with informed consent according to US federal law and are exempt from consideration by the Stanford Administrative Panel on Human Subjects in Medical Research.
Immunoblotting. U251 HIF1KD cells were cultured under hypoxic conditions (0.5% or 2% O2 for 48 hours). Cell extracts were prepared in an Invivo2 400 Hypoxia Workstation (Ruskin) by gently scraping off cells in PBS followed by extraction in 8 M urea buffer containing 1× Complete Mini protease inhibitors (Roche). Western blot analysis was performed with 30 μg protein with anti-human HIF-1α monoclonal antibody (1:1,000) or an anti-GAPDH antibody (1:5,000) as loading control.
Statistics. Statistical analyses were performed by the 2-tailed Student's t test or 1-way ANOVA to determine statistical significance. P values (exact significance) of less than 0.05 were considered statistically significant. Kaplan-Meier curves and the log-rank test were used to compare survival times among the groups. All calculations were performed using Prism 4 (GraphPad).
View Supplemental video 1
We thank G. Bergers (UCSF) for helpful discussions. We are also grateful to G.O. Ahn, C.H. Liao, M.J. Dorie, D. Menke, Z. Varga, and all members of the Brown laboratory for technical assistance and/or advice. This work was supported by NIH grant R01 CA128873 (to J.M. Brown) and awards from the Accelerate Brain Cancer Cure foundation and the James S. McDonnell Foundation (to J.M. Brown).
Conflict of interest: Robert M. Hoffman and J. Martin Brown have significant equity holdings in AntiCancer Inc. and Proacta Inc., respectively.
Reference information: J Clin Invest. 2010;120(3):694–705. doi:10.1172/JCI40283.
See the related article at Resisting arrest: a switch from angiogenesis to vasculogenesis in recurrent malignant gliomas.
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Garcia-Barros M, et al. Tumor response to radiotherapy regulated by endothelial cell apoptosis. Science. 2003;300(5622):1155–1159.
Ogawa K, Boucher Y, Kashiwagi S, Fukumura D, Chen D, Gerweck LE. Influence of tumor cell and stroma sensitivity on tumor response to radiation. Cancer Res. 2007;67(9):4016–4021.
Ceradini DJ, et al. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat Med. 2004;10(8):858–864.
Takahashi T, et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med. 1999;5(4):434–438.
Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med. 2003;9(6):702–712.
Orlic D, et al. Bone marrow cells regenerate infarcted myocardium. Nature. 2001;410(6829):701–705.
Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002;420(6917):860–867.
Condeelis J, Pollard JW. Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell. 2006;124(2):263–266.
Erler JT, et al. Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche. Cancer Cell. 2009;15(1):35–44.
Lyden D, et al. Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts. Nature. 1999;401(6754):670–677.
Lyden D, et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med. 2001;7(11):1194–1201.
Gothert JR, et al. Genetically tagging endothelial cells in vivo: bone marrow-derived cells do not contribute to tumor endothelium. Blood. 2004;104(6):1769–1777.
De Palma M, Venneri MA, Roca C, Naldini L. Targeting exogenous genes to tumor angiogenesis by transplantation of genetically modified hematopoietic stem cells. Nat Med. 2003;9(6):789–795.
Moldovan NI, Goldschmidt-Clermont PJ, Parker-Thornburg J, Shapiro SD, Kolattukudy PE. Contribution of monocytes/macrophages to compensatory neovascularization: the drilling of metalloelastase-positive tunnels in ischemic myocardium. Circ Res. 2000;87(5):378–384.
Capoccia BJ, Shepherd RM, Link DC. G-CSF and AMD3100 mobilize monocytes into the blood that stimulate angiogenesis in vivo through a paracrine mechanism. Blood. 2006;108(7):2438–2445.
Bailey AS, et al. Myeloid lineage progenitors give rise to vascular endothelium. Proc Natl Acad Sci U S A. 2006;103(35):13156–13161.
Ruzinova MB, et al. Effect of angiogenesis inhibition by Id loss and the contribution of bone-marrow-derived endothelial cells in spontaneous murine tumors. Cancer Cell. 2003;4(4):277–289.
Ahn GO, Brown JM. Matrix metalloproteinase-9 is required for tumor vasculogenesis but not for angiogenesis: role of bone marrow-derived myelomonocytic cells. Cancer Cell. 2008;13(3):193–205.
Bertout JA, Patel SA, Simon MC. The impact of O2 availability on human cancer. Nat Rev Cancer. 2008;8(12):967–975.
Du R, et al. HIF1alpha induces the recruitment of bone marrow-derived vascular modulatory cells to regulate tumor angiogenesis and invasion. Cancer Cell. 2008;13(3):206–220.
Sasai K, Brown JM. Discrepancies between measured changes of radiobiological hypoxic fraction and oxygen tension monitoring using two assay systems. Int J Radiat Oncol Biol Phys. 1994;30(2):355–361.
Zips D, et al. Impact of the tumour bed effect on microenvironment, radiobiological hypoxia and the outcome of fractionated radiotherapy of human FaDu squamous-cell carcinoma growing in the nude mouse. Int J Radiat Biol. 2001;77(12):1185–1193.
Rofstad EK, Mathiesen B, Henriksen K, Kindem K, Galappathi K. The tumor bed effect: increased metastatic dissemination from hypoxia-induced up-regulation of metastasis-promoting gene products. Cancer Res. 2005;65(6):2387–2396.
Moeller BJ, Cao Y, Li CY, Dewhirst MW. Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: role of reoxygenation, free radicals, and stress granules. Cancer Cell. 2004;5(5):429–441.
Wang Y, Haider H, Ahmad N, Zhang D, Ashraf M. Evidence for ischemia induced host-derived bone marrow cell mobilization into cardiac allografts. J Mol Cell Cardiol. 2006;41(3):478–487.
Jin DK, et al. Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+ hemangiocytes. Nat Med. 2006;12(5):557–567.
Aghi M, Cohen KS, Klein RJ, Scadden DT, Chiocca EA. Tumor stromal-derived factor-1 recruits vascular progenitors to mitotic neovasculature, where microenvironment influences their differentiated phenotypes. Cancer Res. 2006;66(18):9054–9064.
Grunewald M, et al. VEGF-induced adult neovascularization: recruitment, retention, and role of accessory cells. Cell. 2006;124(1):175–189.
Gerlach LO, Skerlj RT, Bridger GJ, Schwartz TW. Molecular interactions of cyclam and bicyclam non-peptide antagonists with the CXCR4 chemokine receptor. J Biol Chem. 2001;276(17):14153–14160.
Yang M, Reynoso J, Jiang P, Li L, Moossa AR, Hoffman RM. Transgenic nude mouse with ubiquitous green fluorescent protein expression as a host for human tumors. Cancer Res. 2004;64(23):8651–8656.
De Palma M, et al. Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. Cancer Cell. 2005;8(3):211–226.
Jodele S, et al. The contribution of bone marrow-derived cells to the tumor vasculature in neuroblastoma is matrix metalloproteinase-9 dependent. Cancer Res. 2005;65(8):3200–3208.
Song S, Ewald AJ, Stallcup W, Werb Z, Bergers G. PDGFRbeta+ perivascular progenitor cells in tumours regulate pericyte differentiation and vascular survival. Nat Cell Biol. 2005;7(9):870–879.
Bussink J, Kaanders JH, Rijken PF, Raleigh JA, Van der Kogel AJ. Changes in blood perfusion and hypoxia after irradiation of a human squamous cell carcinoma xenograft tumor line. Radiat Res. 2000;153(4):398–404.
Harada H, Kizaka-Kondoh S, Hiraoka M. Optical imaging of tumor hypoxia and evaluation of efficacy of a hypoxia-targeting drug in living animals. Mol Imaging. 2005;4(3):182–193.
Chau NM, et al. Identification of novel small molecule inhibitors of hypoxia-inducible factor-1 that differentially block hypoxia-inducible factor-1 activity and hypoxia-inducible factor-1alpha induction in response to hypoxic stress and growth factors. Cancer Res. 2005;65(11):4918–4928.
Palmowski M, et al. Vessel fractions in tumor xenografts depicted by flow- or contrast-sensitive three-dimensional high-frequency Doppler ultrasound respond differently to antiangiogenic treatment. Cancer Res. 2008;68(17):7042–7049.
Heissig B, et al. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell. 2002;109(5):625–637.
Hattori K, et al. Plasma elevation of stromal cell-derived factor-1 induces mobilization of mature and immature hematopoietic progenitor and stem cells. Blood. 2001;97(11):3354–3360.
Ishizaka S, Kuriyama S, Tsujii T. In vivo depletion of macrophages by desulfated iota-carrageenan in mice. J Immunol Methods. 1989;124(1):17–24.
Prionas SD, Kowalski J, Fajardo LF, Kaplan I, Kwan HH, Allison AC. Effects of X irradiation on angiogenesis. Radiat Res. 1990;124(1):43–49.
Broxmeyer HE, et al. Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. J Exp Med. 2005;201(8):1307–1318.
Rubin JB, et al. A small-molecule antagonist of CXCR4 inhibits intracranial growth of primary brain tumors. Proc Natl Acad Sci U S A. 2003;100(23):13513–13518.
Bronte V, et al. Identification of a CD11b(+)/Gr-1(+)/CD31(+) myeloid progenitor capable of activating or suppressing CD8(+) T cells. Blood. 2000;96(12):3838–3846.
Almand B, et al. Increased production of immature myeloid cells in cancer patients: a mechanism of immunosuppression in cancer. J Immunol. 2001;166(1):678–689.
Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer. 2004;4(1):71–78.
Shojaei F, et al. Tumor refractoriness to anti-VEGF treatment is mediated by CD11b(+)Gr1(+) myeloid cells. Nat Biotechnol. 2007;25(8):911–920.
Pucci F, et al. A distinguishing gene signature shared by tumor-infiltrating Tie2-expressing monocytes, blood "resident" monocytes, and embryonic macrophages suggests common functions and developmental relationships. Blood. 2009;114(4):901–914.
Bergers G, Hanahan D. Modes of resistance to anti-angiogenic therapy. Nat Rev Cancer. 2008;8(8):592–603.
Paez-Ribes M, et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell. 2009;15(3):220–231.
Kawakami K, Kioi M, Liu Q, Kawakami M, Puri RK. Evidence that IL-13R alpha2 chain in human glioma cells is responsible for the antitumor activity mediated by receptor-directed cytotoxin therapy. J Immunother. 2005;28(3):193–202.
Rockwell P, Neufeld G, Glassman A, Caron D, Goldstein NI. In vitro neutralization of vascular endothelial growth factor activation of Flk-1 by a monoclonal antibody. Mol Cell Differ. 1995;3(1):91–109.
Version 1 (February 22, 2010): No description
Version 2 (March 1, 2010): No description


In 1867, the first Wesleyan Methodist Church was built in Leet St and the first Primitive Methodist Church in Don St in 1873 whilst the Strathearn Primitive Methodist Church was built in Elles Rd, opening on 6th November 1887.
In 1994 the decision was made to amalgamate the remaining three congregations and work towards a completely new building on a new site. To meet future needs, rather than being solely a church, this new building would be a community centre.
The first step was to close Central Methodist Church in Jed St, with the final service being held on 19th February 1995, and the members then joining with St Mark's on Exmouth St. This combined congregation worshipped at St Mark's until February 1996 when it in turn joined with the people at St Peter's (Elles Road).
Meanwhile a considerable amount of planning and organisation for a new complex in Lindisfarne St was underway. Building commenced in June 1997 and the work was completed within budget and on time for the opening ceremonies on the weekend of November 29/30th 1997.
The sculpture depicting Christ carrying the cross, was made by a local sculptor, Mr B Walsh. It is made in concrete and was fastened to the St Mark's east wall. It was dedicated at the opening of the new St Mark's Church by the Rev A J Johnston, Chairman of the Otago - Southland Synod District on Saturday, April 10th 1965.
The Strathearn Bell dates back to November 1887 when the Strathearn Primitive Methodist Church was built. Included in this building was an ornamental belfry to house a Capital bell donated by Messers. Luke & Sons Ltd. engineers, shipbuilders and makers of boilers and cooking ranges in Wellington. The Strathearn Bell as it became known, was intended to represent the Methodist people of all Southland. When the church was rebuilt in 1918, the bell was housed in the wooden tower block, and in 1958 the tower was rebuilt in concrete. The Strathearn bell is now in its third home, a lychgate housing.
A much admired feature of Central Church was its stained glass windows, widely acknowledged as masterpieces. Regrettably, as the Lindisfarne Building is not high enough to house the Froggatt and Wesney windows, it was possible to bring only the smaller south wall Myers window depicting John Wesley. It was presented by Mrs B Myers and her daughter Mrs B Holst in 1936.


text:
Aeropuerto de Magdalena  es un aeropuerto que da servicio a Magdalena, una ciudad en el río Itonomas en el Departamento de Beni de Bolivia.

La pista está en el borde occidental de la ciudad. La Baliza no direccional Magdalena (Ident: MGD) se encuentra en el campo.

Referencias

Enlaces externos
OpenStreetMap - Magdalena
OurAirports - Magdalena
SkyVector - Magdalena
Fallingrain - Magdalena Airport

Magdalena
Magdalena

redpajama_set_name: RedPajamaWikipedia


"sales design consultant jobs in longmont co"
... THE JOBnAs a Samsung Experience Consultant, you will:nn Demonstrate, sell, and ... innovation leader in consumer electronics design and technology. Samsung delivers a ... has been the premier outsourced..
... the sell. As an Associate Sales Consultant you will be responsible for ... to competitors. Participate in the design, validation, and presentation of Oracle ... deep technical skills (modernprogramming,..


text:
Q: How do I use ASP.NET Web API Attribute Routing with a complex object parameter? I have a Web API action that looks like the following:
[HttpGet]
[Route("api/query/hello/{query}")]
public HttpResponseMessage Hello([FromUri]Query query)
{
    return null;
}

where the Query class has a public string property named QueryText.  When I hit the following URL, I get a 404 error:
/api/query/hello?QueryText=bacon

This worked before I started using Attribute Routing.  If I have no parameters or primitive type parameters, I can get Attribute Routing to work.  But with a complex parameter, I get 404s.  How does Attribute Routing work with complex action parameters?  Is it compatible with the FromUri attribute?

A: The solution here was that the {query} token in the Route definition was superfluous.  Removing it, as follows, fixed the issue:
[Route("api/query/hello")]

A: The [FromUri] attribute will be needed because you're reading from the URL. Your route should look something like:
public HttpResponseMessage Hello([FromUri]Query query)
{
    //Implement whatever
    return null;
}

/api/{Controller Name}/hello?QueryText=bacon
Should then work correctly.
The model binder will take whatever query parameters you provided then try to bind whatever is inside that Query object. I'd worry about the Route Attribute after you've got it working first.


text:
Home Politics America Michigan Senate Approves 18% Tax on Vaping Products
Michigan Senate Approves 18% Tax on Vaping Products
The Senate of Michigan has recently approved a six-bill package that would impose an 18% tax on nicotine-containing e-liquids and allows the sales of flavoured vaping products.
The latest measure would change the age limit for purchasing tobacco and vaping products from 18 to 21 across the state.
In September 2019, Democratic Governor Gretchen Whitmer had issued an executive order banning the sale of flavoured e-liquids across the State of Michigan. "As governor, my number one priority is keeping our kids safe," said Gov. Whitmer in a statement at the time. However, the proposed ban had been blocked by a state judge, who ruled that the emergency provision short-circuited normal law making procedures.
In response to the block, Gov. Whitmer had taken the matter to the Supreme Court. However the latter had denied Gov. Gretchen Whitmer's request to remove the lower court's block on the ban and also denied her request that the high court consider the case before the Court of Appeals, saying that they were "not persuaded that the question should be reviewed by this court before consideration by the Court of Appeals."
New bill allows flavours
Finally, this new bill would set up an enforcement and licensing system for shops that sell vaping products, but thankfully allows flavours. Additionally, it would change the age limit for purchasing tobacco and vaping products across the state, from 18 years old to 21, and impose a tax rate of 18% on vape products.
"It's regulated. It's enforced," said Senate Minority Leader Jim Ananich, D-Flint, about the measure. Ananich, who was one of the sponsors of the bill, said it would give adults the "choice one way or another" to use the products, and that it aimed to resolve a lack of clarity surrounding federal and state policy on vaping in Michigan.
Read Further: The Detroit News
Michigan: Proposed Bills Would Ban Vitamin E Acetate in E-Liquids
flavour bans
tobacco age limits
Previous articleLebanon Legalizes Marijuana for Medical and Industrial Use
Next articleThe Vaping Weekly Podcast: The Musings of Lindsey Stroud; Australia Soapbox


A decent headset is a must when you're serious about gaming. A good set of speakers is, of course, indispensable for your day-to-day gaming, but when you're playing late at night, or at a tournament, or just getting in a bit of mobile gaming on the bus, it pays to have a quality gaming headset ready for action.
When it comes to picking one, though, the choice can feel a little uninspiring. Search for gaming headsets and you'll come across a massive selection of identical-looking units, all black plastic with black leatherette earpads. And while it's a stylish look regardless of its ubiquity, it doesn't necessarily translate into a comfortable fit. That leatherette may look good at first, but once you've had it clamped around your ears for a few hours you'll probably find it becomes sweaty, sticky, and uncomfortable.
That's why, for its newG433 gaming headset, Logitech G went back to the drawing board to create something that not only delivers best-in-class audio quality, but also looks good and feels amazing to wear, even over extended gaming sessions.
And so, rather than look at existing headsets for ideas, Logitech G instead went to top sports performance brands for inspiration and came up with a headset that uses the latest materials for a supremely comfortable fit, no matter how long you're wearing it. It also comes in a range of color options – Royal Blue, Triple Black, Camo Blue, and Fire Red – so that you'll stand out from the crowd.
The G433's lightweight and breathable sports mesh earpads are a delight to wear and won't get nasty after a few hours of intense gaming. Even after a heavyweight gaming weekend session, you can simply remove them and give them a wash, so that they're spring-fresh when you next need them. And if you don't really get on with sports mesh, that's no problem. The G433 also comes with a set of microfiber earpads so you can go with what feels best to you.
Steel headband sliders make the G433 incredibly durable, while a 90-degree custom swivel means you can rest the ear cups on your shoulder when you don't need them, DJ-style. The hydrophobic stain-resistant finish ensures that the G433 will retain its stunning looks when other headsets are starting to look a bit tatty, too.
There's more to the G433 than its appearance, though. It's packed with technology dedicated to making sure that its sound matches its looks. Central to its performance are Logitech G's own Pro G audio drivers, made with hybrid mesh materials to deliver top-quality audio with clear highs and lows with minimal distortion, so whatever your audio source you'll hear it at its absolute best. And to pump up the sound that little bit more, the G433 features a hidden audio port to boost those mids and lows.
To help you get fully immersed in the game, the G433 also comes with its own USB digital sound card with DTS Headphone:X 7.1 support. It accurately replicates the experience of 7.1 surround sound, complete with positional audio, so that not only can you hear which direction the opposition's coming from, you can also tell just how far away they are. It's the best way to reliably hear your games the way their audio designers want you to.
You can even fine-tune each surround sound channel to your own tastes using Logitech Gaming Software, and set up profiles for all your games so they'll always start up sounding just the way you like them. And, to save you all that trouble, Logitech G is also working with developers to create custom profiles for top games. For instance, EA DICE's audio engineers have created a custom Pro G EQ for Battlefield 1 that recreates the studio environment that the game audio was designed in.
As well as pumping out high-quality audio, the G433 also ensures that all your voice chat is captured with crystal clarity. Its boom mic features a micro filter that eliminates unwanted pops and hisses without the need for a big foam ball on the end, so whether you're talking tactics with your teammates or broadcasting on Twitch, no one's going to miss a word you say. It's removable, too, for when you're playing single-player or out and about, and for those situations the G433 has an extra 3.5mm mobile cable with its own inline mic.
With support for PC, consoles – including Nintendo Switch – and mobile, the Logitech G433 gaming headset is a thoroughly versatile way to hear your games at their best for an audio experience that sets itself apart from the competition. But if you don't quite need all its stunning features there's an alternative in the form of the G233 Prodigy gaming headset.
This more budget-friendly option packs many of the G433's best features, including the Pro G audio drivers, removable boom mic with micro pop filters, and multi-platform support so it's suitable for all your gaming needs. And while it doesn't have the G433's color options, it'll still stand out, with a soft-touch black body and Cyan Blue sports mesh earpads.
Both the G433 and G233 gaming headsets are available now from Logitech G, with free delivery.


text:
package com.graphhopper.gtfs.analysis;

import com.graphhopper.GraphHopperConfig;
import com.graphhopper.config.Profile;
import com.graphhopper.gtfs.GraphHopperGtfs;
import com.graphhopper.gtfs.GtfsStorage;
import com.graphhopper.gtfs.PtGraph;
import com.graphhopper.util.Helper;
import org.junit.jupiter.api.AfterAll;
import org.junit.jupiter.api.BeforeAll;
import org.junit.jupiter.api.Test;

import java.io.File;
import java.util.Arrays;
import java.util.List;

import static org.assertj.core.api.Assertions.assertThat;

public class AnalysisTest {

    private static final String GRAPH_LOC = "target/AnotherAgencyIT";
    private static GraphHopperGtfs graphHopperGtfs;

    @BeforeAll
    public static void init() {
        GraphHopperConfig ghConfig = new GraphHopperConfig();
        ghConfig.putObject("graph.location", GRAPH_LOC);
        ghConfig.putObject("datareader.file", "files/beatty.osm");
        ghConfig.putObject("gtfs.file", "files/sample-feed,files/another-sample-feed");
        ghConfig.setProfiles(Arrays.asList(
                new Profile("foot").setVehicle("foot").setWeighting("fastest"),
                new Profile("car").setVehicle("car").setWeighting("fastest")));
        Helper.removeDir(new File(GRAPH_LOC));
        graphHopperGtfs = new GraphHopperGtfs(ghConfig);
        graphHopperGtfs.init(ghConfig);
        graphHopperGtfs.importOrLoad();
    }

    @AfterAll
    public static void close() {
        graphHopperGtfs.close();
    }

    @Test
    public void testStronglyConnectedComponentsOfStopGraph() {
        PtGraph ptGraph = graphHopperGtfs.getPtGraph();
        List<List<GtfsStorage.FeedIdWithStopId>> stronglyConnectedComponentsOfStopGraph = Analysis.findStronglyConnectedComponentsOfStopGraph(ptGraph);
        List<GtfsStorage.FeedIdWithStopId> largestComponent = stronglyConnectedComponentsOfStopGraph.get(0);

        assertThat(largestComponent)
                .extracting("stopId")
                .containsExactlyInAnyOrder("EMSI", "DADAN", "NADAV", "NANAA", "STAGECOACH", "AMV", "FUR_CREEK_RES", "BULLFROG", "BEATTY_AIRPORT", "AIRPORT");

        List<List<GtfsStorage.FeedIdWithStopId>> singleElementComponents = stronglyConnectedComponentsOfStopGraph.subList(1, 4);
        assertThat(singleElementComponents.stream().map(it -> it.get(0)))
                .extracting("stopId")
                .containsExactlyInAnyOrder("JUSTICE_COURT", "MUSEUM", "NEXT_TO_MUSEUM");
    }

}

redpajama_set_name: RedPajamaGithub


Drinking a lot of uncontaminated water with electrolyte packets.
Appropriate antibiotics are given to kill the bacteria. There are increasing rates of antibiotic resistance throughout the world, so your health care provider will check current recommendations before choosing an antibiotic. Several antibiotics are effective in enteric fever.
Alternative includes Co-trimoxazole (two tablets 400mg of sulpamethoxazole and 80 mg of trimethoprim) 12 hourly).
Increase in number of Salmonella, includes. S.typhi are now resistance to many antibiotics and some are only sensitive to Ciprofloxacin.
a) The third generations under mentioned antibiotics are recommended when the organisms are resistance and the treatment to be continued for a period of fourteens days.:Cephalosporin. in a dose of 500 mg 12-hourly.
In case there is recrudescence of the disease and development of the of a carrier should be treated for a period of 4 weeks with Ciprofloxacin; Cholecystectomy may be necessary in some cases.
Carriers of typhoid must not be allowed to work as food handlers.
Packets in case you get sick.


Idle Gangster v2.3.9 (Mod Apk) Easy, simple and ignorant!
Scout legendary gangsters and make the strongest gang!
New type idle RPG! Just let it be! Boss!
Aren't you sick of middle age fantasy?
Sword and bow, magic? Dragon and Knights? Titans??
Our game isn't such a stereotypical RPG.
Bros, give a try. It will not let you down.
■ Tapping game? No! This is an auto RPG, with a little touch you can enjoy fights.
■ No rush! No need to be hurry or nervous. This is the idle RPG.
■ Earn your golds with gang business and territory.
■ We don't rebirth, we do gang reorganization! Reorganize your gang and make it grow. This is the best incremental RPG.
■ Normal weapons? No, we use tools for fights. Collect your weapons and make a legendary gang, Bro.
■ Let's go for PVP! This is a ladder tournament PVP. How strong is your gang?
Brothers, students are students when they study and gangs are gangs when they aren't afraid to fight.
Play this game and be yourself!
★ You can play the game even when you are disconnected to internet, but some features will be limited.


ShadowFlare is an mission-based PC action/role-playing game. The download is available as an EXE file and the latest version is 2.001.000.
Virus tests ShadowFlare: Episode One is clean of viruses and malware.
User Rating 3.7 / 5.0 (based on 15 votes).
Release status Major release with minor updates and a revised build.
Found with ShadowFlare: Episode One has most often been found with shadowflare episode one, shadowflare episode one walkthrough and shadowflare episode one free download.
EULA Emurasoft, Inc includes an EULA which must be accepted in order to complete installation.


Two years ago, the symposium at Lüneburg explored the role of university writing centers in supporting L2 academic writing, with a focus on writing in English. The next year, the conference in Osnabrück expanded the conversation to include writing competencies in German as a foreign or second language. Both conferences examined challenges of and solutions for incorporating writing excellence into existing academic structures, and invited participants to share the methods and strategies that have been successful in their own universities.
This year, TUM hopes to continue conversations from the last two years to discuss writing excellence and how to teach it. Our focus will still be on the L2 context, but we welcome presentations regarding L1 writing (and comparisons between working with L1 and L2 writers) as well. Educators and administrators from across the German-speaking university landscape will gather to examine and debate the many complexities of learning, teaching, and supporting great writing in a multicultural, multilingual environment. TUM professors, who are themselves academic writers in their fields of expertise, will contribute their own valuable points of view in a special panel discussion.