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Avoidant personality disorder (APD) is a relatively common personality disorder that causes people to be extremely shy and worried about being rejected or embarrassed. This disorder often forces people into isolation, which can prevent them from living productive and enjoyable lives.[1] X Research source It may be possible to recognize many of the symptoms of avoidant personality disorder, but in order to be diagnosed, an individual must be seen by a qualified mental health professional.
Part 1 of 3:
Recognizing Symptoms of APD
Part 1 of 3:
1Look for extreme shyness. One of the most recognizable symptoms of avoidant personality disorder is an intense feeling of discomfort in social situations, beyond what might be considered "normal" shyness. People with this disorder may appear scared or extremely tense whenever they are in a situation that requires them to interact with other people.[2] X Research source
2Pay attention to social relationships. People with avoidant personality disorder often do not have any close friends or romantic relationships. This may be due to the fact that they often consider themselves socially inept.[3] X Research source
- When they do get involved in romantic relationships, people with avoidant personality disorder are likely to still display extreme restraint due to an intense fear of rejection.
- Although they have a difficult time forging close relationships with others, most people with avoidant personality disorder want close relationships and may have detailed fantasies about what it would be like to have them.[4] X Research source
3Notice what kinds of activities are avoided. People who have avoidant personality disorder tend to avoid situations that would involve interactions with other people. This may involve school, work, or recreational activities.
- Many people with avoidant personality disorder also avoid any activities that are new or unfamiliar, for fear of embarrassing themselves.[5] X Research source
4Watch the response to criticism. People with avoidant personality disorder tend to be extremely sensitive to criticism, or even to perceived criticism. They may constantly feel that others are judging them, even when they are reassured to the contrary.[6] X Research source
- Some people with avoidant personality disorder will avoid activities that they believe they will do poorly at in order to avoid being criticized for their poor performance.
- They may expect to be criticized in situations that other people would not take seriously, such as a game.
5Note an exaggerated sense of negativity. People with avoidant personality disorder tend to overestimate the negative aspects of situations. You may notice that they fixate on potential problems and make them seem much more serious than they are.[7] X Research sourceAdvertisement
Part 2 of 3:
Distinguishing APD from Similar Conditions
Part 2 of 3:
1Rule out schizoid personality disorder. Both avoidant personality disorder and schizoid personality disorder can cause people to avoid social interactions with others, but there is a very important difference between the two. People with avoidant personality disorder are typically very troubled by their isolation and want to get involved with others, while people with schizoid personality disorder usually are not distressed by their lack of interaction.
2Consider the possibility of social anxiety disorder. Social anxiety disorder and avoidant personality disorder are extremely similar, so it may be impossible for someone who is not trained in psychology to distinguish one from the other. Typically, individuals with avoidant personality disorder display more symptoms than those with social anxiety disorder, and their symptoms tend to be more inhibiting.[8] X Research source
- Individuals who display only a few of the symptoms of avoidant personality disorder may in fact have social anxiety disorder, but it is important that a trained mental health professional make this diagnosis.
- Some people may be diagnosed with both avoidant personality disorder and social anxiety disorder, which further complicates the distinction between the two conditions.
3Learn about other disorders that can cause a lack of confidence. Avoidant personality disorder is not the only psychiatric condition that can cause individuals to lack self-confidence and feel inadequate. Before assuming that an individual has avoidant personality disorder, consider other, similar personality disorders.[9] X Research source
- Like people with avoidant personality disorder, people with histrionic personality disorder tend to lack self-esteem. The major difference is that people with histrionic personality disorder tend to go out of their way to seek the approval and validation of others, often in an unhealthy or destructive way, while people with avoidant personality disorder instead avoid contact with others.
- Dependent personality disorder is also characterized by a lack of self-worth and fear of abandonment. However, people with dependent personality tend to latch on to a specific individual instead of avoiding all social interactions. People with dependent personality disorder also tend to have a difficult time making decisions on their own, which is not characteristic of avoidant personality disorder.
Part 3 of 3:
Getting a Professional Diagnosis
Part 3 of 3:
1Get a complete physical exam. If you think that you or someone you know may have avoidant personality disorder, the first step in getting a diagnosis is to see a doctor for an exam. The doctor will want to rule out any physical conditions that may be causing the symptoms.[10] X Research source
- This appointment will consist of a physical exam, as well as a detailed examination of the patient's personal and family history.
2See a mental health professional. If no physical conditions are identified, the doctor will most likely refer the patient to a mental health professional, such as a psychiatrist or psychologist. Mental health professionals are specially trained to diagnose personality disorders, including avoidant personality disorder.
- This appointment will consist of a detailed interview. The psychiatrist or psychologist will want to know all about the symptoms the patient is experiencing, when they started, and how they have progressed over time.
- There is not medical test that can be performed to diagnose avoidant personality disorder. A diagnosis is made based on the observations of the patient's behavior and reported symptoms.
- Once a patient is diagnosed, he or she will be encouraged to enter psychotherapy to help overcome the symptoms of avoidant personality disorder.
3Get co-existing conditions diagnosed. Some people with avoidant personality disorder also suffer from other mental health issues, such as anxiety and depression. A thorough psychiatric evaluation should reveal whether any other conditions are complicating the symptoms of avoidant personality disorder.[11] X Research source
- For patients who also suffer from anxiety or depression, medications may be helpful in reducing symptoms. Medications are typically not helpful for patients whose only diagnosis is avoidant personality disorder, however.
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- Most people with avoidant personality disorder begin showing signs of the condition in adolescence, but many are not diagnosed until adulthood because it is difficult to tell if the symptoms are just a passing phase. As patients get older, their symptoms tend to decrease in severity. By the time they are in the 40s or 50s, people with avoidant personality disorder may not be experiencing the worst symptoms of the disorder anymore.[12] X Research sourceThanks!
- ↑ https://www.psychologytoday.com/conditions/avoidant-personality-disorder
- ↑ http://psychcentral.com/disorders/avoidant-personality-disorder-symptoms/
- ↑ https://www.psychologytoday.com/conditions/avoidant-personality-disorder
- ↑ http://psychcentral.com/disorders/avoidant-personality-disorder-symptoms/
- ↑ http://my.clevelandclinic.org/services/neurological_institute/center-for-behavioral-health/disease-conditions/hic-avoidant-personality-disorder
- ↑ https://www.psychologytoday.com/conditions/avoidant-personality-disorder
- ↑ http://my.clevelandclinic.org/services/neurological_institute/center-for-behavioral-health/disease-conditions/hic-avoidant-personality-disorder
- ↑ https://www.verywell.com/avoidant-personality-social-anxiety-disorder-difference-3024444
- ↑ https://www.psychologytoday.com/blog/hide-and-seek/201205/the-10-personality-disorders
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In my previous posts I provide an introduction to working with the Ribbon in CRM 2011 where I demonstrate how to add and remove buttons. In this post I will explain how to edit existing buttons.
Let's start by framing an example. On the Phone Call form we have Ribbon buttons for both "Convert to Case" and "Convert to Opportunity":
You'll note the poor Convert to Case button got the short straw and is presented as a small button, whilst the Convert to Opportunity button appears in full and with a text label. But what if we are implementing CRM for Customer Service? In that scenario we are unlikely to care about Opportunities and would likely prefer these buttons switched around. Here's how we do that…
As described in my previous posts customising the ribbon involves a bit of initial research and then some manual editing of an exported customization XML file. For the research component what we need to do is have a look at the ribbon definition for the Phone Call form. We find that in the SDK here:
[UPDATE: 14 Dec 2011 – The latest SDK did not include the ribbon definition XML files forcing you to build and run an app from source code provided in order to generate them for your self. I provide instructions on this process here]
Open that file in Visual Studio and locate the XML that represents these buttons we want to change. In this case we want to find the form ribbon's Main tab:
Always make sure you are looking at the right Tab as similarly named button may appear on the SubGrid tab and HomepageGrid tabs as well.
The lesson for first timers I want to reiterate here is DO NOT change this XML file. It will have no effect. This file is essentially documentation of the out-of-the-box CRM ribbons it is no way used by CRM to render the ribbons. To change the ribbon we need to make entries into an exported customization.xml file that contains the definition of the Entity involved.
So jump back to CRM, create a Solution called PhoneCall and add the Phone Call entity into that Solution. Export that Solution, unzip the file and then open customization.xml file in Visual Studio. Collapse the EntityInfo and FormXml nodes and you should see the RibbonDiffXml node, this is where we specify changes to the Ribbon:
Replace the CustomActions line with the following XML:
<CustomActions> <CustomAction Id="GT.switch.button.sequence1" Location="Mscrm.Form.phonecall.MainTab.Convert.Controls._children" Sequence="1"> <CommandUIDefinition> <Button Id="Mscrm.Form.phonecall.Convert.Opportunity" Command="Mscrm.Form.ConvertToOpportunity" Sequence="21" Alt="$Resources:MenuItem_ToolTip_ConvertToOpportunity" LabelText="$Resources:MenuItem_Label_ConvertToOpportunity" Image16by16="/_imgs/ribbon/ConvertOpportunity_16.png" Image32by32="/_imgs/ribbon/ConvertOpportunity_32.png" ToolTipTitle="$Resources:Mscrm_Form_Other_MainTab_Actions_Convert_Opportunity_ToolTipTitle" ToolTipDescription="$Resources:Mscrm_Form_Other_MainTab_Actions_Convert_Opportunity_ToolTipDescription" TemplateAlias="o2" /> </CommandUIDefinition> </CustomAction> <CustomAction Id="GT.switch.button.sequence2" Location="Mscrm.Form.phonecall.MainTab.Convert.Controls._children" Sequence="2"> <CommandUIDefinition> <Button Id="Mscrm.Form.phonecall.Convert.Case" Command="Mscrm.Form.ConvertToCase" Sequence="9" Alt="$Resources:MenuItem_ToolTip_ConvertToCase" LabelText="$Resources:MenuItem_Label_ConvertToCase" Image16by16="/_imgs/ribbon/ConvertCase_16.png" Image32by32="/_imgs/ribbon/ConvertCase_32.png" ToolTipTitle="$Resources:Mscrm_Form_Other_MainTab_Actions_Convert_Case_ToolTipTitle" ToolTipDescription="$Resources:Mscrm_Form_Other_MainTab_Actions_Convert_Case_ToolTipDescription" TemplateAlias="o1" /> </CommandUIDefinition> </CustomAction> </CustomActions>
Here's a breakdown of the XML…
This first bit:
Reads as: "I want to perform a Custom Action, lets call my action GT.switch.button.sequence1. I want to change one of the child nodes in the ribbon definition under the node that has the Id: "Mscrm.Form.phonecall.MainTab.Convert.Controls".
Then we have:
Here we are defining a button. Now what I did here was I copy and pasted the definition of the Convert to Opportunity button from phonecallribbon.xml into this customization.xml file. I then changed the properties that I wanted changed. Specifically I changed the Sequence so that this button would be rendered after the Convert to Case button (which has Sequence 20) and I changed the TemplateAlias to o2 so that this button will be rendered as a little button. I think this is important as this button group has been defined as 1 big + 2 small button group and I don't want to defy that. Those are the only 2 items I have changed. Because the Id I have specified for this button matches an existing button CRM knows to merge my definition with its existing definition and overwrite it's definition with mine, rather than creating a new button.
In my XML snippet in addition to the above I have a second Custom Action containing a second button definition where I specify the overrides I want to the Convert to Case button. Each button definition needs to sit under it's own Custom Action header, hence this structure.
That's it. To deploy we simply zip back up the customization.xml file along with it's other solution files and then import that solution zip file back into CRM and Publish.
Here's the end result:
You can follow this same approach to change buttons icons, button labels, etc.
Note: to retain both buttons as large buttons simply set TemplateAlias="o1" on each:

subset: commoncrawl


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Putting a house on the market in Polk County? Contact The Hohman Appraisal Company and make sure you're setting the price right.If you're going to sell your home, you should seriously consider a professional appraisal. Don't think of an appraisal as purely an expense; it pays in the long run! Unless a homeowner studies real estate values on a day to day basis, like a professional appraiser does, it's practically impossible for them to understand the nuances of local real estate values.
It's tempting to want to base your home's value on how much you have invested in your home, or how much you want to sell it for. Rather, an appraisal assesses the true market value of your home. Something is only going to sell for the price another person is going to give you.
Selling on your own?An increasing amount of Florida homeowners are choosing to sell their homes FSBO (For Sale By Owner) and foregoing the help of a real estate agent. But to do it right, quickly and to get the most you can, you need a qualified appraiser. You need an objective opinion, we're human too, we understand its difficult hard to be objective about your own home because of your emotional attachment to it. We will inform you of what you need to know, not just what you want to hear.
Not only can getting a professional appraisal help you set a sound asking price so your home will attract buyers, a professional appraisal can:
Get a professional value opinion before listingYou might have a highly skilled agent, but it's in your best interest to get an analysis of your home's value before signing a listing contract. And that's where The Hohman Appraisal Company comes in. An appraisal can be indispensable in this scenario. You and your agent will get a comprehensive breakdown of the unique features of your property and give you insight into how the local real estate market is performing with a detailed analysis of the latest comparable properties in the area.
We also are available for general consultingNot wanting to get an entire appraisal but would like some information about how the real estate market is doing in your area? We can help solve your real estate dilemmas with services especially tailored for customers with your real estate needs.
This is a little bit of what to expect when you place an order for an appraisal through us...


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Listeriosis is a foodborne disease with a high fatality rate, and infection is mostly transmitted through ready-toeat(RTE) foods contaminated with Listeria monocytogenes, such as gravad/smoked fish, soft cheeses, andsliced processed delicatessen (deli) meat. Food products/dishes stored in vacuum or in modified atmospheresand with extended refrigerator shelf lives provide an opportunity for L. monocytogenes to multiply to largenumbers toward the end of the shelf life. Elderly, pregnant women, neonates, and immunocompromisedindividuals are particularly susceptible to L. monocytogenes. Listeriosis in humans manifests primarily assepticemia, meningitis, encephalitis, gastrointestinal infection, and abortion. In the mid 1990s and early 2000s ashift from L. monocytogenes serovar 4b to serovar 1/2a causing human listeriosis occurred, and serovar 1/2a isbecoming more frequently linked to outbreaks of listeriosis, particularly in Europe and Northern America.Consumer lifestyle has changed, and less time is available for food preparation. Modern lifestyle has markedlychanged eating habits worldwide, with a consequent increased demand for RTE foods; therefore, more RTE andtake away foods are consumed. There is a concern that many Listeria outbreaks are reported from hospitals.Therefore, it is vitally important that foods (especially cooked and chilled) delivered to hospitals and residentialhomes for senior citizens and elderly people are reheated to at least 72C: cold food, such as turkey deli meatand cold-smoked and gravad salmon should be free from L. monocytogenes. Several countries have zerotolerance for RTE foods that support the growth of Listeria.
Among 504 clinical lineage II isolates of Listeria monocytogenes isolated during 1958-2010 in Sweden, 119 pulsed-field gel electrophoresis (PFGE) types (AscI) have been identified based on the number and distribution of all banding patterns in each DNA profile. In this study, these types were further divided into PFGE groups based on the configuration of small bands with sizes <145.5 kb. The 504 isolates included 483 serovar 1/2a isolates distributed into 114 PFGE types and 21 serovar 1/2c isolates distributed into 9 PFGE types; these were further divided into 21 PFGE groups. PFGE group, that is, configuration of small bands below 145.5 kb, and serovars were correlated. L. monocytogenes isolates belonging to PFGE groups A, B, C, E, F, H, K, L, M, S, V, W, Y, and Ö-6 to Ö-12 shared serovar 1/2a, with one exception. PFGE group E also included two PFGE types sharing serovar 1/2c and four PFGE types belonging to either serovar 1/2a or 1/2c. Isolates belonging to PFGE group N shared serovar 1/2c. In contrast to lineage I isolates, small fragments <33.3 kb were visible in all L. monocytogenes isolates belonging to lineage II. In the results from both the present and previous studies, the genomic region of small bands was genetically more conservative than in large bands. The distribution of these small bands established the relatedness of strains and defined a genetic marker for both lineages I and II, while also establishing their serogroup. The division of L. monocytogenes PFGE types into PFGE groups is advantageous as the profile of every new isolate can be identified easily and quickly through first studying the PFGE group affiliation of the isolate based on the smaller band patterns <145.5 kb, and then identifying the PFGE type based on the band patterns >145.5 kb.
Funding Agency:
Stiftelsen Grythytte Akademi Stipendiefond
Microbiological and sensory methods were used to analyze 22 soft cheeses, of which 19 were made from raw milk, one was made from both raw and pasteurized milk and two were made from pasteurized milk. Moderate correlations (r-value 0.5–0.6 and p-value <0.01–0.05) were found between the levels of Enterobacteriaceae 37°C and the intensity of the sensory characteristics "bitter", "metallic", "pungent", "manure" and "ammonia". The present study indicates that it is possible to predict high levels of Enterobacteriaceae in soft cheeses made from raw milk using only the human senses (odor and taste).
The 63 pulsed-field gel electrophoresis (PFGE) types identified among 427 clinical isolates of Listeria monocytogenes that were characterized in a previous study by serotyping and PFGE (AscI) could be further divided into 17 PFGE groups. While the 63 PFGE types, all part of lineage I, were established based on the number and distribution of all bands in each DNA profile, the 17 PFGE groups were based on the configuration of small bands with sizes L. monocytogenes serovar 4b isolates (n=334) were divided into 8 PFGE groups; the 32 PFGE types of serovar 1/2b isolates (n=90) and the serovar 3b isolates (n=3, 1 PFGE type) were divided into 9 PFGE groups. An association was observed between PFGE groups and serovars. L. monocytogenes isolates belonging to PFGE groups I, J, Q, R, X, Z, o-4, and o-5 all shared serovar 4b, whereas isolates from PFGE groups D, G, O, P, T, U, o-1, o-2, and o-3 shared serovar 1/2b. Small fragments L. monocytogenes isolates. From the results of the present study, a procedure for accelerating the identification of PFGE types when analyzing new PFGE profiles can be suggested. Therefore, we propose a stepwise procedure to PFGE profiling by first identifying the PFGE group using the smaller band patterns <145.5 kb, and then determining PFGE types based on the band patterns >145.5 kb.
Släktena Listeria och Erysipelothrix hör till gruppen grampositiva, icke-sporbildande regelbundet formade stavar. Gruppen är mycket heterogen och inkluderar både patogena och apatogena bakterier.
To stem the tide of food-borne illnesses, it is important to understand the source of the problem. This volume examines pathogenic bacteria, viruses, protozoan parasites, moulds, and mycotoxins in food. It discusses food-associated antimicrobial resistance and lessons learned from an actual food-borne outbreak. The book also explores clinical aspects of food-borne illness. The book is intended for medical science students. It will also be useful as a reference book for scientists, technologists, and inspectors in public health-related fields who regularly deal with issues related to food microbiology, food-borne infections and intoxications, and food safety.
Isolates of Listeria monocytogenes (n=932) isolated in Sweden during 1958–2010 from human patients with invasive listeriosis were characterized by serotyping and pulsed-field gel electrophoresis (PFGE) (AscI). Of the 932 isolates, 183 different PFGE types were identified, of which 83 were each represented by only one isolate. In all, 483 serovar 1/2a isolates were distributed over 114 PFGE types; 90 serovar 1/2b isolates gave 32 PFGE types; 21 serovar 1/2c isolates gave nine PFGE types; three serovar 3b isolates gave one PFGE type; and, 335 serovar 4b isolates gave 31 PFGE types. During the 1980s in Sweden, several serovar 4b cases were associated with the consumption of European raw soft cheese. However, as cheese-production hygiene has improved, the number of 4b cases has decreased. Since 1996, serovar 1/2a has been the dominant L. monocytogenes serovar in human listeriosis in Sweden. Therefore, based on current serovars and PFGE types, an association between human cases of listeriosis and the consumption of vacuum-packed gravad and cold-smoked salmon is suggested.
Research Foundation of Ivar and Elsa Sandberg


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Important: There has been an erratum issued for this article. Read more …
Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.
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Rutkowski, M. R., Allegrezza, M. J., Svoronos, N., Tesone, A. J., Stephen, T. L., Perales-Puchalt, A., Nguyen, J., Zhang, P. J., Fiering, S. N., Tchou, J., Conejo-Garcia, J. R. Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer. J. Vis. Exp. (85), e51171, doi:10.3791/51171 (2014).
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Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of αβ and γδ T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.
Breast cancer is the most commonly occurring malignancy in women throughout the world1,2 and the second leading cause of cancer-related deaths2. Complex genetic3,4, histological5, and clinical phenotypes6 are used to characterize the various subtypes of breast cancer and often are used as a means to predict survival. Analysis of a large cohort of women with breast cancer indicated that most (approximately 80%) of the patients that died had recurred within 10 yr post removal of the primary tumor7. For a majority of invasive breast carcinomas, lymphovascular invasion has been shown to be strongly correlated to a poor outcome and more aggressive clinical course of disease8.
Because of the genetic and phenotypic complexity of breast cancer, there is no animal model that recapitulates the entire course of disease. Human breast tumor cell lines have been frequently used as xenograft or orthotopic9 models of invasive and metastatic breast cancer in immune deficient mice. Although informative, these models occur in the absence of immune pressure and because it is a cross species graft, distort the effects of the entire tumor microenvironment. Inducible genetic mutations driven by mammary specific promoters such as murine mammary tumor virus (MMTV) and whey acidic protein (WAP) have contributed a tremendous amount of knowledge about the genetic nature of breast cancer. However, the tissue specific expression of these promoters is compromised by their responsiveness to the endocrine system10-16, resulting in the variable expression of induced genetic mutations that do not mirror the expression of oncogenes typically overexpressed in human breast cancer. To overcome endocrine control of MMTV driven expression of oncogenes, Moody et al. generated a conditional, doxycycline inducible model overexpressing Neu in the breast epithelium17. This model is useful for deinducing Neu after tumor formation to study regression and recurrence, but requires constant doxycycline administration for consistent, long-term oncogene expression. A comprehensive discussion of the many relevant breast tumor models available can be found in the review by Vargo-Gogola et al.10
Our goal was to develop a mouse model of traceable breast cancer on a full C57BL/6 background that, after the permanent induction of mutational events, models the formation of a nascent tumor in the presence of immune pressure. We introduced an adenovirus expressing Cre-recombinase into the mammary ducts of transgenic mice containing floxed alleles of Tp53, and an oncogenic form of K-ras, and YFP. Cre expression ablates Tp53, a frequently mutated gene in many breast cancers18 and induces an oncogenic allele of K-ras in addition to YFP expression specifically in the mammary ductal epithelium. Although mutations in K-ras are infrequent in breast cancer, occurring in only 6.5% of breast cancer patients19,20, the overexpression of upstream kinases such as Her2/neu and EGFR result in constitutive activation of the Ras signaling pathway in human breast tumors21-23. Activation of the Ras signaling pathway in many breast tumor cell lines has also been reported24,25. We will describe the initiation of tumor formation and the technique of intraductal injection of an adenovirus expressing Cre-recombinase into sexually mature, virgin female mice. This model of breast cancer develops overt lesions that grow exponentially after about 8 weeks of slow tumor progression, with lymphovascular invasion and metastasis to the axillary lymph node by 7-8 weeks. Because these mice are on a full C57BL/6 background and YFP-expressing tumor cells are traceable in distal lymph nodes, this model provides a relevant tool to study the cellular and immunological mechanisms of latent metastasis and will help to develop novel therapeutic approaches for the treatment of metastatic ductal breast cancer.
All animal experiments were approved by the Wistar Institute Animal Care and Use Committee.
1. Generation and Maintenance of Transgenic Mice
- Breed LSL-K-rastm4Tyj 26 and Trp53tm1Brn 27 (obtained from NCI mouse models of human cancer consortium on a mixed background) to a full C57BL/6 background 28 by backcrossing at least 10 generations with C57BL/6 mice. To track tumor metastasis, breed B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J (LSL-EYFP, obtained from The Jackson Laboratory on a full C57BL/6 background) with double transgenic LSL-K-rasG12D/+ p53loxP/loxP mice.
Note: Transgenic LSL-K-rasG12D/+ p53loxP/loxP mice have loxP sites flanking a transcriptionally silenced allele of oncogenic K-ras and the endogenous p53 locus, so that upon Cre-mediated excision, overexpression of an oncogenic K-ras mutant and ablation of p53 is achieved.
Note: The LSL-EYFP mouse contains a stop codon flanking a gene for enhanced yellow fluorescent protein (YFP) that upon Cre-mediated excision results in the expression of YFP in the tissues where the YFP stop cassette is excised.
- Breed transgenic mice to obtain LSL-K-rasG12D/+ p53loxP/loxP mice or LSL-K-rasG12D/+ p53loxP/loxP LSL-EYFP mice for intraductal injections.
Note: Mice are bred as homozygous for p53loxP/loxP and heterozygous for LSL-K-rasG12D/+ because mice with a homozygous deletion of K-ras die in utero. Use naïve virgin females at least six-weeks old for intraductal injections.
Note: The primers for genotyping homozygous floxed p53 allele are p53-T010-fwd (5'-AAGGGGTATGAGGGACAAGG-3') and p53-T011-rev (5'-GAAGACAGAAAAGGGGAGGG-3'). They produce a wild type allele at 391 bp and the p53 floxed allele at 461 bp29,30.
Note: The primers to detect the mutant form of K-ras are oIMR8273 (5'-CGCAGACTGTAGAGCAGCG-3') and oIMR8274 (5'-CCATGGCTTGAGTAAGTCTGC-3'). They produce the mutant band detected at 600bp.
Note: For the YFP reporter triple transgenic mice, the primers to detect the ROSA cassette (5'-AAGACCGCGAAGAGTTTGTC-3'), the wild type allele (5'-GGAGCGGGAGAAATGGATATG-3'), and a shared allele (5'-AAAGTCGCTCTGAGTTGTTAT-3') result in the amplification of bands at 320 bp for floxed allele and 600 bp for the wild type allele.
- Breed transgenic mice to obtain LSL-K-rasG12D/+ p53loxP/loxP mice or LSL-K-rasG12D/+ p53loxP/loxP LSL-EYFP mice for intraductal injections.
2. Surgical Preparation
- Clean surgical materials with 75% EtOH and autoclave them before and after all injections.
- Perform surgery on a clean uncluttered laboratory bench in a sanitized room within an animal facility. Wipe down all surfaces including the stage and dials of the surgical microscope with a broad-spectrum disinfectant solution followed by 75% EtOH.
- Weigh and anesthetize mice by intraperitoneal injection of a mix of ketamine (80-100 mg/kg) and xylazine (8-10 mg/kg) in sterile saline.
- Gently place mice back into their cages undisturbed for 5 min while they go under anesthesia. During this time generate virus precipitates (see Protocol 3).
- Verify lack of response to pain by toe pinching. Gently cover the eyes of anesthetized mice with veterinary ointment to prevent excessive corneal drying.
- To prevent hypothermia, place anesthetized mice onto a heating pad set to low heat during the surgical procedure and until they begin to recover.
- For the management of pain, administer mice meloxicam subcutaneously at 1 mg/kg before the surgery and 24 hr after.
3. Generation of Virus Precipitates
CAUTION: Adenovirus vectors, although they have been modified and are unable to replicate, pose the risk of infection. Handle adenovirus with caution. All personnel should be appropriately trained according to the institution's guidelines for handling BSL2 agents After intraductal injection, dispose of adenovirus in accordance with BSL2 guidelines.
- Store adenovirus concentrated virus stocks at -80 °C frozen in aliquots of 4 x 108 pfu each, sufficient for injecting 16 animals with 3 μl of 2.5 x 107 pfu of adenovirus particles.
- Store adenovirus aliquots on dry ice until approximately 15-20 min before beginning the injections.
Note: Avoid repeated freeze thaw cycles, as virus titer drops significantly between each cycle.
Note: Adenovirus precipitates are formed by modifying a protocol described previously 31.
- Reconstitute 504 mg of MEM powder with 50 ml of sterile molecular grade water, supplement with 244 mg of sodium bicarbonate and filter in sterile conditions and store at 4 °C.
- Prepare the calcium chloride solution by adding 1.5 g of calcium chloride to 50 ml of molecular grade water and filter in sterile conditions and store at 4 °C.
- Mix aliquots containing 4 x 108 pfu adenovirus-Cre with sufficient 3% sucrose in sterile water for a final volume of 10 μl. Add 34 µl of MEM to the virus and gently mix. Then add 4 μl of the CaCl2 solution, gently mix, and incubate at room temperature for 15-20 min.
- Store adenovirus on dry ice until ready to form precipitates. Avoid thawing of the adenovirus and storing on ice or room temperature for extended times, unless precipitates are formed.
Note: It is also possible to mix the sucrose, MEM and CaCl2 prior to the surgeries if it is not possible to thaw the adenovirus aliquot and begin making precipitates immediately after removal from -80 °C. This aliquot of sucrose, MEM, and calcium can be saved on dry ice until ready to add the adenovirus.
Note: Virus particles are stable for approximately 1 hr.
- Prior to each injection, gently flick the tube to make sure virus particles are mixed. Draw up 3 μl (2.5 x 107 pfu) of virus particles into the 10 μl syringe and prepare the mouse for the intraductal injection.
4. Intraductal Injection of Virus Particles
- Gently place the mouse on its back onto the illuminated stage of a clean dissection microscope. Illuminate the abdominal side with an extra light source and locate the left 4th or right 9th inguinal mammary gland by the small white patches of fur (visible on C57BL/6 females) surrounding each nipple.
- Rub the nipple gently with a sterile ethanol soaked cotton tipped applicator to clear hair away from the nipple and to sterilize the injection site. If they are difficult to locate, gently apply a thick layer of a depilatory cream or use shears to expose the nipples.
- Remove the keratin plug, a layer of dense dead skin cells, which is covering the nipple. Once the nipple is exposed, the keratin plug should be easily visible under the dissection microscope.
- Secure the nipple with fine surgical forceps and pull up with light force to remove the keratin plug.
- Stabilize the nipple between the forceps.
- Gently insert the needle between the forceps, cannulating the duct canal at 90°. Enter the nipple slightly past the bevel of the needle (not more than 2 mm) to prevent penetration through the mammary tissue and into the serous membranes of the ventral body cavity.
- Do not insert the needle too deep. To ensure proper depth of injection, gently pull the needle up after inserting it into the lumen of the duct, drawing the nipple up along the edges of the needle as it is pulled up.
Note: Visualization of the injection is difficult; therefore practice for this step is recommended using trypan blue.
- Do not insert the needle too deep. To ensure proper depth of injection, gently pull the needle up after inserting it into the lumen of the duct, drawing the nipple up along the edges of the needle as it is pulled up.
- When the needle is appropriately placed into the mammary duct, release the 3 µl of virus precipitates (2.5 x 107 pfu of adenovirus-Cre) by gently plunging the syringe with the thumb of the hand holding the syringe. The nipple should slightly inflate as the liquid is added.
5. Recovery of Mice
- Place the mouse back onto the heating pad after the injection until it begins to recover from the anesthesia.
- Once the mouse is recovered, place it back into a clean cage and monitor for full recovery and movement.
- 24 hr after the intraductal injection, subcutaneously administer meloxicam at 1 mg/kg.
6. Monitoring Tumor Progression
- Palpate the injected mammary gland at day 30 for enlargement and swelling.
- Monitor tumor progression every 5-7 days once a swollen and enlarged mammary gland is observed.
- Measure tumor volumes every 3-4 days for tumor growth kinetics once palpable tumors appear (approximately 50-60 days post adenoviral injection).
- Euthanize mice when tumor volumes exceed 10% of the body weight of the mice.
Representative Results
Successful targeting of the mammary ductal tree can be visualized by preparing whole mounts of the mammary gland as previously described32 after injection of trypan blue (to verify proper injection technique (Figure 1A) or an adenovirus expressing mCherry (to verify proper viral preparation and infection of ductal epithelial cells, Figure 1B).
Figure 1. Intraductal targeting of mammary glands by injection with trypan blue or adenovirus-mCherry. A) A whole mount, as previously reported32, of the mammary gland after injection of mammary gland #4 with trypan blue was prepared 3 hr post injection to visualize/confirm targeting of the entire ductal tree. Images are 4X magnification. B) Infection of the ductal epithelium with adenovirus by injecting 2.5 x 107 pfu of adenovirus expressing mCherry. Mice were injected intraductally, and 4 days post-injection, a whole mount of the mammary gland was prepared to confirm viral infection of the ductal tree. Image is 4X magnification. MG is mammary gland, LD is the lactiferous, or main duct, and TD is the terminal duct. Click here to view larger image.
When tumors are induced in p53loxP/loxP LSL-K-rasG12D/+ transgenic mice, tumors will not be apparent until around day 40 when the mammary glands will become enlarged and swollen. It is necessary to begin palpations when this is observed, monitoring tumor growth every 5-7 days. In our hands, hardening of the mammary gland always precedes the onset of tumor development. Tumors will progress slowly for an additional 2 weeks. Beginning around day 56, tumors will begin to grow exponentially (Figure 2B). At this point, it is critical to measure tumor volumes every 3 days if kinetic studies are desired because there will be slight mouse to mouse variability in tumor progression, which is normal (Figures 2A and 3A). Large abdominal masses will be apparent by day 80 (Figure 2A, 2B, and 3A), after which mice should be euthanized if tumors exceed more than 10% of their body weight. cDNA analysis of three clones from a tumor-bearing mouse revealed expression of mesothelin, cytokeratin-8, Her2/neu, and estrogen receptor-α (Figure 2C).
Figure 2. Tumor development in p53loxP/loxP LSL-K-rasG12D/+ mice injected intraductally with adenovirus-Cre. A) Two examples of tumors 80 days after injection with adenovirus expressing Cre. Mice were given 2.5 x 107 PFU of adenovirus expressing Cre and 80 days post tumor-initiation, large palpable masses can be visualized protruding from the abdominal side of the animal. B) Typical tumor kinetics and palpation schedule for tumors induced in p53loxP/loxP LSL-K-rasG12D/+ mice. C) Characterization of three tumor cell clones derived from the same homogenized tumor of a p53loxP/loxP LSL-K-rasG12D/+ mouse. RNA was extracted and cDNA synthesized for RT-PCR analysis using primers specific to β-actin, mesothelin, cytokeratin-8, Her2/neu, progesterone receptor, estrogen receptor-α, and estrogen receptor-β. Click here to view larger image.
Similar to the cellular microenvironment in human breast cancer, we have observed infiltration of αβ and γδ T cells as well as myeloid derived suppressor cells and macrophages into the tumors (Figure 4). Vasculature draining to the axillary lymph node will begin to engorge before tumors have grown to encompass the entire mammary tissue where the injection was performed (Figure 3A). Lymphovascular invasion and metastasis of tumor cells can be tracked by crossing LSL-K-rasG12D/+ p53loxP/loxP mice with LSL-EYFP mice. After Cre-mediated excision, tumor cells expressing YFP (both high and low) are detected in the tumor and can be traced metastasizing to the draining axillary lymph node (Figure 3B). Metastasis to the distal axillary lymph node was confirmed by successfully culturing a tumor cell line from this site in a tumor-bearing LSL-K-rasG12D/+ p53loxP/loxP mouse (data not shown).
Figure 3. Formation of tumors and latent metastasis to the axillary lymph node. A) Example of three advanced breast tumors with different rates of tumor progression. A solid mass, indicated by the arrowhead, forms and eventually grows to the size of the entire abdominal mammary tissue. The tumor stays confined to the mammary tissue and is not observed to invade or attach to the muscle covering the peritoneal cavity. There is evident engorgement of the superficial epigastric vein between the inguinal and axillary lymph nodes, denoted by white arrowheads. After 7-8 weeks, the axillary lymph node begins to become enlarged due to lymphovascular invasion of the tumor cells, indicated by arrow. B) Metastasis of YFP positive tumor cells can be visualized in the axillary lymph node by flow cytometry. LSL-K-rasG12D/+ p53loxP/loxP LSL-EYFP mice were used to induce tumors and activation of YFP by intraductal delivery of adenovirus-Cre. To verify lymphovascular invasion of tumor cells into the axillary lymph node, 80 days post adenoviral injection, the indicated lymph nodes and organs were harvested and stained for lymphocyte markers and examined for YFP expression. CL represents contralateral nontumor draining lymph node. Results represent gating on CD45 negative tumor cells, indicating the tumor cells are invading the distal axillary lymph node. Numbers represent percent YFP positive cells from total population. Click here to view larger image.
Figure 4. Immune infiltrates in mouse transgenic breast tumors. Mouse breast tumors were homogenized and stained for CD45, CD3, γδTCR, CD11b and GR1. Numbers represent percent of positive leukocytes in entire tumor (63.5), total CD3+ (46.7), total CD3 negative (40.1), total CD3+ γδ+ (γδ T cells, 13), CD3+ γδnegative (24), total GR1 high CD11b (MDSC, 28.6) and total CD11b GR1 low (macrophages, 18.5). Click here to view larger image.
Due to the anatomy of the mouse and technique of intraductal injections, we find targeting of mammary glands 4 and 9 (Figure 5) yields the most consistent results and reliable injections. However, any gland can be targeted depending upon the preference of the technician performing the surgery.
Figure 5. Numbering of mammary ducts. Mammary ducts 4 and 9 are highlighted in red on the diagram and indicated with arrows on the mouse. In our hands, we found that injections were easiest to perform on these mammary ducts, however all other mammary tissues that we targeted developed tumors with similar kinetics. Click here to view larger image.
The success of this procedure hinges on proper technique during intraductal injections, which will be difficult for untrained experimenters. Experienced researchers in our laboratory typically obtain breast tumor masses 79% of the times they administer adenoviral injections. Problems with the injection can result in significantly delayed, variable, or absent tumor development. If the needle is inserted too deep or at an inappropriate angle, the ductal canal may be missed. It is important to enter the nipple slightly past the bevel of the needle (not more than 2 mm), to prevent penetration through the mammary tissue and into the serous membranes of the ventral body cavity. Also, too shallow placement of the needle or the injection of greater than 3 ml of virus precipitates can result in spillage of viral prep outside the mammary gland and the induction of unintentional tumors. One way to overcome these issues is to insert the needle into the nipple slightly deeper than 3 mm, and slowly draw the syringe back up out of the duct until 2 mm from the tip of the bevel. This will ensure that mammary tissue is targeted instead of the muscle surrounding the peritoneal cavity of mice (Figure 1A). This will also stretch the nipple along the edges of the syringe so that when the virus is expelled into the duct, there is no spillage and loss of viral precipitates.
Visualization of the injection is difficult and practice for this step is recommended. We have observed an increase in successful injections following practice, resulting in a higher penetrance of tumor development. Because this technique uses nonlactating virgin females, it is critical to remove the keratin plug covering the nipple to reveal the underlying duct canal. We recommend practicing this step by injecting trypan blue or some other sterile traceable dye inside of the duct and preparing whole mammary mounts to confirm targeting of the ductal tree. Additionally, other protocols describing intraductal injection of reagents have been published33,34, which may be useful for developing proper technique. Issues with viral preparation or infection of the ductal lumen can also be investigated by using a mCherry expressing adenovirus. Nontransgenic mice can be used for each of these purposes until the injection technique is optimized.
Although any mammary gland can be used to initiate tumors, we have achieved the most consistent growth rates by targeting mammary gland 4 or 9, which we believe is because it is easier to perform proper injections on these glands, resulting in more efficient targeting of the duct. The proximity of the left 4th or right 9th inguinal mammary glands to the draining inguinal lymph node is also useful to examine anti-tumor immune responses during different temporal points of tumor progression. To model and track latent metastasis to distal sites, transgenic mice were crossed with LSL-EYFP mice. As the tumor progresses, vasculature connecting the tumor to the axillary lymph node will become slightly engorged at approximately 5 weeks, before the tumor begins to grow exponentially (Figure 3A). Eventually, after 7-8 weeks, lymphovascular invasion will result in tumor growth within the axillary lymph node (Figures 3A and 3B). Using reporter mice and Cre-loxP technology, incorporation of YFP creates a platform to track tumor cells metastasizing to distal sites throughout tumor progression. This can facilitate studies aimed at elucidating the cellular and epigenetic mechanisms that promote latent metastasis. In our hands, breast tumor cell lines expressed cytokeratin-8, mesothelin, estrogen receptor-α, and Her2/neu, confirming targeting of the ductal epithelium. However, depending upon the mutations induced and due to the difficulty and the variability of injections, we recommend histological characterization of tumors once the model is well established in the laboratory.
Because breast cancer is such a deadly and pervasive disease1,2, it is important to use animal models that accurately recapitulate the complex interplay between tumor and host. Here we describe a fully backcrossed C57BL/6 murine model of breast tumor. First, by inducing tumors from native cells, we allow the tumor to evolve naturally in a full immune microenvironment. The immune microenvironment in the advanced mouse breast tumors recapitulates the populations of αβ and γδ T cells, myeloid derived suppressor cells, and macrophages commonly observed in human breast cancer (Figure 4). As we have published previously using adenovirus-Cre to induce ovarian tumors, we found that the viral injection had negligible effect on the corresponding tumor infiltrates and tumor progression28. Second, endocrine independent expression of oncogenes ensures that tumor cells have persistently high levels of target gene expression. Third, by taking advantage of latent mutations, we can control the timing of tumorigenesis to facilitate precise temporal tracking of tumor evolution. Applications of this model include research on tumor cell biology, studies on factors in the tumor microenvironment, anti-tumor immune responses, and even efficacy evaluation of new therapeutics. Through the availability of the Cre-loxP system, this technique can be used as a platform for investigating a diverse array of additional mutations in the initiation and progression of breast tumors. We hope that the use of this model will enhance the understanding of breast cancer biology and eventually lead to new therapeutics aimed at treating metastatic breast cancer.
The authors declare no competing financial interests.
This work was supported by NCI Grants RO1CA157664 and RO1CA124515, and a Breast Cancer Alliance award. We would like to thank Jeffrey Faust, David Ambrose, and Scott Weiss from the Wistar Flow Cytometry core facility, James Hayden from the Wistar Imaging facility, and the entire staff of the Wistar Institute Animal Facility for their invaluable technical support.
|Catalog Number
|Trp53tm1Brn Transgenic mice
K-rastm4Tyj Transgenic mice
|Obtained from NCI mouse models of human cancer consortium
|Mice were backcrossed ten times to a full C57BL/6 background
Gt(ROSA)26Sortm1(EYFP)Cos/JTransgenic mice
|Jackson Labs
|Primers p53loxP/loxP
|Integrated DNA Technologies
|Primers LSL-K-rasG12D/+
|Integrated DNA Technologies
|Primers for LSL-EYFP to detect Rosa promoter
|Integrated DNA Technologies
|Primers for detection of Mesothelin expression
|Integrated DNA Technologies
|Primers for detection of Progesterone Receptor expression
|Integrated DNA Technologies
|Primers for detection of Cytokeratin 8 expression
|Integrated DNA Technologies
|Primers for detection of Erbb2 expression
|Integrated DNA Technologies
|Primers for detection of Estrogen Receptor A expression
|Integrated DNA Technologies
|Primers for detection of Estrogen Receptor B expression
|Integrated DNA Technologies
|Primers for detection of B-Actin expression
|Integrated DNA Technologies
|Gene Transfer Vector Core from the University of Iowa
|Store aliquots of virus (4 x 108 pfu/aliquot) at -80 °C to avoid repeated freeze thaw cycles.
|Gene Transfer Vector Core from the University of Iowa
|Store aliquots of virus (4 x 108 pfu/aliquot) at -80 °C to avoid repeated freeze thaw cycles.
|Hamilton syringe
|Hamilton company
|10 μl syringe, RN series.
Autoclave before and after each use. Clean with PBS and 75% ethanol.
|Custom needle
|Hamilton company
|33 G 0.5 in long RN needle, with a 12° bevel. Autoclave before and after each use. Clean with PBS and 75% ethanol.
|Surgical forceps
|Dumostar No. 5 forceps. Clean with 75% ethanol after each use, followed by autoclaving
|MEM powder
|Store at 4 °C in powder and reconstituted form
|Sodium Bicarbonate
|Add to MEM and filter stearilize
|Calcium Chloride
|Minimum 96%, anhydrous
- Youlden, D., et al. The descriptive epidemiology of female breast cancer: an international comparison of screening, incidence, survival and mortality. Cancer Epidemiol. 36, 237-248 (2012).
- Siegel, R., Naishadham, D. Cancer statistics, 2012. Cancer J. Clin. 62, 10-29 (2012).
- Sorlie, T., et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc. Natl. Acad. Sci. U.S.A. 98, 10869-10874 (2001).
- Gatza, M., et al. A pathway-based classification of human breast cancer. Proc. Natl. Acad. Sci. U.S.A. 107, 6994-6999 (2010).
- Bastien, R., et al. PAM50 breast cancer subtyping by RT-qPCR and concordance with standard clinical molecular markers. BMC Med. Genom. 5, 44 (2012).
- Montagna, E., et al. Heterogeneity of triple-negative breast cancer: histologic subtyping to inform the outcome. Clin. Breast Cancer. 13, 31-39 (2013).
- Karrison, T. G., Ferguson, D. J., Meier, P. Dormancy of mammary carcinoma after mastectomy. J. Natl. Cancer Inst. 91, 80-85 (1999).
- Rakha, E., et al. The prognostic significance of lymphovascular invasion in invasive breast carcinoma. Cancer. 118, 3670-3680 (2012).
- Kim, I., Baek, S. Mouse models for breast cancer metastasis. Biochem. Biophys. Res. Commun. 394, 443-447 (2010).
- Vargo-Gogola, T., Rosen, J. Modelling breast cancer: one size does not fit all. Nat. Cancer. 7, 659-672 (2007).
- Archer, T., et al. Steroid hormone receptor status defines the MMTV promoter chromatin structure in vivo. J. Steroid Biochem. Mol. Biol. 53, 421-429 (1995).
- Cato, A., Henderson, D., Ponta, H. The hormone response element of the mouse mammary tumour virus DNA mediates the progestin and androgen induction of transcription in the proviral long terminal repeat region. EMBO J. 6, 363-368 (1987).
- Schoenenberger, C., Zuk, A., Groner, B., Jones, W., Andres, A. Induction of the endogenous whey acidic protein (Wap) gene and a Wap-myc hybrid gene in primary murine mammary organoids. Dev. Biol. 327-337 (1990).
- Li, Y., et al. Deficiency of Pten accelerates mammary oncogenesis in MMTV-Wnt-1 transgenic mice. BMC Mol. Biol. 2, 2 (2001).
- Martelli, C., et al. In vivo imaging of lymph node migration of MNP- and (111)In-labeled dendritic cells in a transgenic mouse model of breast cancer (MMTV-Ras). Mol. Imaging Biol. 14, 183-196 (2012).
- Klover, P. J., et al. Loss of STAT1 from mouse mammary epithelium results in an increased Neu-induced tumor burden. Neoplasia. 12, 899-905 (2010).
- Moody, S. E., et al. Conditional activation of Neu in the mammary epithelium of transgenic mice results in reversible pulmonary metastasis. Cancer Cell. 2, 451-461 (2002).
- Banerji, S., et al. Sequence analysis of mutations and translocations across breast cancer subtypes. Nature. 486, 405-409 (2012).
- Miyakis, S., Sourvinos, G., Spandidos, D. A. Differential expression and mutation of the ras family genes in human breast cancer. Biochem. Biophys. Res. Commun. 251, 609-612 (1998).
- Malaney, S., Daly, R. J. The ras signaling pathway in mammary tumorigenesis and metastasis. J. Mammary Gland Biol. Neoplasia. 6, 101-113 (2001).
- Loboda, A., et al. A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors. BMC Med. Genomics. 3, 26 (2010).
- von Lintig, F. C., et al. Ras activation in human breast cancer. Breast Cancer Res. Treat. 62, 51-62 (2000).
- Downward, J. Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer. 3, 11-22 (2003).
- Eckert, L. B., et al. Involvement of Ras activation in human breast cancer cell signaling, invasion, and anoikis. Cancer Res. 64, 4585-4592 (2004).
- Hollestelle, A., Elstrodt, F., Nagel, J., Kallemeijn, W., Schutte, M. Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines. Mol. Cancer Res. 5, 195-201 (2007).
- Jackson, E., et al. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev. 15, 3243-3248 (2001).
- Jonkers, J., et al. Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nat. Genet. 29, 418-425 (2001).
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Formal Correction: Erratum: Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer
Posted by JoVE Editors on 10/20/2014. Citeable Link.
A correction was made to Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer. Protocol sections 3.1, 3.3.2, 3.4, and 4.7 were updated. The materials table was also updated.
Protocol section 3.1 was updated from:
3.1) Store adenovirus concentrated virus stocks at -80°C frozen in aliquots of 4 x 108 pfu each, sufficient for injecting 16 animals with 3 ml of 2.5 x 107 pfu of adenovirus particles.
3.1) Store adenovirus concentrated virus stocks at -80°C frozen in aliquots of 4 x 108 pfu each, sufficient for injecting 16 animals with 3 µl of 2.5 x 107 pfu of adenovirus particles.
Protocol section 3.3.2 was updated from:
Mix aliquots containing 4 x 108 pfu adenovirus-Cre with sufficient 3% sucrose in sterile water for a final volume of 10 ml. Add 34 ml of MEM to the virus and gently mix. Then add 4 ml of the CaCl2 solution, gently mix, and incubate at room temperature for 15-20 min.
Mix aliquots containing 4 x 108 pfu adenovirus-Cre with sufficient 3% sucrose in sterile water for a final volume of 10 µl. Add 34 µl of MEM to the virus and gently mix. Then add 4 µl of the CaCl2 solution, gently mix, and incubate at room temperature for 15-20 min.
Protocol section 3.4 was updated from:
Prior to each injection, gently flick the tube to make sure virus particles are mixed. Draw up 3 ml (2.5 x 107 pfu) of virus particles into the 10 ml syringe and prepare the mouse for the intraductal injection.
Prior to each injection, gently flick the tube to make sure virus particles are mixed. Draw up 3 µl (2.5 x 107 pfu) of virus particles into the 10 µl syringe and prepare the mouse for the intraductal injection.
Protocol section 4.7 was updated from:
When the needle is appropriately placed into the mammary duct, release the 3 ml of virus precipitates (2.5 x 107 pfu of adenovirus-Cre) by gently plunging the syringe with the thumb of the hand holding the syringe. The nipple should slightly inflate as the liquid is added.
When the needle is appropriately placed into the mammary duct, release the 3 µl of virus precipitates (2.5 x 107 pfu of adenovirus-Cre) by gently plunging the syringe with the thumb of the hand holding the syringe. The nipple should slightly inflate as the liquid is added.
Item 15 in the Materials table under Comments was updated from:
10ml syringe, RN series Autoclave before and after each use. Clean with PBS and 75% ethanol.
10µl syringe, RN series Autoclave before and after each use. Clean with PBS and 75% ethanol.
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Full Gazette BISE Lahore Board 9th Class Result 2020
The Board of Intermediate and Secondary Education is going to announced the Matric / SSC Part 1 {09th, Nine, Ninth} Class Annual Exams Result 2020 on the official government website at biselahore.com on Friday, 21st August, 2020. He is a first biggest board of Pakistan where more than 02 millions students appear every year in the annual exams of matriculation and intermediate. Every year he taken the examinations of matric and inter in starting month of March and end of April after the exams the result will be declared by the board about 03 months later in these days he prepared the final results of candidates who appear in the last year examinations through this board of Pakistan.
He is located in the second biggest city of Lahore in 86 Mozang Road and daily hundreds candidates visit board building and solve his issues like correction of date of birht, roll number slips, obtain result cards, rechecking of paper marks, for addmission inquery and also other many reasons and solved them here without any problem becuase the great educated teachers and staff is reamined to coperate for all candidates. He is awarded the many boys and girls who get the top position in the annual exams of the division and district level. PM (Prime Minister) and CM (Cheif Minister) aslo provide the gift prizes and scholarships (wazifa's) for the top position holders boys and girls in the shap of gift prizes of money and other prizes.
Online Check Lahore Board Matric Part 1 Result 2020
The Board of Intermediate and Secondary Education BISE Lahore Board will be announced the Matric / SSC Part 1 / 09th Class Result 2020 on dated Friday, 21st August, 2020 (21/08/2020) of board website. All the students free download and online check the nine class result 2020 at this webpage easily. You can keep touch with this webpage for latest updates of coming results of Lahore 09th Class Result 21-08-2020 uploads the our Biseworld.com site for our visitors come here. His jurisdiction is which are Kasur, Nankana Sahib, Sheikhupura and Lahore.
BISE Lahore Board 9th Class Result 2020
Dated: Friday, 21st August, 2020
Coming Soon Here !!!


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Berkeley Skeleton Watch
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Produced by Paul Ryan
This story originally aired on Jan. 28, 2006.
In 1995, a bodybuilder who sold steroids and was involved in a love triangle in Fort Myers was gunned down inside his home. Police eyed an associate of the victim as a suspect but he disappeared before officers could make an arrest.
Years later and thousands of miles away, the murder of a police officer would stun a nation and reveal to investigators that the two cases were linked.
"48 Hours" correspondent Susan Spencer reports on the investigation, in cooperation with Granada Media and True North Productions.
On Dec. 26, 2003, in Leeds, England, police officer Ian Broadhurst lay dying in the street beside his patrol car. The search for his killer - one of the biggest manhunts in British history - had just begun.
Ian's mother Cindy remembers the day had started so peacefully. It was a holiday, the day after Christmas, what the British call "Boxing Day," and Ian and his wife were visiting.
"They actually came for Boxing Day breakfast and we had a lovely breakfast and a lot of laughs. We sat together and we watched the film and we laughed," she says.
Ian, 34, was a traffic cop in the city of Leeds and, holiday or not, he had to go to work.
"Boxing Day is generally a quiet day. Looking for what we normally look for, stolen cars, anybody that is doing something that draws us attention," says Neil Roper, 43, who had been Ian's partner for only a few months.
The two men had grown close.
"He was my mate, not just a policeman. He was just a genuine fella that got on with everybody," says Roper.
In the short time they were partners, Broadhurst and Roper had developed an almost uncanny ability to spot stolen cars, and this day after Christmas would be no different.
That afternoon the officers turned onto a small side street to check out a BMW that was parked at an odd angle.
"I just basically saw this black 3-series BMW parked up on the causeway in a - how can I put it - a peculiar position," Roper says. "We went slowly past the passenger side of the vehicle. I looked and saw just this white man reading a racing post."
The officers approached the vehicle and then radioed in. Their hunch was right: the car was stolen.
The driver - a very big man - was making Roper nervous.
"In the police car there is a button that you press which gives you the facility, obviously, to record anything that's being said in the car. This is the first time I've ever done this," he says.
The chilling record of what happened next was all caught on tape.
On the tape, one can hear the man saying he was from Leeds but that his country of birth was Canada. He also told officers, "Just to let you know I did not steal the car."
Roper was growing more wary by the minute and decided he should handcuff this suspect and got out of the car to call for backup, leaving Broadhurst alone.
The BMW was towed away. Moments later, Officer James Banks arrived. Broadhurst now got out of the patrol car.
Like most British police, none of the three officers carried a gun.
"I said to James, 'When I'm cuffing him, can you just watch me back,' " says Roper. "As I've looked forward, I've just seen this gun coming up to my face and what can I say from there. I've just shouted, he's got a gun."
Then gunshots could be heard on the tape.
Though Roper was hit in the shoulder and stomach, he somehow made it to a nearby building and radioed for help.
"I've been shot twice. I don't know about Ian, he's down on the floor," Roper radioed.
Neil Roper was critically wounded and James Banks was saved only because the bullet hit his police radio. But Ian Broadhurst died on his way to the hospital.


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NY SportsMed is New York City's leading one-stop clinic providing urgent and orthopaedic care, sports medicine, and physical therapy services that meet the needs of active people. They serve the growing market of people looking for efficient and personalized options through a retail clinic model — offering urgent care, sports medicine, physical therapy, orthopaedic surgery, and X-Rays to active New Yorkers.
NY SportsMed has been providing state-of-the-art services to athletes and active lifestyle enthusiasts for 7 years, seeing over 50,000 patient visits annually. They currently operate out of 3 offices, with an expert staff made up of 65 employees, including 4 doctors and 20 physical therapists. Last year alone, they saw a growth rate of over 25%. The team is working to open up an additional 4 satellite clinics and rapidly expand their clientele. They have raised $1 million to date.


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Coach Vicky
please contact Vicky
at email@example.com. [/button]
About Me
My biggest passions in life have always been helping others and fitness, so it is no surprise to people who know me that almost everything I do in my life circles around these two concepts. I graduated from The College of New Jersey in May of 2013 with a Bachelor's degree in English and Secondary Education which incited my career as a High School English teacher at Mendham High School. While education became my main focus in college, fitness was never far behind and after moving to Morristown, I found CrossFit and instantly fell in love with the sport! In January of 2017, I received my CrossFit Level 1 and began coaching at CrossFit 13 Stars. Hoping to further my passion of helping others, in October of 2017 I decided to take the Level 1 Nutritional Coaching Institute Certification to help others achieve their goals by optimizing their nutrition.
About Nutritional Coaching
I provide two different nutrition coaching experiences
• Macro based approach
This means that you'll be expected to weigh and measure the food you eat in a day
All information can be recorded in a food tracking app called MyFitnessPal
• • Client will receive customized goals for daily protein, carb and fat intake in addition to suggestions for:
• Hydration
• Supplements
• Food timing to make sure that you feel great while working out
• Habit based approach
Coach and client work together to create custom goals
Habits are developed based on nutrition, sleep, lifestyle, wellness, etc.
Habits will be tracked in a custom tracker for accountability and feedback
My two most important coaching philosophies are communication and education, so you have 24/7 access to text or email me with questions. I really want you to learn from the experience, and learn the "ins and outs" of the process so that at some point, you are able to go off on your own and continue succeeding.
I offer nutrition coaching for:
• Weight Loss
• Strength gains/improved fitness performance
• Overall improvement of health
Nutrition Blog
I will be posting regular articles relating to nutrition in a variety of ways. Please take a look, and feel free to reach out with any questions that you had while reading.
My Macro Story
After being an athlete my entire childhood, I gained about 30 pounds in college, which on my 5'2 frame is a lot. My senior year, I decided to get my butt in shape, joined a Gold's Gym and started doing the South Beach Diet. I was able to lose 25 pounds, but gained most of it back once I graduated from The College of New Jersey in May of 2013. Flash forward to to March of 2015, I did a Paleo Challenge here at the gym. Very similar to South Beach, I was the thinnest I have ever been, BUT old habits die hard and the weight slowly started to creep back, once I finished the Paleo challenge. In November of 2016, I started working with my macro coach, Laurie from iN3 Nutrition. By working with her for over a year, I was seeing huge results in the gym and feeling the best I have in my body. Laurie, suggested that I take the Nutritional Coaching Institute certification to become a nutrition coach because she sensed my passion and inquisitiveness about nutritional coaching. So here I am in 2018, working with clients and still LOVING the flexible dieting lifestyle. This is the only way of eating where I know that I CAN still "have my cake and eat it too" (as long as it's weighed and measured, of course).
Testimonials[tabs type="horizontal"] [tab title="Stella"]
"Vicky was incredibly helpful during one of the most stressful times in my life: wedding planning. Macro tracking became the most consistent thing in my life and Vicky was there every step of the way to coach me, physically and mentally. We tested different combinations of macro numbers to get me to where I wanted to be for the wedding.
I appreciated the weekly email check in because it not only kept me accountable but allowed me to remind myself at the progress I have made throughout the weeks. Vicky made herself available 24/7 (except for 8 hours because that's how many hours you need to feel rested ya'll!) via text.
She also shares with her clients about her own macro journey, meal prep tips, and motivation throughout the roughest of cuts. I am happy to call Vicky a coach and a friend!" – Stella[/tab] [tab title="CK"]
"Sometime in February I realized my weight had steadily crept to an unacceptable level. I regularly went to CrossFit and even excelled at the strength training component at the gym. However, still the weight crept up. It was clearly diet more than anything else, but aside from calorie-counting or being unrealistically restrictive on what I ate, there was no way as far I knew to just figure out how eat in a way that gave me energy for they but where I lost weight.
I tried a variety of approaches to diet in order to lose and/or maintain weight. But most of them often failed because they tended to be too restrictive and often ran too counter to the practicalities of everyday existence; for example, the Paleo life is unsustainable where you can't eat pizza, rice, yogurt, milk, beans, or bread. Sure, moderating them or cutting back by a lot seems fair, but I tended to fail miserably at sum-zero, rigid diets. Macros provides me with the necessary goal-posts to know how much to cut back on some things and how much to eat on others.
Working with Vicky has really helped me create a lifestyle that reconciles the two impulses everyone has with food: to both eat in ways that were nutritionally sound while also wanting to enjoy the full versatility that food has to offer. Vicky achieves this through two really important pillars to her consultation: she is very detailed-oriented and will give you precise parameters on success and will empathize with the social and emotional pratfalls that come with trying to find the right food balances. Rather than telling you to break off your relationship with foods that you love, she gives you a road map for re-framing your relationship with those foods. She often does this by example: both Vicky and I are doughnut enthusiasts, and we often discuss strategies and ways in which we can have those things in our lives (infrequently and moderately!) without losing out on results.
Since February I have lost twenty pounds. I have learned that I need more vegetables in my life and way, way more protein than I would've ever thought was acceptable (like, a lot more). I've learned to care about fiber and drink an ungodly amount of water. But it isn't zero sum, because in the midst of all that I have also occasionally had pizza or doughnuts or fried foods. (An aside: What you may find when you start eating the right amounts of your macros is that your craving for these things dulls. My deep, abiding love for pizza has settled into a strong, Platonic, yet distant friendship. On the flip side, my cravings for protein are way more acute.)
Vicky provides you with balance in your diet, and she gives a lot of great advice about how to fit it to your specific needs. The only thing obnoxious about this service is that I didn't really come around to this approach sooner." – Chris[/tab] [tab title="JENNA"]
"Working with Vicky has been awesome. It's been a slow and steady process, which is great because I eased into it, never feeling deprived, and have been progressively reaching my nutrition goals since October. One of my goals was to fit in size 10 jeans and I reached that goal within the first two months. I am so happy with my progress and feel great. I have more energy, I'm sleeping better, working out harder, and seeing visible results in the mirror, which is super motivating to me. It's a flexible way of eating and I never feel like I am restricted. My progress photos speak for themselves!" – Jenna[/tab] [tab title="GREG"]
"I've struggled with nutrition for most of my adult life, particularly with a stressful, office job. I've never responded well to overly strict diets because they weren't sustainable. Vicky has helped me find an adaptable and sustainable way to manage my nutrition. Managing your diet through tracking macronutrients focuses on what you should eat (and sometimes where you can indulge), which makes it less stressful than "dieting." Vicky helped me look at how my body was responding to my diet, my sleep patterns and my exercise schedule. If you feel like you have hit a plateau with your fitness, nutrition or health goals, working with Vicky is a good way to re-think your approach and get to the next level. " – Greg[/tab] [tab title="RAE"]
"I highly recommend working with Vicky. As a coach, Vicky is always positive and encouraging. I appreciate her balance and understanding." – Rae[/tab] [/tabs]


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I'm flabbergasted.
Looks like there's going to be a PSP game based on the line up of Robot Spirits toyline. Battle Robot Spirits. Eeek! From the looks of it, gameplay is going to be like those arcade mecha shooter/fighting games. Like Virtual On or those unofficial Gundam and Magical Girl games. Heroman, despite being merely Incredible Hulk sized, is gonna be scaled up so he's actually hulking compared to the other mechs. The Full Metal Panic guys will include Bonta! You know what? I bet Doraemon's gonna be a secret character.
And leading the charge? The Tamashii Nations-bot.
Oh, and the Gundams are conspicuous by their absence, but hey, who cares about Gundams when you've got Aura Battler Dunbine and the "Galactic Pretty Boy" Tauburn?


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Both of our courses would normally be offered at a rate of £925 per person for four days training plus online support plus all materials. However they are available FREE to schools/ staff that are:
1. In 'The South' (see picture) AND Ofsted graded 3 or 4.
2. In an Opportunity Area OR Category 5/6 Local Authority (see below for list of LAs).
3. No funded places are available to schools in category 1 or 2, or schools outside of the specified local authorities (ie category 5 and 6). TBT training is still available for schools that are not eligible for the TLIF funding, but this would be charged at a commercial rate. Please contact TBT for a quote.
5. Tom Bennett can, subject to availability, design a unique training service for your school: one day training, targeted groups, leadership consultancy, new teacher training, key notes. Please contact us for more details and prices.
Category 6 Local Authorities
(Opportunity Areas in bold)
- Breckland
- Corby
- Crawley
- Daventry
- East Cambridgeshire
- East Northamptonshire
- Fenland
- Forest Heath
- Gosport
- Great Yarmouth
- Hastings
- Huntingdonshire
- Ipswich
- Isle of Wight
- Kettering
- King's Lynn and West Norfolk
- Northampton
- Norwich
- Peterborough
- Portsmouth
- Waveney
- Wellingborough
- West Somerset
- Weymouth and Portland
- Worthing
West Somerset
Fenland and East Cambridgeshire
You are more likely to be admitted if your school is located in one of the prioritised local authority districts from Category 5 and/or 6 (opportunity areas):
- West Somerset
- Norwich
- Fenland and East Cambridgeshire
- Ipswich
- Hastings


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Past Relationships Help Us To Grow
If I am a sapling today, what will I be tomorrow or when I draw my last breath?
- What does "learning to grow in a relationship mean"?
- Do I grow from past relationships?
- Do I make meaningful strides to improve myself both during and after the relationship ends?
- Do I take lessons learned and apply them to my life to improve?
- Do I become more aware and create a better version of myself?
I have always believed that in this life journey, we are in constant development and constant preparation. Life is a journey of personal growth regardless of who we choose to bring into our lives. In those moments between past relationships, we have the chance to reflect, discard unwanted baggage and embrace our full potential. Each relationship that you experience is relevant so we should always be learning to grow in a relationship. While not every relationship will last forever, each is an opportunity to learn, grow and improve.
I do not mean to minimize the value of past relationships, even if they were significant, like a deep love or long-term marriage. For one reason or another, however, that relationship ended. Either you or the other was not ready to be in the relationship as it had been defined. That does not mean that you did not share powerful and memorable experiences, and even experience a love that will live in your heart forever. Yet, the relationship ended. Do the question still stands: did you grow in a relationship?
It is healthy to think fondly of your past relationships and embrace those qualities that sustained your lives together, because each relationship has brilliant moments. Of equal importance, I do not look at the ending of the relationship as a failure but as a learning opportunity for growth. If one can get beyond the immediate pain and brokenness of the ending, there are certainly valuable lessons to be learned. One of the partners either was not ready to be or was not ready to stay in the relationship at that moment. It is not wrong to take the approach that was each new relationship we grow and learn and prepare ourselves for the next great thing. One door always opens when another door closes.
This is part of the optimism of living the single life: the door was shut for a purpose, and it is up to us to find both the reason and the meaning behind the ending.
Do you believe there is purpose to the ending of your previous relationship?
Regardless of how the past relationship ended, did you walk away with some learning point that made you better?


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I need enable TCP connections to my system, for use graphical applications in marionnet, network simulation system, in debian squeeze.
In older versions of debian it is achieved with System|Administration|Login Window -> uncheck "Deny TCP connections to the Xserver". but in squeeze I have no idea.
EDIT: I have the answer: take a look here


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By Shane Thomas
Jessica Ennis. The face of London 2012. Such a nice girl, isn't she? Wouldn't it be lovely if more people were like her?
Let's stop this one-sided view of Britain's latest sporting icon. Behind the courteous persona and schoolgirl demeanour lies a soul-deep core of competitive steel. The nation's sweetheart? I doubt her opponents in the heptathlon think so.
That's not to say Ennis' down-to-earth disposition is a falsehood. Far from it. But it's only a part of who she is. I know plenty of agreeable people, and not one of them would have what it takes to be Olympic Champion - especially when the whole nation is demanding nothing less.
It will be easily forgotten now, but Ennis did not come into the heptathlon competition as the favourite. She lost her World title to Tatyana Chernova last year, and then a few months ago, surrendered her World Indoor crown to the (then) reigning, Olympic Champion, Natalya Dobrynska. On both occasions, Ennis produced strong displays, but not her very best. This brought into sharp focus that only her maximum would do to succeed in London.
And how she produced. Personal bests in the 100 metre hurdles and the javelin - her weakest event. The hurdles wasn't only Ennis's quickest ever time, but the quickest time in the history of the heptathlon.
Her hurdles performance is important to note here. It's the first event of the competition, the one to lay down a marker and set the pace - especially if it's one of your stronger events. All the pressure, all the build-up has led to this. No more talking, time to go out and perform.
The one emotion that seldom gets associated with Ennis is anger. But don't think that after those aforementioned defeats to Chernova and Dobrynska, that Ennis wasn't angry. She doesn't compete to win silver medals, and would have had a storm raging inside her as she lined up on the track. Hell hath no fury...
The time of 12.54 was a blitzkrieg to her rivals, a run of outright rage that singed every one of her competitors. Chernova and Dobrynska would not have known what hit them, and it looked to have a residual effect, as both women had wretched first days in the competition.
Along with her gold medal, what will likely have pleased Ennis the most is how she responded to adversity. A mediocre high-jump and solid display in the shot-put was followed up a searing run in the 200 metres to end Day 1. Ennis was in silver medal position going into the race, and knew how vital it was that she regain the lead before Saturday morning's long jump. It was the hallmark of Ennis - every time she was (figuratively) pushed, she pushed right back.
Day 2 always sets the nerves jangling for Ennis fans. She has a record of being inconsistent in the long jump and woeful in the javelin, sometimes leaving her with too much ground to make up in the 800 metres.
We needn't have worried. She produced a super display of 6.48 metres to cement her lead, before giving that personal best of 47.49 metres in the javelin to make the 800 metres a procession. But Ennis isn't The Queen, she doesn't do processions. She could have finished 12 seconds behind then-second placed Austra Skujyte and still have won gold.
However, Ennis is driven by a fierce competitive nature. This was no time to relax. Roared on by the home crowd, she took the initiative from the gun and raced into the lead. But the heptathlon takes such a physical toll on the body. You could see the pain etched on Ennis' face as she dragged herself around the two laps of the track.
Chernova, who could still win a medal, passed Ennis with 250 metres to go, and "our Jess" was in third going into the home straight. No matter, third is fine, she'd still take the gold.
But no, it wasn't fine. All the hype, the advertising billboards, the television adverts, the raucous home support, and most importantly, Ennis' own voracious drive for excellence would not allow it to end like this. She would win, and do it the right way. Summoning a final burst of energy that her rivals couldn't match, Ennis regained the lead, crossed the line, and the hopes of a nation were fulfilled.
You'll have to forgive me, but I'm about to steal a line from the journalist, Simon Barnes. Before the heptathlon started, Barnes made the astute statement that the reason we wanted Ennis to win so desperately, why we chose her to be the frontwoman of these Olympics is that, much like Cathy Freeman in the Sydney Games, Ennis represents the type of country we want modern Britain to be. Our national symbol; Polite, decent, unassuming, mixed-race, and a woman. And not only that, the heart of a champion. Ruthless and determined. She's both a nice person and a hard-nosed warrior.
After her disappointments of the past 12 months, Ennis knew she would have to produce the very best of herself to fulfil her dream of Olympic gold. And in her glorious success, the achievement enshrined us all.
"The Greatest Events in Sporting History" is available to download from http://www.simplysyndicated.com/shows/sportinghistory/, e-mail us at firstname.lastname@example.com and you can follow us on Twitter @TGEISH


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Boot windows 8 or windows 8.1 DVD
- Type cd: and hit Enter
- Type cd windows hit Enter
- Type cd system32 hit Enter
- Type ren utilman.exe utilman_bak.exe
- Type copy cmd.exe utilman.exe
- Type exit
Restart Windows and let it boot normally.
Once at the login Window Click on Ease of Access.
net user
look for Administrator account. Lets enable it with the next command.
net user administrator /active:yes
now lets reset the admin password using the following.
control userpasswords2
Now use the following window to reset the Administrator password.
Reboot the computer and you should now see the Administrator account.


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Today was vet day. Despite being still unwell, I had to take my mum's dog, Daisy, to the vet for her annual jab. Together with my sis, Stef and her hubby, Kok, and their 2 dogs, we went to pick Daisy up at my mum's early this morning.
As I was late when Stef and hubby came to pick me up, I hurriedly pulled on my tank top and skort (skirt + shorts underneath) and went downstairs to the waiting car. All the way to the car I was feeling quite uncomfortable in my skort and kept picking on my underwear 😛 Then when we got to my mum's and I collected Daisy, on the ride down in the elevator I realised why I kept picking on my underwear: I had my skort the wrong way round!
The moment we stepped out of the elevator and I saw Stef, I told her about it and we laughed. I told her I had to go change it around and she said "Can't you just turn it around?" Um…no, sis, I can't because it's a skort and it has shorts underneath that are attached to the skirt. Then I saw the tinted windows of the backseat of the car and made a decision. Kok was ordered out of the car…and the skort got turned around 😉
The trip to the vet was uneventful, even with 3 dogs in the car. Poor Daisy was so stressed that she shed lots of fur in the back of the car. She didn't flinch when she had the jab but her tail was permanently down and hooked onto the edge of the observation table, as if hanging on to dear life LOL But at least she's subdued when stressed or afraid and sits quietly. The other two small dogs were something altogether, barking, growling and jumping about.
Did Daisy get any treat for being a good girl? Well, if deworming tablets can be considered as a treat, then yes, she did 😛


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The X Latin American Robotics Symposium
From 21 Oct, 2013 17:05 until 27 Oct, 2013 19:05
Categories: Chapter Events
The X Latin American Robotics Symposium LARS, the XII Latin American Robotics Competition LARC and the I School Robotics Olympiad will be held in Arequipa, southern Peruvian city, in October 21-27, 2013.
The event congregates researchers and students from Latin American countries and the world to present recent advances in the field of robotics as well as to participate in the different robotics competitions. Previous versions of LARS-LARC show that they are a robotics festival plenty of technology, modernity and friendship.
Arequipa, also known as the White City, is a special metropolis where tradition and modernity came together to create a peaceful and friendly environment. It is located in the Andean mountains close to Cuzco city and Machu Picchu, Inca Empire lost city, which is one of the New Wonders of the World.
More information regarding the call for papers and competitions can be found at http://ucsp.edu.pe/lars2013
Easy Links
RAS is a volunteer driven society with over 13,000 members worldwide.
Students are future of robotics and automation.
IEEE International Conference on Automation Science and Engineering
IEEE/RSJ International Conference on Intelligent Robots and Systems
IEEE International conference on Robotics and Automation


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The first ever International cat show will be held here at the land below the wind, Sabah! Witness a world class pedigree cats on a two day show. Yeayy!! Wife is super happy..
Venue : Sabah Trade Centre, Likas, Kota Kinabalu Sabah
Date : 23rd & 24th August 2008
Rings : 4 Rings All Breed
Judges : Rod U'ren (Australia), Becky Orlando (USA), Wayne Trevathan (USA), Diana Rothermel(USA)
Master Clerk : Dick Kallmeyer(USA)
organiser : Mr. Ben Hsu, Borneo Cat Club
En. Ken Wee Kwong Ho (Entry Clerk) at : +6012 6804045
En. Nor Arisham Bakar (Asst. Entry Clerk) at : +6016 278298
email your entry at : email@example.com or you can also fill up the online entry form here
Hurry up! Entry limits will be 255 entries and the closing date is 31 July 2008.


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The best Swiss Replica Watches, all Replica Watches are Swiss Engineering and sold at wholesale price.
Copyright © 2020 Swiss Replica Watches Store. Powered by ok-replicas.org
Watches Categories
Home- Panerai- Submersible- Panerai Luminor Submersible 1950 3 Days Automatic PAM 671 Clone Panerai P.9000 Automatic Bronzo Case with Blue Dial Dot Markers and Brown Leather Strap - 1:1 Origianl(KW)


text: Family Residential Courses FSC family residential holiday courses are based at FSC field centres throughout the UK, picked for their diverse flora and fauna ranging from the majestic Scottish islands to the beautiful South Devon coast. Birthday Parties These exciting Birthday Parties run after school, at weekends and in school holidays (depending on availability and centre). A range of outdoor activities with an indoor space for your party food. Family Day Activities FSC family day activities are designed for unaccompanied children and family groups. A range of fun outdoor activities for children of different ages and interests. Real Family Holidays: Escape To The OutdoorsEscape to the outdoors and enjoy nature with your family, on an affordable family holiday this summer.Discover More