text:
using content::BrowserThread;

namespace extensions {

namespace settings = settings_namespace;
namespace util = settings_test_util;

namespace {

// To save typing ValueStore::DEFAULTS everywhere.
const ValueStore::WriteOptions DEFAULTS = ValueStore::DEFAULTS;

// Creates a kilobyte of data.
scoped_ptr<Value> CreateKilobyte() {
  std::string kilobyte_string;
  for (int i = 0; i < 1024; ++i) {
    kilobyte_string += "a";
  }
  return scoped_ptr<Value>(Value::CreateStringValue(kilobyte_string));
}

// Creates a megabyte of data.
scoped_ptr<Value> CreateMegabyte() {
  ListValue* megabyte = new ListValue();
  for (int i = 0; i < 1000; ++i) {
    megabyte->Append(CreateKilobyte().release());
  }
  return scoped_ptr<Value>(megabyte);
}

}

class ExtensionSettingsFrontendTest : public testing::Test {
 public:
   ExtensionSettingsFrontendTest()
      : storage_factory_(new util::ScopedSettingsStorageFactory()),
        ui_thread_(BrowserThread::UI, MessageLoop::current()),
        file_thread_(BrowserThread::FILE, MessageLoop::current()) {}

  virtual void SetUp() OVERRIDE {
    ASSERT_TRUE(temp_dir_.CreateUniqueTempDir());
    profile_.reset(new util::MockProfile(temp_dir_.path()));
    ResetFrontend();
  }

  virtual void TearDown() OVERRIDE {
    frontend_.reset();
    profile_.reset();
    // Execute any pending deletion tasks.
    message_loop_.RunUntilIdle();
  }

 protected:
  void ResetFrontend() {
    storage_factory_->Reset(new LeveldbSettingsStorageFactory());
    frontend_.reset(
        SettingsFrontend::Create(storage_factory_.get(), profile_.get()));
  }

  base::ScopedTempDir temp_dir_;
  scoped_ptr<util::MockProfile> profile_;
  scoped_ptr<SettingsFrontend> frontend_;
  scoped_refptr<util::ScopedSettingsStorageFactory> storage_factory_;

 private:
  MessageLoop message_loop_;
  content::TestBrowserThread ui_thread_;
  content::TestBrowserThread file_thread_;
};

// Get a semblance of coverage for both extension and app settings by
// alternating in each test.
// TODO(kalman): explicitly test the two interact correctly.

TEST_F(ExtensionSettingsFrontendTest, SettingsPreservedAcrossReconstruction) {
  const std::string id = "ext";
  ExtensionServiceInterface* esi =
      extensions::ExtensionSystem::Get(profile_.get())->extension_service();
  static_cast<extensions::settings_test_util::MockExtensionService*>(esi)->
      AddExtensionWithId(id, Manifest::TYPE_EXTENSION);

  ValueStore* storage = util::GetStorage(id, frontend_.get());

  // The correctness of Get/Set/Remove/Clear is tested elsewhere so no need to
  // be too rigorous.
  {
    StringValue bar("bar");
    ValueStore::WriteResult result = storage->Set(DEFAULTS, "foo", bar);
    ASSERT_FALSE(result->HasError());
  }

  {
    ValueStore::ReadResult result = storage->Get();
    ASSERT_FALSE(result->HasError());
    EXPECT_FALSE(result->settings()->empty());
  }

  ResetFrontend();
  storage = util::GetStorage(id, frontend_.get());

  {
    ValueStore::ReadResult result = storage->Get();
    ASSERT_FALSE(result->HasError());
    EXPECT_FALSE(result->settings()->empty());
  }
}

TEST_F(ExtensionSettingsFrontendTest, SettingsClearedOnUninstall) {
  const std::string id = "ext";
  ExtensionServiceInterface* esi =
      extensions::ExtensionSystem::Get(profile_.get())->extension_service();
  static_cast<extensions::settings_test_util::MockExtensionService*>(esi)->
      AddExtensionWithId(id, Manifest::TYPE_LEGACY_PACKAGED_APP);

  ValueStore* storage = util::GetStorage(id, frontend_.get());

  {
    StringValue bar("bar");
    ValueStore::WriteResult result = storage->Set(DEFAULTS, "foo", bar);
    ASSERT_FALSE(result->HasError());
  }

  // This would be triggered by extension uninstall via a DataDeleter.
  frontend_->DeleteStorageSoon(id);
  MessageLoop::current()->RunUntilIdle();

  // The storage area may no longer be valid post-uninstall, so re-request.
  storage = util::GetStorage(id, frontend_.get());
  {
    ValueStore::ReadResult result = storage->Get();
    ASSERT_FALSE(result->HasError());
    EXPECT_TRUE(result->settings()->empty());
  }
}

TEST_F(ExtensionSettingsFrontendTest, LeveldbDatabaseDeletedFromDiskOnClear) {
  const std::string id = "ext";
  ExtensionServiceInterface* esi =
      extensions::ExtensionSystem::Get(profile_.get())->extension_service();
  static_cast<extensions::settings_test_util::MockExtensionService*>(esi)->
      AddExtensionWithId(id, Manifest::TYPE_EXTENSION);

  ValueStore* storage = util::GetStorage(id, frontend_.get());

  {
    StringValue bar("bar");
    ValueStore::WriteResult result = storage->Set(DEFAULTS, "foo", bar);
    ASSERT_FALSE(result->HasError());
    EXPECT_TRUE(file_util::PathExists(temp_dir_.path()));
  }

  // Should need to both clear the database and delete the frontend for the
  // leveldb database to be deleted from disk.
  {
    ValueStore::WriteResult result = storage->Clear();
    ASSERT_FALSE(result->HasError());
    EXPECT_TRUE(file_util::PathExists(temp_dir_.path()));
  }

  frontend_.reset();
  MessageLoop::current()->RunUntilIdle();
  // TODO(kalman): Figure out why this fails, despite appearing to work.
  // Leaving this commented out rather than disabling the whole test so that the
  // deletion code paths are at least exercised.
  //EXPECT_FALSE(file_util::PathExists(temp_dir_.path()));
}

TEST_F(ExtensionSettingsFrontendTest,
       QuotaLimitsEnforcedCorrectlyForSyncAndLocal) {
  const std::string id = "ext";
  ExtensionServiceInterface* esi =
      extensions::ExtensionSystem::Get(profile_.get())->extension_service();
  static_cast<extensions::settings_test_util::MockExtensionService*>(esi)->
      AddExtensionWithId(id, Manifest::TYPE_EXTENSION);

  ValueStore* sync_storage =
      util::GetStorage(id, settings::SYNC, frontend_.get());
  ValueStore* local_storage =
      util::GetStorage(id, settings::LOCAL, frontend_.get());

  // Sync storage should run out after ~100K.
  scoped_ptr<Value> kilobyte = CreateKilobyte();
  for (int i = 0; i < 100; ++i) {
    sync_storage->Set(
        ValueStore::DEFAULTS, base::StringPrintf("%d", i), *kilobyte);
  }

  EXPECT_TRUE(sync_storage->Set(
      ValueStore::DEFAULTS, "WillError", *kilobyte)->HasError());

  // Local storage shouldn't run out after ~100K.
  for (int i = 0; i < 100; ++i) {
    local_storage->Set(
        ValueStore::DEFAULTS, base::StringPrintf("%d", i), *kilobyte);
  }

  EXPECT_FALSE(local_storage->Set(
      ValueStore::DEFAULTS, "WontError", *kilobyte)->HasError());

  // Local storage should run out after ~5MB.
  scoped_ptr<Value> megabyte = CreateMegabyte();
  for (int i = 0; i < 5; ++i) {
    local_storage->Set(
        ValueStore::DEFAULTS, base::StringPrintf("%d", i), *megabyte);
  }

  EXPECT_TRUE(local_storage->Set(
      ValueStore::DEFAULTS, "WillError", *megabyte)->HasError());
}

// In other tests, we assume that the result of GetStorage is a pointer to the
// a Storage owned by a Frontend object, but for the unlimitedStorage case, this
// might not be true. So, write the tests in a "callback" style.
// We should really rewrite all tests to be asynchronous in this way.

static void UnlimitedSyncStorageTestCallback(ValueStore* sync_storage) {
  // Sync storage should still run out after ~100K; the unlimitedStorage
  // permission can't apply to sync.
  scoped_ptr<Value> kilobyte = CreateKilobyte();
  for (int i = 0; i < 100; ++i) {
    sync_storage->Set(
        ValueStore::DEFAULTS, base::StringPrintf("%d", i), *kilobyte);
  }

  EXPECT_TRUE(sync_storage->Set(
      ValueStore::DEFAULTS, "WillError", *kilobyte)->HasError());
}

static void UnlimitedLocalStorageTestCallback(ValueStore* local_storage) {
  // Local storage should never run out.
  scoped_ptr<Value> megabyte = CreateMegabyte();
  for (int i = 0; i < 7; ++i) {
    local_storage->Set(
        ValueStore::DEFAULTS, base::StringPrintf("%d", i), *megabyte);
  }

  EXPECT_FALSE(local_storage->Set(
      ValueStore::DEFAULTS, "WontError", *megabyte)->HasError());
}

TEST_F(ExtensionSettingsFrontendTest,
       UnlimitedStorageForLocalButNotSync) {
  const std::string id = "ext";
  std::set<std::string> permissions;
  permissions.insert("unlimitedStorage");
  ExtensionServiceInterface* esi =
      extensions::ExtensionSystem::Get(profile_.get())->extension_service();
  static_cast<extensions::settings_test_util::MockExtensionService*>(esi)->
      AddExtensionWithIdAndPermissions(id, Manifest::TYPE_EXTENSION,
          permissions);

  frontend_->RunWithStorage(
      id, settings::SYNC, base::Bind(&UnlimitedSyncStorageTestCallback));
  frontend_->RunWithStorage(
      id, settings::LOCAL, base::Bind(&UnlimitedLocalStorageTestCallback));

  MessageLoop::current()->RunUntilIdle();
}

}  // namespace extensions


text:
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Tag: James Ossuary
The James Ossuary
By Sara Fawley in Ancient Mysteries
Sara Fawley
Hello my name is Sara Fawley. I was born and raised in San Diego, Ca Oct 1965. I lived there until I was 19, then moved with my now ex-husband to Texas where I lived for the next 25 years. I have two grown sons who are married and have lives and families of their own. My current husband and I now live in a small town in Arkansas with our dachshund Shabar. I owned and antiques and collectibles shop but closed it down in September 2013 after having a heart attack. I still buy and sell antiques and collectibles and make handmade natural gemstone jewelry which we sell at shows and festivals. I don't recall ever not knowing that there was more than just us out there. My first experience happened when I was a young child and was visited by my godfather "Grandpa Clyde" at the time of his death and for three weeks after that. I have seen one other full bodied apparition in my life and had several other minor experiences that left me scratching my head.I am not an investigator but have a huge thirst for knowledge. I am always looking up this or that legend, myth or story I hear or sometimes I just have random thoughts and look them up to see if anyone else thinks the same way. I am very analytically minded and always like to get to the bottom of things.
Latest posts by Sara Fawley (see all)
What is a Full Spectrum Camera? - January 31, 2017
Three Knocks! Should I Be Scared??? - January 11, 2017
The Dingonek, West Africa's "Jungle Walrus" - August 5, 2016
By Sara Fawley
What is an Ossuary?
Before we talk about the specifics of this artifact, let's first talk a little bit about what an ossuary is. Ossuaries are small stone (usually limestone) chests in which the bones of the dead were placed after the flesh had decayed. They were very popular in the Jewish community during the Second Temple perior (40 BCE – 135 CE.) These "bone boxes" are generally 2 ½ feet long in order to accommodate the longest bone in the human body ( the adult leg bone.) They taper slightly at the bottom, have flat or vaulted lids and have designs chip-carved or chisled on the outsides. Some do have a red of yellow wash of paint but most decorations ( plants, buildings, animals) were carved. Many contain inscriptions carved on the side in either Hebrew, Aramic or Greek. The incription generally consisted of the name and status in the family of the person whose bones were interred. .
The emergence of the James Ossuary
In 2001 Oded Golan, a Tel Aviv antiquities collector, announced that he had possesion of a stone ossuary which bore the Aramic inscription "Yaakov bar Yosef akhui di Yeshua" which translates in English to "James, son of Joseph, brother of Jesus". Many biblical scholars and archeaologists went wild. Finally there was solid proof (albiet circumstancial) of the existence of Jesus of Nazereth's existence. If then inscription held true, then this was the "bone box" of "James the Just" Apostle and brother to Jesus.
The inscription was validated by two of the world's leading paleographers (experts in authenticating and dating inscriptions based on the shape and stance of the letters) Andre Lemaire of the Sorbonne and Ada Yardeni of the Hebrew University. The ossuary and inscription were also examined and authenticated by the Geological Survey of Isreal(GSI) and Father Joseph Fitzmyer ( the world's leading expert in Aramic).
In October of 2002 the ossuary was displayed at the Royal Ontario Museum in Canada. Thousands of sholars and the general public flocked to the museum to see it.
In 2003, Oded Golan and four other gentlemen were arrested and charged with fraud and forgery of antiquities including the James Ossuary. The ossuary was seized by the Israel Antiquities Authority and tested by a panel of experts appointed by the authority. This panel included Professor Yuval Goren clay expert at the University of Tel Aviv. Goren reported that he had found fake patina added to the "brother of Jesus" portion of the inscription and that it had been added to the ossuary at a later date than the original portion of the inscription. This was disputed by Mr Golan's supporters.
In 2005 the trial of Oded Golan for fraud and forgery of many antiquites including the James Ossuary began. The trial 138 witnesses, more than 400 exhibits and over 12,000 pages of testimony. Both sides provided many experts for and against the authenticity of inscription. In 2012, after 7 years of trial, Oded Golan was aquitted of all forgery charges. He was convicted and fined for some lesser charges such as selling antiquities without a license.
So does this mean the James Ossuary is without a doubt the "bone box" of James the Just? Not necessarily. In the judgement handed down it was made clear that the judgement was in no way authenticating any of the artifacts in question. The judgement simply provides that the prosecution did not prove by a preponderance of evidence that Oded Golan participated or was aware of any forgery or fraud in connection with the named antiquities. Both sides of the issue still have their experts and scholars who stand firmly behind their argreement or objection to the aunthenticity of the artifact.
The bottom line is even if the inscription were proven to be 100 % authentic this does not necessarily mean that the James inscribed on the box is actually James the Just. James, Joseph and Jesus were all very common names in that time period. Many argue that the odds of there being another James who had a father named Joseph and a brother named Jesus in the same time period are astronomical but the fact remains that it is still a very real possibility. Without a provenance of where the box came from (Golan aquired it in the 1970's but has always been evasive as to from where) we may never know.
http://www.livescience.com/50444-james-ossuary-talpiot-tomb…
http://www.biblicalarchaeology.org/…/is-the-brother-of-jes…/
http://members.bib-arch.org/publication.asp…
http://www.jewishvirtuallibrary.org/…/ejud_0002_0015_0_1524…
http://jamesossuarytrial.blogspot.com/
ancient mysteries, Articles and Submissions, James Ossuary
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redpajama_set_name: RedPajamaCommonCrawl


text:
El Douglas XB-31 (Douglas Model 332) fue un diseño presentado por Douglas tras una solicitud de las Fuerzas Aéreas del Ejército de los Estados Unidos por un bombardero muy pesado, la misma solicitud que condujo al Boeing B-29 Superfortress, Lockheed XB-30, y Consolidated B-32 Dominator.

Diseño y desarrollo

Alrededor de 1938, el General Henry H. "Hap" Arnold, el jefe del Cuerpo Aéreo del Ejército de los Estados Unidos, empezó a alarmarse ante la posibilidad de una guerra en Europa y en el Pacífico. Deseando estar preparado para los requerimientos a largo plazo de la Fuerza Aérea, Arnold creó un comité especial presidido por el General de Brigada W. G. Kilner; uno de sus miembros fue Charles Lindbergh.

Después de una gira por las bases de la Luftwaffe, Lindbergh se convenció de que la Alemania nazi estaba muy por encima de las otras naciones europeas. En un informe de 1939, el comité hizo un número de recomendaciones, incluyendo el desarrollo de nuevos bombarderos pesados de largo alcance. Cuando la guerra estalló en Europa, Arnold solicitó estudios de diseño, a varias compañías, de un Bombardero de Muy Largo Alcance capaz de viajar 5000 millas (8000 km). Se concedió la aprobación el 2 de diciembre de 1939.

El diseño XB-31 fue rechazado (junto con el XB-30) en favor de los B-29 y B-32, debido a que el USAAC encontró los B-29 y B-32 superiores a los diseños de Douglas y Lockheed.

Especificaciones (según diseño)

Aeronaves relacionadas

Véase también

 Anexo:Aeronaves de la Fuerza Aérea de los Estados Unidos (históricas y actuales)

Referencias

Bibliografía

 Francillon, René J. McDonnell Douglas Aircraft since 1920. London: Putnam & Company Ltd., 1979. ISBN 0-370-00050-1.
 Jones, Lloyd S. U.S. Bombers: B-1 1928 to B-1 1980s. Fallbrook, California: Aero Publishers, Inc., 1974. ISBN 0-8168-9126-5.

Enlaces externos

 Página de Aviones Bombarderos de Estados Unidos de Joseph F. Baugher.

B-31
Aeronaves de ala alta
Aviones tractores de cuatro motores
Bombarderos de los años 1930 de Estados Unidos
Proyectos cancelados de aeronaves militares de los Estados Unidos

redpajama_set_name: RedPajamaWikipedia


Apparition and Sony Pictures Worldwide Acquisitions Group have announced that they have decided to expand The Boondock Saints II: All Saints Day "nationwide to additional markets" on November 13th 2009. The film grossed over $500,000 this past weekend in just four markets: New York, Los Angeles, Boston, and Philadelphia, with a per theater average of over $8,000. Because of the demand/fan base, the studio has announced that they will be opening the movie wider on November 13 to additional cities including Seattle, San Francisco, San Diego, Chicago, Detroit, Dallas, Atlanta, Houston, and more.
The Boondock Saints: All Saints Day will opens wide in theaters on Friday, November 13.


Except the Will that says to them: "Hold on!"
I used to be able to recite "IF" and tried to remember it here...I couldn't quite! But isn't it wonderful...no wonder it was voted the Nation's favourite.
Magpie and Reynard came to stay this week and as the weather was being particularly "April'ish" we decided it was high time we took ourselves off to Batemans, the home of Rudyard Kipling.
I must congratulate the National Trust ~ I know they are making efforts to ensure that visits are less "You can look but don't touch!" and they seem to be succeeding here. I particularly liked the old wireless in Elsie's sitting room that you can "tune" and listen to Ralph Fiennes reading extracts of Kiplings' work.
The whole house is fascinating and we whiled several hours away.. outside it thundered, hailed, rained and shone in equal turns.
Enjoying those April showers wherever you are?
PS This is for Kay, Georgia Girl With An English Heart...I seem to remember you asking me to visit?
Looks like a beautiful place to visit, how lucky the family were to have it as their home.
I know odd lines from 'If' but don't think I've ever read the whole thing before. DH and I visited Batemans a few years ago and really liked it. DH is a huge fan of Kipling and I think he has every book by or about him ever written!
Oh gosh - so lovely.
Sarah-Jane, You Doll! I can't believe you remember me saying that to you! I am so happy to see these photos. Thank you, you have made my day!
We NEED April showers, I have my fingers crossed!
What a beautiful post I would love to visit it. My great uncle had an antiques shop and when Rudyard Kipling and his wife, visted Bath they would usually call in and buy antiques from them. I wonder if any items are still in his house!


text:
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Karlee Smith's drive to create sexy and supportive post-op bras started, of course, with her own breast surgery experience. As every woman wants in this intimate period of her life, Karlee wanted her post op support bra to feel like a fun and fashionable addition to her wardrobe, not unbearable obligation.
It all started with Karlee's determination as a high school student to intern with VF Corporation, one of the best intimates companies in the industry. She soon after submitted a stellar scholarship application that awarded her full tuition entrance to The Art Institute of Fort Lauderdale. Karlee's passion was always for intimates, activewear, and swim although she spent most of her internship in college either as an editor at Vicious Magazine and working on more general clothing items at VF Corporation in their sourcing division; after graduating, brands like Fruit of the Loom and Victoria's Secret could all see the potential for a great innovator in bra fashion.
After receiving awards for her fashion concepts in marketing & merchandising at New York Fashion Week and Elle Girl Magazine, plus opportunities with Fortune 500 companies like VF Corporation, Fruit of the Loom, and Victoria's Secret, Karlee decided to develop her own brand to leave women feeling supported and confident no matter the circumstances.
Karlee is now very proud of her decision to focus on post-op bra design, loving any and every story she hears from confident KS women. She strives to grow and evolve her line after the many empowered women who now wear her brand and from the activities she enjoys the most, like fitness, travel, music, art, and of course, fashion. (Even if she admittedly has had a few ugly bumps in the road).


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If you go on the Internet, you can discover more about this company by checking out GetAPlumberNow. They are market leaders in the Chicago location, a business that is suggested by those that have actually used to their services, plainly showing that they are one of the very best. brand name is an excellent business to contact and will be more than pleased to use their expert services for you and your household. They have years of experience in this industry, and will have the ability to not only repair work or replace your warm water heating system for you, but can also offer you great pointers on the best ways to ensure the problem does not occur once again. Their suggestions will likewise extend into the area of helping you make a hard option if it is economically based, showing you how much cash you might be losing because of your existing warm water tank, and what does it cost? cash you will conserve in the long run. This normally makes it possible for people to make that leading option in using the services of this plumbing business in Call A Plumber Streamwood for hot water heating system repairs and replacements.


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text:
Tumor Biology
A Critical Step in Metastasis: In Vivo Analysis of Intravasation at the Primary Tumor
Jeffrey B. Wyckoff, Joan G. Jones, John S. Condeelis and Jeffrey E. Segall
Jeffrey B. Wyckoff
Departments of Anatomy and Structural Biology [J. B. W., J. S. C., J. E. S.], Pathology [J. G. J.], and the Analytical Imaging Facility (J. S. C.), Albert Einstein College of Medicine, Bronx, New York 10461
Joan G. Jones
John S. Condeelis
Jeffrey E. Segall
DOI: Published May 2000
Detailed evaluation of all steps in tumor cell metastasis is critical for evaluating the cell mechanisms controlling metastasis. Using green fluorescent protein transfectants of metastatic (MTLn3) and nonmetastatic (MTC) cell lines derived from the rat mammary adenocarcinoma 13762 NF, we have measured tumor cell density in the blood, individual tumor cells in the lungs, and lung metastases. Correlation of blood burden with lung metastases indicates that entry into the circulation is a critical step for metastasis. To examine cell behavior during intravasation, we have used green fluorescent protein technology to view these cells in time lapse images within a single optical section using a confocal microscope. In vivo imaging of the primary tumors of MTLn3 and MTC cells indicates that both metastatic and nonmetastatic cells are motile and show protrusive activity. However, metastatic cells show greater orientation toward blood vessels and larger numbers of host cells within the primary tumor, whereas nonmetastatic cells fragment when interacting with vessels. These results demonstrate that a major difference in intravasation between metastatic and nonmetastatic cells is detected in the primary tumor and illustrate the value of a direct visualization of cell properties in vivo for dissection of the metastatic process.
Metastasis leads to poor prognosis in many cancer types. Metastasis of adenocarcinomas involves leaving the primary tumor either via lymphatics or blood vessels, transport to and arrest in a target organ, and growth of metastases in the target organ (1) . Each of these steps is a multicomponent process, with potentially different tumor cell properties and molecules playing critical roles in different steps (2) . As novel molecular methods are being developed to identify new genes and proteins that could contribute to specific steps, it is important to develop more detailed methods for the analysis of metastasis at the cellular level to accurately evaluate the roles of specific gene products in individual steps of metastasis. Similarly, as new therapies are developed, the effects of specific treatments on the individual steps in the metastatic cascade need to be evaluated.
The most common assays for metastatic ability in vivo have been end point assays. For example, i.v. injection of tumor cells (often termed "experimental metastasis"), followed by determination of the number of metastases in a target organ such as the lung, is a simple method for evaluation of arrest and growth of tumor cells in target organs (3) . Detailed studies using this assay have demonstrated that extravasation per se tends not to be rate limiting, but that growth of metastases is inefficient (4 , 5) . However, this assay is limited by the introduction of a bolus (typically 100,000 cells) of in vitro cultivated cells in a nonphysiological manner. A more physiological approach to analysis of tumor cell metastasis makes use of the injection of tumor cells into an appropriate (orthotopic) tissue, followed by growth of a primary tumor as the source of tumor cells for metastasis. Such "spontaneous metastasis" assays are more accurate models of human disease in that they rely on growth of a primary tumor, and the tumor cells themselves then must actively leave the primary tumor and enter the vasculature (6, 7, 8) . Cell lines specifically selected for high metastatic ability through use of the experimental metastasis assay are not necessarily highly metastatic in the spontaneous metastasis assay (9) . Thus, for a detailed comparison of all of the various steps of metastasis, an assay such as the spontaneous metastasis assay must be used.
However, analysis of metastasis using the spontaneous metastasis assay typically measures only the growth of the primary tumor and the number of metastases that form in a target organ. The relative efficiencies for various steps in this assay have never been directly determined. Such an analysis is crucial because of the heterogeneity of tumors and tumor cell lines. Human primary tumors show extensive variation in all properties, ranging from growth and morphology of primary tumors through tumor cell density in the blood and then formation and growth of metastases. Similarly, tumor cell lines show broad variation in formation of a primary tumor and metastatic ability. As specific cell lines are manipulated to express particular activated or inhibitory oncoproteins, the effects on metastatic abilities will need to be interpreted in terms of the particular steps in the metastatic cascade that are affected. Growth of the primary tumor is simple to quantify and has been an extremely useful assay for determining proteins that are important for tumor formation. However, entry of tumor cells into the circulation is the critical first step in the metastatic cascade, and although it has been assayed in various ways (10, 11, 12) , it has not been observed directly.
Because for certain tumor types and conditions there are high levels of circulating tumor cells (13 , 14) , it has sometimes been assumed that entry into the circulation is not a critical step in tumor cell metastasis and that formation of metastases in target organs is rate limiting. Some studies using cell-based assays indicate that tumor cell burden in the blood can correlate with poor prognosis (15 , 16) . However, PCR or antibody-based assays for tumor cells in the blood may not show as strong a correlation (17, 18, 19, 20) . Animal studies using well-characterized cell lines and controlled conditions are needed to evaluate the role of intravasation.
In this study, we describe a straightforward procedure for comparing intravasation, extravasation, and growth in target organs during metastasis. For two mammary adenocarcinomas, the metastatic MTLn3 and poorly metastatic MTC cell lines, we use this procedure to demonstrate that intravasation is important. We then use in vivo time lapse confocal microscopy of MTLn3 and MTC primary tumors expressing GFP 3 to compare the behavior of metastatic and nonmetastatic cells relative to blood vessels. We find that although both metastatic and nonmetastatic cells are motile in the primary tumor, metastatic cells are polarized toward blood vessels and nonmetastatic cells tend to fragment during intravasation. These results provide new insights into the differences between metastatic and nonmetastatic cells and have implications for the types of assays that should be developed for prognosis.
Cell Lines.
MTLn3-GFP cells were used as described before (21) . MTC-GFP cells were created using retroviral transfection of parental MTC cells with a GFP expression construct. The GFP sequence was excised from pEGFPN1 (Clontech) using NsiI and EcoRI and subcloned into the BamHI/EcoRI site of pLXSN. This construct was then transfected into Phoenix cells [supplied by Vaughn Latham and Rob Singer (AECOM, Bronx, NY)] using standard methods (22 , 23) and allowed to grow overnight. The supernatant was collected, spun at 1000 rpm for 5 min, and 1 ml was overlaid on a confluent plate of MTC cells. Positive clones were selected by neomycin selection and GFP fluorescence. Stable cells were cultured the same as parental cells, as described previously (24) . Cell growth rate and morphology of transfected cells was determined to be the same as parental cells, and fluorescence was shown to remain constant for 30 passages (data not shown). Extensive histopathology studies for the MTLn3-GFP cells (21) and the MTC-GFP cells were done to confirm that their metastatic potentials were similar to the parental cell lines.
Blood Burden, Single Cells in the Lung, and Metastases.
Tumor cell blood burden was determined by placing a rat with a 6-week-old tumor under isoflurane anesthesia and removing 4 ml of blood from the right atrium via heart puncture. The blood was then spun at 5000 rpm for 5 min, and the serum layer and buffy coat region were plated into α-MEM growth medium. The following day, plates were rinsed twice with Dulbecco's PBS (Life Technologies, Inc.) to remove RBCs, and regular growth medium was added. After 6 days, all clones in the dish were counted. To test cell viability in the collection process, blood was drawn as above from noninjected rats. Cultured MTLn3-GFP and MTC-GFP cells were removed from growth dishes using trypsin/EDTA. Ten, 100, and 1000 tumor cells were added to 1 ml of blood and 1 ml of medium, respectively. The mixtures were then centrifuged, plated, and counted as described above for blood samples derived from rats bearing tumors. There was approximately 50% recovery of tumor cells from mixtures with blood as compared with mixtures with growth medium for both cell lines.
For visualization of single cells near the surface of the lungs, the lungs were removed after blood removal and euthanization of the rat. The lungs were then placed in matTek dishes (MatTek Corporation, Ashland, MA) with 1 ml of L15 medium (Life Technologies, Inc.) to keep them moist. Ten fields from each side of both major lobes were then visualized using a ×60 objective on a Nikon inverted microscope. All whole single cells visible in these fields were counted. Tumor cells were shown to be countable by confirming their GFP fluorescence in the fluorescein channel and lack of fluorescence in the rhodamine channel of the Bio-Rad MRC-600 confocal microscope. A field is the 1.1-mm diameter visible area through the microscope oculars.
For measurement of metastases, excised lungs were placed in 3.7% formaldehyde, mounted in paraffin, sectioned, and stained with H&E. Slices were viewed using a ×20 objective, and all visible metastases in a section containing more than five cells were counted.
Imaging of Living Tumors and Their Vasculature.
Tumor imaging was performed as described previously (21) . Briefly, 1 × 106 cells were injected under the second nipple anterior from the tail of a Fischer 344 rat and allowed to grow for 2.5 weeks. After 2.5 weeks, the rat was placed under isoflurane anesthesia and the tumor was exposed using a simple skin flap surgery, with as little disruption of the surrounding vasculature as possible. The animal was then placed onto a Bio-Rad MRC-600 confocal microscope, using a ×20 objective and imaged in time lapse, with a single image being taken every minute. On average, about three different fields of each tumor were imaged for 20–30 min each.
For visualizing vasculature, 200 μl of rhodamine-dextran (2 M dalton; Sigma Chemical Co.) at 20 mg/ml in Dulbecco's PBS was injected into the tail vein of the rat after anesthesia, but before surgery. The vasculature in the tumor was then visualized using the rhodamine channel of the Bio-Rad confocal.
Quantification of Images.
Time lapse movies were reconstructed using NIH Image. Movies then were viewed and evaluated for the following criteria: (a) cell extension and retraction; (b) cell locomotion (21) ; (c) cell orientation to the vasculature; and (d) host cell locomotion. Fields are defined as the visible area for each optical condition and are 2.4 × 105 square microns, unless indicated otherwise. For statistical purposes, an experiment is defined as a single tumor, and data are reported as the percentage of time lapse sequences for which a recorded event happens during an experiment. The velocity of cell locomotion was determined by the distance the cell moved between image frames.
Percent orientation in Table 2 ⇓ was determined as the percentage of blood vessels in an imaging field with four or more directly adjacent cells polarized toward the vessel. Percent orientation was corrected for randomly polarized fields of cells by subtracting from the above value the percentage of blood vessels with both adjacent and nonadjacent cells polarized toward the vessel.
Entry into the Bloodstream Is an Important Step in Metastasis.
To provide a quantitative comparison between blood burden and lung metastasis during spontaneous metastasis, we made use of mammary adenocarcinoma cells stably expressing GFP. The metastatic MTLn3 and nonmetastatic MTC cell lines are both derived from the Fisher rat 13762 NF mammary adenocarcinoma (25) . They were transfected with GFP expression vectors and the neomycin resistance selection marker, as described by Farina et al. (21) and in" Materials and Methods." The cells are uniformly fluorescent, and expression of GFP does not alter their metastatic properties (see Ref. 21 and below).
Cells were injected into the mammary fat pads of Fisher 344 rats and allowed to grow for 6 weeks. To determine blood burden before filtration by any capillary bed, but with minimal perturbation of the primary tumor, the rats were anesthetized, and 4 ml of blood was withdrawn from the right atrium of the heart. The cells in the serum and buffy coat were plated in growth medium and allowed to grow for 1 week. Tumor cell colonies were identified by cell morphology and GFP fluorescence. The blood of rats carrying MTLn3 primary tumors formed about 23 colonies/4 ml of blood, which was significantly more than the blood burden of <1 colony/4 ml for rats carrying MTC primary tumors (P < 0.002; Table 1 ⇓ , column 2). Control experiments using defined numbers of cells mixed with blood showed a 50% plating efficiency for both cell lines. Thus, there was a significantly larger number of viable tumor cells present in the blood of rats carrying MTLn3 tumors compared with the blood of rats carrying MTC tumors. This difference was not due to larger MTLn3 tumors (Table 1 ⇓ , column 1). MTC tumors tended to be larger than MTLn3 tumors.
In vivo tumor cell distributions
For MTLn3, 15 rats received injections and for MTC, 8 rats received injections. Tumor cell distributions were measured, means and SEs of the mean were calculated, and statistical significance was evaluated using the nonparametric Wilcoxon test.
GFP fluorescence was used to identify single cells present in the lungs of the same rats used to measure blood burden. After removal of the blood, the animals were euthanized and the lungs were dissected. The lungs were viewed using a confocal microscope, and the number of fluorescent cells present in 40 high power fields was determined for each animal. Only cells showing fluorescence in the GFP channel with no fluorescence in the rhodamine channel were counted. Rats carrying MTLn3 tumors had significantly more tumor cells present in the lungs than rats carrying MTC tumors (P < 0.003; Table 1 ⇓ , column 3). In addition, for rats carrying MTLn3 tumors, comparing blood burden and fluorescent cells in the lungs in each animal there was a significant correlation (correlation coefficient, 0.86; P < 0.0004). However, there were no correlations with the size of the primary tumor (correlation coefficient, 0.23- 0.5; P > 0.05).
To evaluate metastases in the lungs of animals, sections of lungs were stained with H&E and evaluated for metastases. Rats carrying MTLn3 tumors had significantly more metastases than rats carrying MTC tumors (P < 0.003; Table 1 ⇓ , column 4). In addition, for rats carrying MTLn3 tumors there was a significant correlation between blood burden and metastases (correlation coefficient, 0.96; P < 0.0001).
In summary, from this analysis there was a clear correlation between tumor cell density in the blood and either numbers of single tumor cells in the lungs or metastases. This correlation was present both when comparing metastatic and nonmetastatic cell lines (MTLn3 and MTC) and when comparing MTLn3 tumors on the level of individual animals. To our knowledge, this is the first time that such a comparison has been performed on a single animal basis. The presence of a correlation between blood burden and both single cells, as well as metastases in the lungs, indicates that entry into the blood is important for metastasis of cells derived from the 13762 NF mammary adenocarcinoma. These data suggest that a more detailed analysis of cell behavior in the primary tumor is important for determining the properties that enable MTLn3 cells to gain access to the circulation. To perform such studies, we made use of the GFP expression of these cell lines to perform intravital imaging of the primary tumors.
Intravital Imaging of MTLn3 and MTC Primary Tumors
Metastatic Cells Are Oriented toward Blood Vessels.
As reported previously (26) , MTLn3-GFP cells form loose clusters of rounded, nonpolarized cells in the primary tumor, whereas MTC-GFP cells are elongated and polarized in tight sheets. This was confirmed by both histopathology sections and intravital imaging. However, around blood vessels, MTLn3-GFP cells were elongated and polarized toward blood vessels. In Fig. 1A ⇓ , MTLn3-GFP cells (green) are elongated and polarized (arrows) toward the vessel (red), whereas cells away from the vessel are rounded (arrowheads). Fig. 1B ⇓ shows MTC-GFP cells (green) that are elongated throughout the field of view, with cell elongation occurring independent of the vessel positions. Overall, for MTLn3 tumors, 57 +/− 6% of all vessels had polarized cells near them, compared with 24 +/− 4% for vessels in MTC-GFP tumors (Table 2 ⇓ , column 1). This suggests that there is a vessel-mediated effect on MTLn3 cells that results in orientation toward the vessels.
MTLn3-GFP cells orient toward blood vessels, whereas MTC-GFP cells do not. Orientation of MTLn3-GFP cells (A) and MTC-GFP cells (B) to blood vessels in the primary tumor. A, MTLn3-GFP cells (green) near the vessel (red) are seen to orient themselves toward the vessel in an elongated fashion (arrows) as opposed to those away from the vessel (arrowheads). B, MTC-GFP cells (green) randomly associate with the vessel (red), and remain elongated away from the vessel. Bar, 25 μm.
In vivo analysis of tumor cell behavior
The percentage of tumor time lapse sequences with cells showing the specified behavior is given, together with SE of the mean. Orientation was determined as the percentage of blood vessels in an imaging field with four or more adjacent cells polarized toward the vessel (see" Materials and Methods"). For MTLn3 tumors, a total of 122 fields from 45 tumors containing 163 vessels was analyzed. For MTC tumors, a total of 63 fields from 24 tumors with 97 vessels was analyzed.
The differences in cell locomotion (translocation) and protrusiveness between metastatic and nonmetastatic cells are much smaller in the primary tumor. During a time lapse sequence, cells within the primary tumor can be detected extending and retracting processes, as well as translocating. Both protrusiveness and translocation were reported previously for MTLn3-GFP cells in the primary tumor (21) , and similar phenomena were observed in the MTC-GFP primary tumors. For the MTLn3-GFP tumors, cell protrusion and retraction occur in 62% of tumors compared with 47% for MTC tumors (Table 2 ⇓ , column 2). Fig. 2 ⇓ shows the protrusive activity of an MTC-GFP tumor cell. The cell extends a small protrusion (Fig. 2B) ⇓ , which then enlarges (Fig. 2C) ⇓ . The movement of the leading edge of this cell was 3.8 μm/min during maximum rate of extension, consistent with the average rate of movement seen for MTLn3 and MTC cells (see below). For MTLn3-GFP cells, the most obvious shape changes occur at the periphery of vessels where the cells are elongated, whereas for MTC cells protrusion and locomotion occur randomly relative to vessels.
MTC-GFP cells are able to move in a primary tumor by first sending out a thin leading edge protrusion through a small space. Protrusive movement of a whole MTC-GFP cell within the primary tumor viewed in a single optical section in situ. A, initial position of cell. B, initial extension of protrusion. C, further protrusion. The outline shows starting position. The images are 5 min apart. Bar, 25 μm.
Cell translocation occurs much less frequently in both tumor types. As opposed to protrusive activity, only a small fraction of the cells within a primary tumor translocate at a given time. MTLn3-GFP cell locomotion within the primary tumor, as reported previously and observed with more tumors here, is seen as a linear movement. The cell shown in Fig. 3 ⇓ moves on a linear path at a speed of 3.6 μm/min. This type of movement is seen in 17% of all MTLn3 tumors and 10% of MTC tumors (Table 2 ⇓ , column 3). The MTC-GFP cell shown in Fig. 4 ⇓ moves at a rate of 3.6 μm/min. The average velocities of movement of MTLn3 (3.4 μm/min, 12 cells) and MTC (3.7 μm/min, 7 cells) were similar.
MTLn3-GFP cells exhibit linear translocation in situ. Movement of a MTLn3-GFP cell in a single optical section of the primary tumor. The cell moved over a distance of 22 μm. A, the cell seen in its starting position (outlined, with arrow). B, cell has moved to a new position 6 min later (arrow). Its starting position is outlined. The cell moved at an average velocity of 3.6 μm/min. Bar, 25 μm.
MTC-GFP cells exhibit linear translocation in situ. Movement of a MTC-GFP cell in a single optical section of the primary tumor. A, cell is shown outlined in its starting position (arrow). B and C, images showing a progressively linear movement (arrows). The outline shows the original position. The cell moved at an average speed of 3.6 μm/min. The images are 4 min apart. Bar, 25 um.
Nonmetastatic Cells Demonstrate Extensive Fragmentation.
MTC-GFP tumor cells show a significantly greater number of cell fragmentation events compared with MTLn3-GFP tumor cells. Fragments of cells are visualized breaking from cell protrusions and moving away at an accelerated rate of speed (Fig. 5) ⇓ . In Fig. 5 ⇓ , a fragment is seen attached to a cell extension (A), then breaks off and moves away from the cell (B and C). The initial rate of protrusion of the extension was 2.8 μm/min, but the fragment speed of movement is 11–15 μm/min. This was observed in 32% of MTC-GFP tumors compared with 6% of MTLn3-GFP tumors (Table 2 ⇓ , column 4). In a number of cases, the fragmentation was seen to occur in blood vessels. The higher velocity of the fragment is consistent with passive flow in the blood because the velocity is too high for cell locomotion (maximum about 4μ m/min) and close to the velocity of blood flow in small vessels.
The protrusions of MTC-GFP cells in situ are seen to break off from the cell body and move away rapidly. A, cell protrusion before being broken off (arrow). B and C, fragment broken off from the protrusion (arrows). The outline shows starting position. Speed of fragment after breaking away from protrusion suggests movement into vascular flow (large hash marks delineate vessel). The initial speed of protrusion is 2.8 μm/min; after release it increases to 11–15μ m/min. The images are 1 min apart. Bar, 25 μm.
More Motile Host Cells Are Visualized in Metastatic Tumors.
During fluorescent imaging of GFP-tagged tumor cells, we have observed nonfluorescent cells moving against the fluorescent tumor cell background as they block and scatter fluorescence from the tumor cells (Fig. 6) ⇓ . It is likely that these are host immune system cells for the following reasons: (a) the cells are not fluorescent; (b) the cells are smaller than the tumor cells; and (c) the speed of movement is relatively high (on the order of 10 μm/min). Although host cells were observed at similar frequencies in MTLn3 and MTC tumors (28% and 23%, respectively; Table 3 ⇓ , column 1), there was a significant difference in the number of host cells observed. For MTLn3-GFP tumors, there was an average of 11 host cells observed per field, compared with 2 host cells/field for MTC-GFP tumors (Table 3 ⇓ , column 2). The increased numbers of host cells may aid in either polarizing the tumor cells toward blood vessels or in generating pores through which tumor cells can intravasate.
Host cells can be visualized as shadows on top of white fluorescent cells in the primary tumor. Host cell movement over MTLn3-GFP cells in a single optical section of the primary tumor viewed in situ. A-C, host cells are seen as black shapes (arrows) moving over fluorescent tumor cells (MTLn3-GFP). The outline shows the starting position. Host cells move at 7.7 μm/min, and the images are 7 min apart. Bar, 25 μm.
Host cells in tumor
The percentage of tumor time lapse sequences with cells showing the specified behavior is given, together with SE of the mean. For MTLn3 tumors, a total of 122 fields from 45 tumors was analyzed. For MTC tumors, a total of 63 fields from 24 tumors was analyzed.
Movies of the data from which the figures were derived are available. 4
We present here the combination of two novel analyses of tumor cell metastasis: (a) a steady-state analysis of tumor cell distributions in individual animals; and (b) in vivo imaging of cell behavior in primary tumors. The steady-state analysis of tumor cell distributions indicates that entry into the vasculature is an important step that contributes to differences in metastasis between MTLn3- and MTC-derived tumors. To define the mechanisms behind this difference, we used confocal microscopy of GFP-transfected cells in anesthetized animals. MTLn3 tumor cells were more highly oriented around blood vessels compared with MTC tumor cells. Protrusive activity and random cell movement were similar. However, MTC tumor cells were much more likely to form fragments. In addition, more host cells were detected in MTLn3 tumors compared with MTC tumors.
Steady-State Analysis of Metastasis.
We have used a steady-state analysis of tumor cell distributions in individual animals to develop a general method for comparing intravasation with growth in the lungs during metastasis. We have found that although the primary tumor sizes formed are similar, rats carrying the more metastatic MTLn3 tumors have about 90 times more cells in the blood than rats carrying MTC tumors. Although previous studies had demonstrated that MTC cells injected i.v. form few metastases in the lungs (27) , this is the first demonstration that MTC cells are also unable to effectively enter the circulation.
There are two limitations in our estimate of tumor cell density in the blood. First, because we are using colony counts, each clump of tumor cells would be counted as a single colony-forming unit, or cell (10 , 12 , 14) . Second, the cells must be able to grow in vitro. Given that these tumor cell lines grow well in vitro before injection into the animal to form a tumor, the latter limitation is not significant. Reconstruction experiments in vitro indicate that exposure to blood per se produces a roughly 50% reduction in plating efficiency for both cell lines. Thus, our estimate of tumor cell density in the blood may be an underestimate of the true number.
To determine whether metastasis of the MTLn3 cells could be dependent on intravasation as well, we compared the tumor cell density in the blood with single cells and metastases in the lungs for each rat carrying MTLn3 tumors. We found a significant correlation between blood density and single cells or metastases in the lungs. This result suggests that entry into or survival in the vasculature is an inefficient step for MTLn3 cells as well as for MTC cells. In addition, for animals carrying MTLn3 tumors, the number of lung metastases is less than would be expected given the single cell density in the lungs. This indicates that growth of metastases in the lungs is also inefficient, as has been well recognized (28, 29, 30, 31, 32) .
In Vivo Imaging of the Primary Tumor.
Given the results above indicating that intravasation is an important difference between metastatic and nonmetastatic tumors, a more detailed analysis of the cell behavior in the primary tumor becomes important. A number of researchers have made use of GFP (5 , 21 , 33, 34, 35) or lacZ (36, 37, 38, 39, 40) expression to increase the sensitivity of observations of disseminated tumor cells in target organs. We made use of techniques developed to follow individual tumor cells in primary tumors with an intact blood supply. By using a confocal scanning microscope to image cells that stably express green fluorescent protein, we are able to follow cell movements over 30 min. These studies provide the first comparison of in vivo cell behavior at the primary tumor with metastatic ability.
There are three major differences between the MTLn3 and MTC primary tumors: increased cell orientation toward blood vessels in MTLn3 tumors, increased fragmentation of cells in MTC tumors, and increased numbers of putative host cells in MTLn3 tumors. There are not as dramatic differences in protrusive activity and random cell translocation between these tumors.
Increased cell orientation of MTLn3 cells toward blood vessels could increase the efficiency with which they can then intravasate. The increased orientation could result in directed movement into the blood vessel and more cells moving into the blood. The orientation could be induced by chemoattractant diffusing from the blood vessel. Growth factors, including EGF or platelet-derived growth factor, are present in platelets and smooth muscle cells (e.g., see Refs. 41, 42, 43, 44 ). Release of growth factors from these cells or endothelial cells could provide a gradient that would produce a chemotactic response. MTLn3 cells are chemotactic toward EGF in vitro, whereas MTC cells are not (45) . MTLn3 cells express more EGF receptors than MTC cells (46) , and expression of the EGF receptor in MTC cells increases chemotactic responses to EGF and metastatic ability (47 , 48) . Expression of the EGF receptor and other EGF receptor homologues, such as ErbB2, have been correlated with poor prognosis. It is possible that chemotactic signaling mediated by the EGF receptor is important in enhancing metastatic capability in addition to the well characterized effects of EGF receptor signaling on mitogenesis.
The increased fragmentation of MTC cells compared with MTLn3 cells provides an additional mechanism by which viable cell number in the blood might be low for MTC cells. In some cases, we have identified fragmentation occurring during intravasation. If the shear forces in the blood vessels are causing MTC cells to fragment as they enter the blood vessel, that event will eliminate the possibility of metastasis. The increased susceptibility to fragmentation of MTC cells could reflect a lack of orientation toward the blood vessel and slow traversal of the endothelium due to a reduced chemotactic response, as suggested above. Morris et al. (49) have reported fragmentation of tumor cells injected i.v. and then viewed during extravasation into microvascular beds. At the extravasation stage of metastasis, fragmentation of both metastatic and nonmetastatic cells may occur if cells do not rapidly polarize and exit the blood vessel.
The observation of fragmentation of poorly metastatic cells has important implications for biochemical or molecular-based assays of blood. Given that cell fragments would contain tumor protein and DNA, immunological assays for tumor proteins or PCR-based assays for tumor DNA would be positive, although the fragments would be unable to metastasize. In that event, PCR- or protein-based assays of blood samples might not be useful for predicting metastases. A cell-based assay that evaluates the size of cells may be a more powerful predictor of poor prognosis.
An unexpected dividend of the GFP-based imaging was the ability to image motile nonfluorescent cells against the background of fluorescent cells. We have observed small, rapidly moving cells that are likely to be host immune cells. H&E sections of the primary tumor confirm that host immune cells are present in the primary tumor (data not shown), supporting this interpretation. Increased numbers of immune system cells could contribute to increased metastasis by production of chemotactic factors or degradation of extracellular matrix barriers (e.g., see Refs. 50, 51, 52, 53, 54 ).
Breast cancer both in humans and in animal models including the MTLn3 cells (21 , 55) spreads to lymph nodes, indicating that the lymphatic circulation can also be important for tumor cell metastasis. In our in vivo studies, in cases in which tumor cells are oriented or exiting the tumor in the absence of a labeled blood vessel, it is possible that the cells are interacting with lymphatics. Comparing the in vivo behavior of tumor cells around lymphatics with their behavior around blood vessels awaits development of an appropriate in vivo marker for lymphatics.
In summary, we have demonstrated a method for providing a more detailed analysis of metastatic ability of tumor cells. The method is straightforward and should be valuable in determining the contributions to metastasis of specific proteins expressed in tumor cells. In addition, our in vivo analysis of tumor cell behavior has indicated that cell orientation toward blood vessels may be a characteristic of metastatic cells and that poorly metastatic cells can fragment during intravasation. These distinctions may prove important in choosing appropriate assays for predicting disease progression.
We gratefully acknowledge the staff of the Analytical Imaging Facility at Albert Einstein College of Medicine for assistance in intravital imaging and image analysis, Dr. Maryse Bailly for aid in the generation of rat tumors, and the laboratories of Drs. John S. Condeelis and Jeffrey E. Segall for scientific and critical input.
↵1 Supported by grants from the Department of Defense Army Breast Cancer Program [DAMD17-94-J-4314 (to J. E. S.) and DAMD17-96-1-6129 (to J. S. C.)] and a New York State Department of Health Empire Award (J. S. C.). J. E. S. is supported by an Established Scientist Award from the New York City affiliate of the American Heart Association.
↵2 To whom requests for reprints should be addressed, at Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: (718) 430-4237; Fax: (718) 430-8996; E-mail: segall{at}aecom.yu.edu
↵3 The abbreviations used are: GFP, green fluorescent protein; EGF, epidermal growth factor.
↵4 http://www.aecom.yu.edu/asb/segall/segall.htm.
Received August 27, 1999.
Accepted March 14, 2000.
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Lin, W. C., Pretlow, T. P., Pretlow, T. G., and Culp, L. A. Development of micrometastases: earliest events detected with bacterial lacZ gene-tagged tumor cells. J. Natl. Cancer Inst., 19: 82: 1497–1503, 1990.
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Kan Z., Liu T. J. Video microscopy of tumor metastasis: using the green fluorescent protein (GFP) gene as a cancer-cell-labeling system. Clin. Exp. Metastasis, 17: 49-55, 1999.
Hoffman R. M. Orthotopic transplant mouse models with green fluorescent protein- expressing cancer cells to visualize metastasis and angiogenesis. Cancer Metastasis Rev., 17: 271-277, 1998.
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Abstract LB-318: Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE study): Reproducibility assessment of an automated multiplexed immunofluorescence slide staining, imaging, and analysis workflow
Abstract LB-312: Interleukin 6 transsignaling is a candidate mechanism to drive progression of human DCCs during periods of clinical latency
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Democrats Need a Winning Message in Georgia. Bernie Sanders Says Fight for $15 Minimum Wage
The Vermont senator noted that "47% of workers in Georgia make less than $15 an hour and 71% of voters in Georgia support increasing the federal minimum wage."
November 11, 2020 Kenny Stancil Common Dreams
"If Democrats take back the Senate," Sen. Bernie Sanders (I-Vt.) said on Monday, November 9, 2020, "we will increase the minimum wage from a starvation wage of $7.25 an hour to a living wage of at least $15 an hour.", (Photo: Erik McGregor/LightRocket via Getty Images)
For Democrats to win both runoff races planned for January 5 in Georgia and secure a Senate majority, they're going to need a winning campaign message.
Sen. Bernie Sanders (I-Vt.) shared a suggestion on social media: "If Democrats take back the Senate," he said on Monday afternoon, "we will increase the minimum wage from a starvation wage of $7.25 an hour to a living wage of at least $15 an hour."
Sanders' tweet implied that vocally fighting for a higher minimum wage could be the key to victory for candidates Jon Ossoff and Raphael Warnock in Georgia, where 47% of workers make less than $15 an hour and 71% of voters support increasing the federal minimum wage.
As Common Dreams reported last week, voters in Florida—despite casting roughly 370,000 more ballots for outgoing President Donald Trump than President-elect Joe Biden—approved a ballot measure to establish a $15 minimum wage with support from nearly two-thirds of the state's electorate.
After Floridians passed a minimum wage increase by a margin of 61% to 39% while Biden lost the state by capturing only 47.8% of the vote compared to Trump's 51.2%, progressives criticized the Democratic Party for what some characterized as an inadequate embrace of progressive positions, ineffective communication, or both.
After all, critics noted, it is Biden, not Trump, who actually supports the $15 minimum wage policy that will give nearly 2.5 million low-income workers in Florida a much-needed raise.
With a major potential victory for progressives in sight, Sanders offered a campaign message for candidates Ossoff and Warnock that could prevent the Democrats from coming up short in Georgia and handing Senate control to the Republicans.
His messaging idea, which seeks to excite people about the possibility of a Democratic-led Senate delivering a minimum wage hike, reflects his desire to see Ossoff and Warnock champion a living wage policy that can generate enthusiasm amongst voters and spark a strong turnout for the special Senate elections in Georgia.
As TIME reported last week, Atlanta's above-average turnout among young voters (18-29 years old), and particularly young Black voters, 90% of whom voted for Biden, was instrumental in securing victory for the president-elect. If Democrats are to win a Senate majority, replicating a high turnout of voters who oppose the GOP's reactionary agenda will be crucial.
Data from Oxfam America and the Economic Policy Institute suggests that strongly advocating for a living wage, which researchers have pointed out would substantially reduce racial inequality, could propel a high turnout among working class populations that are electorally significant but often underrepresented at the polls.
In Georgia, 69.4% of Hispanic, 59.4% of Black, 41.9% of Asian American, and 39.9% of white workers make less than $15 an hour. Furthermore, of the workers in the state who make less than $15 an hour, 24% are under 25 years old while 36% are between the ages of 25 and 39.
In other words, Sanders is suggesting that many of the same Atlanta residents and other people around the state in young and nonwhite demographic categories who voted overwhelmingly for Biden are probably more likely to support Ossoff and Warnock in January 2021 if the candidates' campaigns make it clear that low-income workers in Georgia stand to benefit from a Democratic Senate majority.
As HuffPost reported last week, organized labor played a key role in successfully rejecting Trump at the ballot box in 2020, with union leaders encouraging members and other working class households, particularly Black and Hispanic voters in urban areas of swing states, to get to the polls to support a Biden administration, which organizers said "would protect their healthcare and do more to raise their wages."
Sanders is now highlighting how a Democratic-led Senate could pass a $15 minimum wage and advocating for the party to make the case to Georgians that they'll benefit from helping to defeat the Republican Party and Senate Majority Leader Mitch McConnell.
Kenny Stancil is a staff writer for Common Dreams. Follow him on Twitter: @kenny_stancil
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In blender combine chipotle, garlic, cumin, coriander, 3 ounces canola oil. Puree. Paint salmon fillets with puree, reserving 1 tablespoon, let sit 10 minutes. In very hot cast iron skillet, sear 2 minutes each side. Remove fish fillets and reserve. In a separate, clean, very hot skillet, place 1-ounce of canola oil. Add red onion, red bell pepper, and scallions. Season with salt and pepper. Cook 2 minutes. Add remaining tablespoon of blender puree and cilantro. Cook 2 minutes more. Remove from skillet. In same skillet, add 1 tablespoon extra olive oil, chard, garlic, hot pepper sauce and jalapeno. Cook for 3 minutes. While that is cooking, in a small pot heat beans with roasted red peppers, onions, chili and butter.
Assembly: Toast tortillas lightly in skillet and put on plate. Mash salmon fillets with fork and place in toasted tortilla, top with red onion/scallion/cilantro mixture, and sprinkle with lime juice. Roll up into burrito and serve with cooked chard and black beans on the side, as well as limes wedges and sour cream.


Bishop Renison was a prolific and talented writer in addition to his work as a church man. Fluent in written and spoken Cree, he wrote a Cree Hymnal. This folder contains a draft of part of the Cree Hymnal. 16 pages textual record.


Alice started Guitar lessons last Monday; using teacher's full size guitar in pic to left. This Monday she will have her own guitar, a small sized Fender Squire Mini, which is much easier for her to manage. The teacher, Tracy, also teaches voice lessons so we're getting her to do both for us, intermixing as she and Alice see fit.


DURHAM, N.C. — Despite deflated assets resulting from last year's plunge in Red Hat stock, the foundation created by executives of the Durham-based publisher of the Linux open-source operating system has expanded its focus.
The Center for the Public Domain, formerly the Red Hat Center, aims to champion efforts to balance the rights to intellectual property with public access to knowledge.
"Since it's very important to support the marketplace, it is equally important to understand the value of publicly accessible information and to allow both sides to flourish," says Laurie Racine, the foundation's president.
The $5 million-asset foundation was endowed with $30 million in Red Hat stock pledged when shares were near their peak. But the foundation did not get the shares until they had lost most of their value.
The foundation, which has since sold the stock, initially focused on promoting the free and open exchange of information, and making technology accessible and easy to use, manipulate and understand.
In addition to roughly half of the $4 million it pledged over four years to ibiblio, a digital library it is developing with the University of North Carolina at Chapel Hill, the foundation handed out $615,000 in grants last year and has awarded $556,000 this year.
The foundation, which also sponsors original research, conferences and collaborative programs, is giving $280,000 over two years to help create a fellowship program in intellectual property and the public domain at the School of Law at Duke University in Durham — and $150,000 for a related scholarly conference at the law school in November.
The foundation also will convene major funders this fall to explore intellectual property issues, and is creating a Web site to feature public-domain resources in the areas of law, culture, technology, science and advocacy.


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Peter "Pete" Tong (ur. 30 lipca 1960 w Hartley) – angielski DJ i producent muzyczny, znany głównie z prowadzenia audycji Essential Mix oraz Essential Selection, na antenie BBC Radio 1.

Życiorys
Kształcił się w Rochester. W wieku 15 lat zadebiutował jako DJ, grając na weselu. W 1997 został dziennikarzem czasopisma Blues & Soul. Na koncie ma występy w wielu klubach na całym świecie. Jest założycielem wytwórni FFRR Records.

Przypisy

Brytyjscy didżeje
Brytyjscy producenci muzyczni
Urodzeni w 1960


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Pixel Perfect Gaming News!
Mobile/Other
Indie Highlight
Atelier Ryza: Ever Darkness & the Secret Hideout Review
featured, Nintendo, Reviews
Jordan Wharton
Atelier Ryza: Ever Darkness & the Secret Hideout is the newest game in the Atelier series and follows brand new characters along their journey of exploration. Like previous Atelier games, one of the main protagonists takes up Alchemy. The main focus of this title is to explore the world, learn and improve your alchemy skills and to grow your party together as friends. It's a large world filled with a range of creatures to fight and materials to collect.
It was admittedly my first Atelier game, so I went in near completely blind to the series. In many ways, this fresh outlook allowed me to critique and appreciate the game from a different perspective. The story and gameplay were not completely new for me, as I've played many similar titles. However the approach certainly was refreshing. Regarding the story, there may be brief spoilers for the beginning of the game. If you do not wish to read them, then please take care when reading the below paragraphs.
Ryza is a girl who seeks adventure. This is made clear from the very first scene of the game. The story is very much so a focus on this need for adventure, with each area providing a new feeling of wonder. Ryza aims to explore outside of the main island she lives on. After taking a boat from a hidden port, she and her two friends Lent and Tao set out to explore the world. The first actual adventure comes along sooner than expected, as the group venture into a place called Pixie Forest. As the group venture further through the forest, they find a girl in need of saving. After a quick battle, the girl is saved.
The girl, who is carrying some sort of box, thanks the team for their help and is escorted out of the forest. The girl then reveals her name is in fact Klaudia Valentz. A fitting name for a character that seems sophisticated and almost princess like. Before being able to escape the forest, the team is ambushed by an unbeatable opponent and all fall in battle. Just as they're about to be struck down for good, two mysterious characters appear and save them from their demise.
After the team are saved by what looks like an explosion, Ryza gains an obsession of what power the mysterious strangers used. These mysterious characters reveal to Ryza that the power was in fact alchemy. The group then move towards the shore to depart the island. Klaudia's father, Lubart, is there to meet them. After his brief introduction, he introduces the mysterious characters. The male being called Empel, and the female being called Lila. All newly introduced characters agree to stay in the same village as Ryza, and so are all set for future interactions with each other.
The story follows a very expected path of Ryza learning alchemy. It's a very calm and slow paced story that focuses on providing a fun and an easy to understand experience. The above introduction is just a brief summary of the very first part of the game. After the initial story developments, the story can be slow paced for long periods of time. This is not necessarily a negative thing, as it helps add focus on the gameplay aspects of Atelier Ryza: Ever Darkness & the Secret Hideout. This story is truly about the main characters finding out about themselves and adventuring into not only the outside world, but into life.
Exploring in Atelier Ryza: Ever Darkness & the Secret Hideout is one of the main selling points of the game. With such a range of environments, enemies, ingredients, items and more to find, there is near infinite potential in finding everything in the game. Navigating the world is fairly easy too, thanks to the mini map that is always on screen. Other points of interest appear on the map too, which can be additional help when navigating places like forests.
Many areas will feature resting areas that allow the team to restore HP. These resting areas take the shape of a large flower with a bright light. An important note to remember is that these resting areas can usually only be used once within a certain time frame. After resting at a flower, they will require some time to recover before they'll become available again.
After various areas have been visited, it's possible to visit them via guideposts. These noticeboard type structures allow the player to move around without having to walk absolutely everywhere. An incredibly useful tool to use when hunting for materials. Early on in the game, it is also possible to travel straight back to Ryza's room using the – button on the Nintendo Switch Joycon. This proves useful for saving the game in a hurry, or for dropping off unneeded items.
As far as battling goes, it's introduced out of the blue. In some ways the introduction to battles felt forced, but it does get the basics out of the way very early into the game. In terms of breaking down each area of the UI and each action, the tutorial does an excellent job. It is clear and easy to understand, pointing out each option on screen and giving a brief description of everything that is or can be used.
At the start of the game there are three main options during battle. Attacking, moving or using a skill. Using a standard attack will allow your chosen character to gain an AP point and do average damage to the opponent. Moving will allow your chosen character to reposition themselves for a better strategic advantage.
Skill uses one of the characters acquired skills, which range from a powerful attack to healing. As mentioned earlier, characters gain AP points for using a standard attack. These AP points are shared between all the characters in the current party. AP points can also be spent in battle too, so it's important to balance spending them and saving them for various other uses.
One such use and another important growing level in battle is the Tactics Level. This Tactics Level raises by earning the max AP and then consuming it all at once. Raising this Tactics Level provides a range of benefits during the battle. An example of the advantages of raising the Tactics Level is, after levelling it up from 1 to 2, characters gain the ability to attack twice with normal attacks. This upgrades once again at level 3, allowing the player to strike the enemy 3 times in a row. Such a small upgrade might prove important when fighting multiple enemies or stronger foes, so the Tactics Level is a mechanic to keep an eye out for.
The balance between AP usage and Tactics Level are is extremely important to winning battles and requires at least some level of strategy and skill. The higher the Tactics Level, the higher the max AP, meaning the Tactics Level won't always be raising every battle. Taking these two gameplay elements into account along with the other elements such as skills makes the battling feel like a somewhat separate game. That's not an entirely negative thing, but it does take away from the calm and relaxing atmosphere all the other experiences provide.
The battling system does provide two different modes to somewhat lessen the amount of thought or concentration required. Aggressive Mode is a mode in which other party members consume AP to use their skills. This could be an advantage against weaker enemies when wanting to get the battle over as soon as possible. The other mode is called Passive mode, in which the party members will simply use normal attacks. This is great for reserving AP for various reasons, but may take a little longer to take down the enemy.
Alchemy has two major stages in this game. The first and most time consuming part is the gathering of the materials/ingredients. Whilst it can be fun to explore various areas to search for required materials, it's also extremely tedious due to the lack of information given. One of the first goals is to create a bomb. In the guide, the bomb item is listed along with the materials needed to create the item. This is extremely helpful for knowing the materials needed, but not helpful for finding these materials. Crimson Ore, Gunpowder, Fuel and Supplement are the materials needed, but when searching for more information on where to find them, there is none.
This leads to a large problem later on in the game when trying to create or upgrade various items, as it leads to extremely repetitive gameplay that forces you to search and run around for long periods of time for little reason. The game aims to provide a calm and relaxing experience, so the large amount of running around can be either seen as encouraging this peaceful focus, or making the game in some ways more stressful than it needs to be.
The second stage of the Alchemy is mixing the ingredients together in a cauldron. This is how items are synthesised. The easiest way of approaching this extremely complicated task is by simply choosing an item you'd like to create and choosing the materials you've gathered. Various other elements can come in to play, such as skills, stat related materials and the four material types. However, the game doesn't pace out explaining all of these and throws them all at once to the player.
I personally found the explanations to be far too drawn out and confusing, so much so that it put me off the Alchemy portion of the game completely. I simply pressed A to quickly create items for the most part, to save myself the trouble of having to calculate each individual step. I feel that if the game slowly introduced the alchemy portion of this game, it might have been more approachable and easy to understand. Unfortunately this was not the case and combined with the tedious gathering aspect completely takes away from the calm and relaxed gameplay atmosphere that is attempted with this game.
The Nintendo Switch is known for having the lowest graphical ability of the current generation consoles, but that didn't stop Atelier Ryza: Ever Darkness & the Secret Hideout from looking incredible. Whilst some areas and textures in the environment looked poor, the majority of the surroundings fitted the aesthetic. The character models were all beautifully created with love and care and truly encapsulated each characters personality.
Two major problems I faced were during the cutscenes of the game. Whilst I personally have no problem with the letterbox effect in cutscenes, it did bring me out of the immersion at times when a cutscene was sudden. This was not a major problem however. During near every cutscene in the game, the mouth of the character speaking did not match the speed of which they spoke. I'd expect this if the game had multiple audio options for dialogue, but it did not.
This out of sync talking made it very hard to take the game seriously at times and made it difficult to enjoy the story. Another problem during cutscenes was the running animations. Ryza seemed to run too fast compared to the speed she actually moved along the scene, so she appeared to run on the spot many times throughout the game. This once again made the cutscenes a somewhat silly experience, which was a shame, as they had a lot to offer in terms of story and quality.
User Interface and Navigation
The user interface and menu navigation is one of the most important parts of any game, as it defines how easy the player will be able to find what they're looking for and interact with the world. Atelier Ryza: Ever Darkness & the Secret Hideout is a game that does an excellent job at making a lot of information easy to navigate. With the wealth of information given in this game, each menu had to be unique and stand out. That's exactly what players were given, which increased ease of gameplay dramatically.
The equipment screen for example showed stats, equipped items, roles, level and more. The amount of information on this one screen was utterly insane and yet it was all displayed in an easy to understand way. Not only that, but navigating into different displays was just as easy. It was possible to jump from one screen to another without having to go back every time. This saved so much time and really made me appreciate the simplicity of the overall menu user interface.
This ease of use continued into things like the monster information screens. Each monster has their very own screen that details their name, status, a picture, a description, element adaptions, drop items and habitat. All of these pieces of information are absolutely vital for a game about acquiring items and staying alive, so these kinds of screens are something that are much needed.
My main focuses are on the habitat and picture. Finding what you need in this game can at times be incredibly difficult, so these simple additions can make a huge difference to the enjoyment of the game. There is also an option to see related journal entries. This option to see related entries is not restricted to only monster pages. It is a useful tool throughout the entirety of the game and makes near everything easier to find and do. I adore the user interface and menu design of Atelier Ryza: Ever Darkness & the Secret Hideout. It was honestly one of the best I've ever seen in any game.
Music and Audio
Since the very beginning of the game, the fun and fitting pieces of music accompanied the tone perfectly. The use of upbeat music in the very comedic areas of dialogue between characters made the whole experience feel more refreshing and calm. The majority of areas in game also had their own piece of music, which helped with navigation. The use of music being helpful to the player as well as setting the mood is vital in a game about exploring.
Audio is fitting in most areas, adding to the feeling that the world is alive. Various ambient noises accompany the music in a blend that feels fitting. The dialogue carries on this feeling with extremely well done voice acting work. Emotions of the characters gets across the feelings of the characters, despite the game only featuring Japanese voice acting.
Whilst Atelier Ryza: Ever Darkness & the Secret Hideout may have some drawn out gameplay segments and can be tedious at times, the characters and music keep players involved. With such a large amount of space to explore, many things to create and many enemies to face, the game provides a large range of content whilst still being, for the most part, easy to understand. As my first Atelier game, it was difficult to get into the alchemy initially, but i'd fully recommend it to newcomers who are looking for a relaxing and enjoyable game. It's certainly worth getting past the first hurdle. The game has an incredible amount of DLC planned in the future, from character costumes to new areas. For more information you can check out the Official Site.
The screenshots used in this review are from a Nintendo Switch console. For more reviews and gaming news you can keep up to date at Informed Pixel. Want to speak to the team and interact with other gamers? You can do so over on the Informed Pixel Facebook page or on the Informed Pixel Twitter account. You can also find my work on my Facebook page or on my Twitter.
Summary: Whilst Atelier Ryza: Ever Darkness & the Secret Hideout may have some drawn out gameplay segments and can be tedious at times, the characters and music keep players involved. With such a large amount of space to explore, many things to create and many enemies to face, the game provides a large range of content whilst still being, for the most part, easy to understand. As my first Atelier game, i'd fully recommend it to newcomers who are looking for a relaxing and enjoyable game.
Atelier Ryza: Ever Darkness & the Secret Hideout - 83%
User Rating: 0% (0 votes). You rated: __
Atelier Ryza: Ever Darkness & the Secret Hideout Review Jordan Wharton November 12, 2019 Whilst Atelier Ryza: Ever Darkness & the Secret Hideout may have some drawn out gameplay segments and can be tedious at times, the characters and music keep players involved. With such a large amount of space to explore, many things to create and many enemies to face, the game provides a large range of content whilst still being, for the most part, easy to understand. As my first Atelier game, i'd fully recommend it to newcomers who are looking for a relaxing and enjoyable game.
Atelier Ryza: Ever Darkness & the Secret Hideout is the newest game in the Atelier series and follows brand new characters along their journey of ...
Atelier Series
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