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Our workshop is based around the idea of movement being our medicine, and art as our therapy.
The movement is dance, but it's the kind of dance you do when no one is watching.
A dance that you only know you're doing it right when it feels good.
We call it movement - because it could just be as simple as swaying your hips.
We create art, but this is no art class.
This is the art we created when we were children.
We use our hands and we spread the paint across the paper.
There's no such thing as doing it wrong.
Using the tools of art and creativity, combined with dance and moving our body in a way that feels good, we explore the expression of our wisdom and wild self to find our freedom.
We will journey through movement and dance, and ground into our bodies through the practise of Qoya, led by Melanie.
We will explore the expression of our creativity, and release our inhibitions through the medium of art, guided by Daisy.
Interweaving these two therapeutic practises will help heal and ground us as we journey with our true wild self. We will release and create. We will find the true meaning of what it is to be wise, wild and free.
Together we entwine the relationship between expression of movement and creativity. We dance with our inner wild woman, we face our authenticity in the ink that spills across the paper and we find freedom to run with the colours of our soul beneath our feet as we dance and create art.
I am a London based creative, with an Arts background in design and illustration (BA), and have trained in Reiki Healing in more recent years.
Having hosted drawing workshops at festivals, and for Art students in schools, I have a passion to share the freedom of expression and exploration through art.
Using art as a therapeutic practise enables women to explore their emotions and assist in healing. By creating we release, and by releasing we create.
I am a Modern Day Alchemist and help women to get in touch with their authentic self, find out where their blocks are and work through them so that they can live their best life.
Among my alchemical tools of Goddess Healing, fire medicine and energy work is the tool of Movement Medicine - Qoya.
Qoya is a wellness practise that blends yoga, dance and sensual movement to be used as a portal to remembering.
Photo by Olena Zaskochenko


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Welcome to FindLaw's Cases & Codes, a free source of state and federal court opinions, state laws, and the United States Code. For more information about the legal concepts addressed by these cases and statutes, visit FindLaw's Learn About the Law.
(a) On the request of a petitioner, an advisory committee shall give advice concerning the options for medical care and treatment of an individual with a life-threatening condition.
(b)(1) The advisory committee shall make a good faith effort to notify a patient, a patient's immediate family members, a patient's guardians, and an individual with a power of attorney to make a decision with a medical consequence for a patient, of the individual's right:
(i) To be a petitioner;
(ii) To meet with the advisory committee concerning the options for medical care and treatment; and
(iii) To receive an explanation of the basis of the advisory committee's advice.
(2) Any information or document that indicates the wishes of the patient shall take precedence in the deliberations of the advisory committee.
(c) An advisory committee or a member of an advisory committee who gives advice in good faith may not be held liable in court for the advice given.
(d) A person that assists one or more hospitals or related institutions in the establishment of an advisory committee may not be held liable in court for any advice given in good faith by that person, the related institution, the advisory committee, or any member of the advisory committee and the committee and its members may not be held liable for any advice given in good faith.
(e)(1) The proceedings and deliberations of an advisory committee are confidential as provided in § 1-401 of the Health Occupations Article.
(f) A hospital or related institution may not be held liable in a civil action for failing to carry out the advice of an advisory committee concerning a patient's medical care if the advice given is inconsistent with the written policies of the hospital or related institution.
Cite this article: FindLaw.com - Maryland Code, Health-General § 19-374 - last updated December 31, 2021 | https://codes.findlaw.com/md/health-general/md-code-health-gen-sect-19-374/
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domain: law


The Zionist Organization of America (ZOA) has applauded recent comments by Secretary of State Hillary Clinton that the Obama Administration will not permit Iran to obtain nuclear weapons. In an interview on NBCs Meet the Press program, Secretary Clinton said that What we want to do is to send a message to whoever [in Iran] is making these decisions that if youre pursuing nuclear weapons for the purpose of intimidating, of projecting your power, were not going to let that happen You have a right to pursue the peaceful use of civil, nuclear power You do not have a right to obtain a nuclear weapon. You do not have the right to have the full enrichment and reprocessing cycle under your control. But theres a lot that we can do with Iran if Iran accepts what is the international consensus (David E. Sanger, Clinton Says Nuclear Aim of Iran Is Fruitless, New York Times, July 27 2009).
Secretary Clintons reassurances that the Obama Administration would not permit Iran to obtain a nuclear weapon came following earlier indications from her that the Obama Administration might be willing to acquiesce in Iran obtaining a nuclear weapons capacity. Only last week, Secretary Clinton implied that the U.S. would accept Iran becoming a nuclear power and was planning to meet this challenge by extending a missile shield over the Middle East: We want Iran to calculate what I think is a fair assessment: that if the United States extends a defense umbrella over the region, if we do even more to develop the military capacity of those (allies) in the Gulf, it is unlikely that Iran will be any stronger or safer (Barak Ravid, Israel slams Clinton statement on nuclear Iran, Haaretz, July 22, 2009).
ZOA National President Morton A. Klein said, We applaud Secretary Clintons statement declaring that the Obama Administration will not permit Iran to become a nuclear power because clarity is urgently needed regarding the determination of this Administration to deal with the looming Iranian nuclear threat. Not only were there Secretary Clintons remarks of last week suggesting that an Iranian nuclear weapons capacity was to be accepted, but various statements by senior Administration officials have indicated that the Obama Administration would do less than everything to stop Iran or else condition what it did on Israel making concessions to the unreconstructed, terror-supporting Palestinian Authority.
As we said at the time, Arab states that are genuinely threatened by the looming Iranian nuclear threat would need no inducement to cooperate with America in our efforts to prevent Iran from manufacturing these weapons. As President Obama has said several times, Iran poses a serious security threat to the U.S., Israel and the world. It is therefore a vital American interest to prevent Iran obtaining nuclear weapons. There is no linkage between the Arab/Israeli negotiations and the threat posed by Iran except that a nuclear Iran would increase the dangers of that conflict. In her statement affirming that the Obama Administration will not permit Iran to become a nuclear power, Secretary Clinton has implicitly acknowledged this fact and we applaud her statement.


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On the sunny day of Wednesday 24th October, a group of abolitionists descended on Bishopthorpe Palace in York, not to talk about the legacy of Wilberforce and his colleagues, but instead to ask how the church can act today to prevent and expose modern slavery.
The Clewer Initiative, the Church of England response to modern slavery, are helping churches to respond to modern slavery in their communities, and raise awareness of how this crime has taken root on our high streets. This was their first Northern Province networking event, bringing together representatives from 10 of the 12 northern dioceses.
In her welcome, Bishop Alison White, the Bishop of Hull, thanked the group for giving up their time to be there and spoke about how momentous the occasion could be for all involved. Speaking of the church’s role in ending modern slavery, she said: “our presence has the potential to make a difference”.
The unique position of the church to shed light on this issue was brought up again and again by speakers at the event. Bishop Alastair Redfern, the founder of The Clewer Initiative, spoke about the potential of Christians to “go the extra mile”, and work with the police to find modern slavery by spotting evidence that “only we can notice”. Professor Gary Craig from the University of Newcastle, reflecting on his 20 years of researching and action in the modern slavery sector, said that “the church is uniquely placed to reach people in the smallest and most remote communities”.
The theme of what the church can do was then explored in several break out sessions, one on the vulnerability of homeless people (www.theclewerinitiative.org/homeless) to modern slavery, one on the training packages available to equip churchgoers, and a further session for attendees who were not from the church, designed to give them a roadmap for working in partnership with faith groups.
After a networking lunch, the group was joined by DI John Freer, Modern Slavery Lead for North Yorkshire Police, who shared some of the challenges that his team face when dealing with apparent victims of modern slavery who don’t want to see themselves as ‘victims’. Some may be earning £30 for a 12 hour day in a car wash, and others might be exploited in sex work, being controlled by a pimp, but when visited by police, their mantra will nevertheless be ‘I’m fine’. Bishop Alastair later encouraged people to see the role of the church in giving isolated victims an opportunity to ”participate in the experience of community”.
The final session of the day grouped neighbouring dioceses together, asking how they could be working in partnership with local communities and law enforcement to combat modern slavery in their area. When feeding back, groups highlighted that they wanted their responses to be simple and achievable locally and to centre on storytelling to draw people in, helping people to understand a complex and difficult issue on a human level.
Speaking about the day Revd Gemma Sampson said “when the task is so huge – like trying to end modern day Slavery – it is really easy to feel totally overwhelmed and not know quite what to do. Coming together with fellow ‘freedom fighters’ is inspiring and energizing and leads to a simple, but deeply held, belief that together we might be able to work to end this dreadful crime against humanity. Partnerships and networks are developing and people across statutory, voluntary and faith sectors are uniting to make a significant difference. May this lead to the end of oppression and make a pathway to freedom for some of Gods most vulnerable, precious, children.”
For more information about The Clewer Initiative go to www.theclewerinitiative.org

domain: sociology


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Attorney General demands update in Cummings’ trial – Liberia news The New Dawn Liberia, premier resource for latest news – The New Dawn Liberia Attorney General demands update in Cummings’ trial – Liberia news The New Dawn Liberia, premier resource for latest news.
If convicted on indictment, the person could additionally be imprisoned for five years and fined $25,000.00. Section 56.5 provides that in addition to any time restrict imposed by any enactment, the judge could refuse to grant reduction in any case during which the choose considers that there was unreasonable delay earlier than making the application.
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Colorectal cancer (CRC) organoids can be derived from almost all CRC patients and therefore capture the genetic diversity of this disease. We assembled a panel of CRC organoids carrying either wild-type or mutant RAS, as well as normal organoids and tumor organoids with a CRISPR-introduced oncogenic KRAS mutation. Using this panel, we evaluated RAS pathway inhibitors and drug combinations that are currently in clinical trial for RAS mutant cancers. Presence of mutant RAS correlated strongly with resistance to these targeted therapies. This was observed in tumorigenic as well as in normal organoids. Moreover, dual inhibition of the EGFR-MEK-ERK pathway in RAS mutant organoids induced a transient cell-cycle arrest rather than cell death. In vivo drug response of xenotransplanted RAS mutant organoids confirmed this growth arrest upon pan-HER/MEK combination therapy. Altogether, our studies demonstrate the potential of patient-derived CRC organoid libraries in evaluating inhibitors and drug combinations in a preclinical setting.https://doi.org/10.7554/eLife.18489.001
Recent technical advances mean that miniature replicas of many tissues can be grown in the laboratory. These so-called organoids provide scientists with model systems that are not as limited as simple, two-dimensional sheets of cells growing in a petri dish, and less labor and resource intensive than studies using laboratory animals. In particular, organoids grown from tumor cells from cancer patients have been suggested as having numerous advantages over both laboratory-grown cancer cells and mice when it comes to testing potential new anticancer drugs.
Mutations in a gene called KRAS are common in many types of cancer including colon cancer. Tumors with these mutations are difficult to treat and so far virtually all attempts to generate compounds that selectively interfere with the KRAS protein encoded by the mutant gene have failed. Instead, drugs that indirectly inhibit this protein’s effects by targeting other proteins in the same signaling pathway are currently being tested on patients. However, there is still a need for better ways to pre-test whether these drugs will be effective in humans without having to expose the patient to side effects or an ineffective drug.
Now, Verissimo, Overmeer, Ponsioen et al. have tested clinically-used KRAS pathway inhibitors and drug combinations against normal colon organoids and colon cancer organoids derived from patients with colon cancer. Gene editing techniques were used to introduce KRAS mutations into some of the normal organoids grown from healthy tissue, and into cancer organoids grown from tumors that had a normal copy of the KRAS gene. In all cases, only those organoids with mutant forms of the KRAS gene were resistant to the treatments. Furthermore, when organoids with the KRAS mutation were treated with some combination therapies that are currently being tested in clinical trials, the tumors stopped growing but the tumor cells failed to die. Similar drug treatments on mice carrying human colon cancer organoids confirmed these results, which is in line with previous studies where tumor tissue from human patients was transplanted into mice.
These findings show that collections of tumor organoids from multiple patients could help researchers to quickly identify and optimize targeted anticancer therapies before they are incorporated into clinical trials. In the future, clinical studies are needed to verify how accurately the testing of cancer drugs on organoids predicts whether the drug will or will not work in patients.https://doi.org/10.7554/eLife.18489.002
One of the great challenges in targeted cancer treatment has been the development of effective RAS-targeting drugs. RAS mutations occur in about 15% of all human tumors (Bos, 1989) and so far all attempts to selectively interfere in mutant RAS signaling have failed in the clinic (Stephen et al., 2014; Cox et al., 2014). Progress has long been impeded by the fact that the currently used model systems to pre-test drugs are insufficient: cell lines, on the one hand, have very limited genetic diversity, while mouse models on the other hand, may not represent human tumors (Sachs and Clevers, 2014; Gould et al., 2015). Moreover, until recently, personalized medicine required large-scale in-vitro screening on short-term cultures of tumor sections (Centenera et al., 2013), or alternatively, resource-intensive in-vivo screens using xenotransplantation of tumors into immunodeficient mice (Jin et al., 2010; Tentler et al., 2012). Recently, stem-cell based organoid technology was introduced to establish long-term cultures of both normal and tumor tissues from various organs (Sato et al., 2009, 2011; Bartfeld et al., 2015; Boj et al., 2015; Huch et al., 2015; Karthaus et al., 2014; Gao et al., 2014). The advantage of this technology is that it can capture the genetic diversity of both normal and tumor tissues. Indeed, for colorectal cancer (CRC) a genetically diverse Biobank of patient-derived CRC organoids was established and used to integrate genomic data and monotherapy drug responses at the level of individual patient-derived organoid lines (van de Wetering et al., 2015).
We employed this biobank to further explore potential strategies to target mutant RAS, including the combination therapy of pan-HER and MEK inhibition, which is currently tested in clinical trials. We confirm the strong correlation between the presence of mutant RAS and resistance towards EGFR inhibition. Our data reinforce the notion that an oncogenic mutation in RAS is sufficient to confer this resistance independent of cellular status, whether it concerns normal or tumorigenic cells. Moreover, real-time imaging of the resistant drug response at the cellular level reveals predominant cell-cycle arrest in RAS mutant organoids, in contrast with the complete induction of cell death in CRC organoids with WT RAS. In vivo drug response of xenotransplanted RAS mutant CRC organoids confirmed the arrest in tumor growth upon dual inhibition of the EGFR-MEK-ERK pathway. Finally, efficient inhibition by dual targeting of the mutant RAS pathway strongly sensitizes for the induction of cell death, as illustrated by minimal addition of BCL inhibition. Our studies demonstrate the strong potential of patient-derived CRC organoid libraries in evaluating inhibitors and drug combinations in a preclinical setting.
To explore drug responses of patient-derived CRC organoids towards combination therapies of targeted inhibitors of the EGFR-RAS-ERK pathway, we applied a drug sensitivity screen using EGFR-family and MEK inhibitors (EGFRi and MEKi resp.) either as mono or combination therapy on two cancer organoids from a previously established biobank of CRC organoids (van de Wetering et al., 2015). To start, we chose cancer organoids from the individuals P8 and P26, which share a similar composition of frequent cancer mutations such as functionally inactive APC and TP53. However, they differ in their KRAS status. P8T contains wild-type (WT) KRAS, while P26T contains an oncogenic mutant version of KRAS (G12V).
3D-organoids were challenged with drugs for 72 hr and drug responses were determined by quantifying cell viability through measurements of ATP levels using CellTiter-Glo (van de Wetering et al., 2015). We observed the expected sensitivity of P8T towards afatinib (irreversible EGFR/HER2 inhibitor) and insensitivity of KRAS mutant P26T (Figure 1A). Selumetinib (MEKi) as a monotherapy showed little efficacy in both P8T and P26T, but combination therapy confirmed previous findings that MEKi sensitizes RAS mutant tumor cells to EGFR/HER2 inhibition (Figure 1A) (Sun et al., 2014). However, the KRAS mutant P26T organoids were less sensitive to the combination therapy than the KRAS WT P8T organoids.
To monitor drug response on a cellular level, we stably introduced DNA constructs encoding fluorescently-labeled H2B and performed real-time confocal imaging on the 3D-organoids for 72 hr in the presence and absence of drugs. We performed EGFR-RAS-ERK pathway inhibition with relatively high concentrations of afatinib (1 µM) in combination with selumetinib (1 µM). In P26T (mutant KRAS) we only observed cell cycle arrest with very limited cell death induction. This was in stark contrast with the very rapid induction of cell death in P8T (WT KRAS) (Figure 1B, Video 1). When we repeated these imaging experiments using much lower drug concentrations, we noticed a general shift to resistance for both organoid lines. Under these conditions, also P8T predominantly showed cell cycle arrest rather than cell death, and the cancer cells in P26T organoids even continued to proliferate (Figure 1—figure supplement 1, Video 2). Taken together, our data indicate that 72 hr of combination treatment with afatinib and selumetinib (EGFRi/HER2i and MEKi) effectively kills KRAS WT P8T organoids, while the mutant KRAS P26T organoids are significantly less sensitive.
In order to validate the observed drug response of in vitro cultured organoids in an in vivo model, we xenotransplanted P18T and P26T tumor organoids in immunodeficient mice. In line with a previous report where only engineered tumor progression organoids with increasing number of cancer mutations (APC, KRAS, P53 and/or SMAD4) showed efficient engraftment (Drost et al., 2015), we only obtained reliable engraftment using P26T CRC organoids. We initially started using concentration schedules of afatinib and selumetinib that had previously been reported (Sun et al., 2014), but we observed no significant effect of the drug combination on tumor growth over time (Figure 2A). To exclude that the tumors had acquired resistance during the in vivo drug treatment, we isolated the tumors to re-establish secondary organoids and subjected these to identical drug tests. Dose-response curves on these secondary organoids were identical to the parental organoid line P26T, independent of the type of drug treatment that the tumors underwent in the mice (Figure 2—figure supplement 1). Indeed, in agreement with lower drug concentrations that proved to be ineffective in blocking proliferation in vitro (Figure 1—figure supplement 1, Video 1), we speculate that the in vivo drug concentrations were insufficient to effectively block the EGFR-MEK-ERK pathway. To confirm this hypothesis, we further increased the drug levels to high but tolerable doses. This indeed induced significant growth stabilization (but no regression) of P26T xenotransplanted tumor in mice (Figure 2B), in agreement with loss of proliferative activity as was also detected in vitro (Figure 1B). The fact that in vivo xenografted CRC organoids yields similar drug responses as in vitro organoid cultures and identical to previous reported drug response of KRAS mutant PDX models of CRC (Sun et al., 2014), validates the testing and evaluation of targeted inhibitors in CRC organoids.
P8T and P26T CRCs are microsatellite-stable (MSS) and belong to the same molecular subtype classification based on RNA expression data (TA, also referred to as canonical CMS2 according to consensus classification) (van de Wetering et al., 2015; Guinney et al., 2015). Genomic characterization of these patient-derived CRC organoids in comparison to their matched normal tissue revealed many additional mutations within the protein coding sequence of the genome (van de Wetering et al., 2015). For P8T and P26T, 230 and 163 of such cancer specific mutations were detected respectively (van de Wetering et al., 2015). To exclude potential contributions of all these additional mutations to the effect that oncogenic KRAS imposes on drug responses, we introduced an oncogenic KRAS mutation in patient-derived CRC organoid P18T via CRISPR/Cas9-mediated homologous recombination (Drost et al., 2015). Like P8T, original P18T is WT for the entire downstream EGFR signaling pathway. P18T-KRASG12D mutant cells were generated as reported previously for normal colon organoids (Drost et al., 2015) and genotyping of clonally expanded organoids confirmed that the clones contained the KRASG12D mutation (Figure 3A), as well as a Cas9-mediated inactivation of the second allele by introducing an 86 bp deletion. Upon addition of oncogenic KRAS, no overall differences in morphology or growth rates were observed during normal culture conditions.
To investigate the exclusive effect of oncogenic KRAS on a combination therapy that targets the EGFR-RAS-ERK pathway, we performed a full matrix screen of 14 drug concentrations over a 5 nM to 5 µM range of both the targeted inhibitors afatinib (EGFR/HER2i) and selumetinib (MEKi) (Figure 3B). Notably, their combined administration is currently used in a clinical trial for patients with RAS mutant CRCs (NCT02450656). While the original P18T demonstrated high sensitivity to EGFR/HER2 inhibition by monotherapy, a single introduced oncogenic point mutation in KRAS provided resistance to EGFR/HER2 inhibition. Moreover, we analyzed combination effects using the Bliss independence model. Positive Bliss scores indicate combinatorial effects that exceed additive effects. The heat map of Bliss scores for P18T and P18T-KRAS shows that a large range of concentrations for both compounds show positive scores, but that presence of oncogenic KRAS renders the loss of viability and positive Bliss range towards higher drug concentrations indicating resistance (Figure 3—figure supplement 1).
Next, we again studied the cellular drug response by real-time imaging. Reminiscent of the patient-derived CRC organoid with an endogenous RAS mutation (P26T), we noticed that the introduction of oncogenic KRAS renders a CRC organoid less sensitive to the afatinib/selumetinib combination therapy (Figure 3C, Video 1). More specifically, quantifications of all mitotic and apoptotic events during the filmed drug response revealed both loss of proliferation and apoptosis induction in P18T, while P18T-KRASG12D only showed reduced proliferation but unchanged apoptosis rates (Figure 3D and Figure 3—figure supplement 2).
Despite the phenotypic difference in drug response, pERK levels in both tumor organoids were severely reduced (Figure 3—figure supplement 3). Since suboptimal suppression of ERK activity might permit tumor growth in BRAF mutant cancers (Bollag et al., 2010; Corcoran et al., 2015), we determined the cellular effects of drug response when lowering drug concentrations. Since significant differential effects were observed between P18T and P18T-KRASG12D during the matrix screen around 33 nM afatinib + 200 nM selumetinib (Figure 3B), we repeated real-time imaging of drug response using these lower drug concentrations. As with P8T and P26T, we noticed a general shift from sensitivity towards resistance when drug concentrations were reduced. More specifically, the RAS WT cancer organoids showed cell cycle arrest rather than cell death, while the RAS mutant organoids appeared unaffected and sustained proliferation (Figure 3—figure supplement 4, Video 2).
Considering the isogenic CRC organoids P18T and P18T-KRASG12D as our gold standard to reveal the specific effects of KRASG12D on drug responses, we expanded our focus at targeting the linear EGFR-RAS-ERK pathway with the ultimate aim to find a targeted therapy that is specifically effective against RAS mutant CRCs. Multiple targeted inhibitors against identical targets were used to exclude artifacts and to increase the mechanistic significance behind the rationale of potential therapies (Figure 4A; and Figure 4—source data 1 and Supplementary file 1 for all dose-response curves) of which few combination therapies are in clinical trial (Figure 4B).
First, we noticed a much lower sensitivity of P18T-KRASG12D for pan-HER inhibitors afatinib, lapatinib and dacomitinib in contrast to the parental P18T (much lower IC50, Figure 3—figure supplement 1). Second, within P18T hardly any additive sensitivity could be observed when EGFRi was complemented with MEK or ERK inhibition (Figure 4—figure supplement 1). In contrast, dual-targeting strategies strongly enhanced efficacies in P18T-KRASG12D regardless which specific inhibitor combination was used (Figure 4—figure supplement 1). Nevertheless, all tested combinations that included EGFR inhibition revealed stronger negative effect on cellular viability in P18T than in P18T with mutant KRAS (positive ΔIC50’s, Figure 4—figure supplement 1). In contrast, most mono- and combination therapies against MEK and/or ERK that excluded EGFRi showed on average similar efficacies in P18T-KRASG12D as in P18T (Figure 4—figure supplement 1).
In parallel, we tested dual-targeting strategies involving PI3K-AKT and EGFR-RAS-ERK pathways considering the interconnectivity between these pathways (Figure 4—figure supplement 2A). Like MEK or ERK inhibition, we observed that pharmacological inhibition of PI3K or AKT in combination with anti-EGFR therapy did not enhance efficacy in a KRAS mutant background (Figure 4—figure supplement 2B). In line with this, clinical studies focusing on combining MEK inhibitors with PI3K, AKT or mTOR inhibitors in KRAS mutant CRCs did not yield satisfactory results (Shimizu et al., 2012).
Next, we aimed to further establish whether the effects of oncogenic KRAS on drug response is dependent on a tumorigenic background or could manifest independent of cellular state. We therefore used normal colon organoids and a derivative of that line in which the oncogenic KRASG12D mutation was introduced via similar CRISPR/Cas9-mediated genome-editing strategy as in P18T (Drost et al., 2015). In analogy with mouse studies (Snippert et al., 2014), we observed no morphological alteration nor induction of senescence upon introduction of oncogenic KRAS (Figure 5—figure supplement 1). Strikingly, drug response profiles of normal organoids to targeted inhibitors against the EGFR-RAS-ERK pathway (Figure 5 and Figure 5—figure supplement 2) revealed a similar trend as in CRC organoid P18T (Figure 4 and Figure 4—figure supplement 1). Thus, the effect that oncogenic KRAS imposes on drug response appears independent of cellular status and the presence of additional cancer mutations.
Next, we aimed to extend our analyses towards a wider collection of CRC organoids that is more representative for the clinic. We screened 10 additional patient-derived CRC organoids for combinatorial therapies against the EGFR-RAS-ERK signaling pathway. Since all the organoid lines are fully characterized in terms of genome information, we could select CRC organoids with and without a mutant RAS pathway. Based on EGFR/HER2 dual inhibition by afatinib, we could clearly discriminate the organoid panel in two types of responders, namely the sensitive versus the resistant ones (Figure 6A, green versus red lines respectively). Indeed, drug sensitivity towards all tested EGFR inhibitors clearly correlated with the mutational status of KRAS. However, there were two notable exceptions (Figure 6A and Figure 6—figure supplement 1). The first was P25T, which, although WT for KRAS, turned out to contain an oncogenic mutation in NRAS (Q61H), thereby fully explaining the resistant behavior. The second exception was organoid line P19bT that, unlike the other CRC organoids in our panel, is characterized as microsatellite instable (MSI) including the hyper-mutator phenotype (van de Wetering et al., 2015). Most importantly, P19bT tumor contains a BRAF (V600E) mutation, providing resistance towards the targeted drugs (Di Nicolantonio et al., 2008). Thus, these two cases underscore that drug screening on human organoids is able to evaluate the functionality of entire oncogenic pathways beyond the scope of the most frequent candidate mutations.
Next, we aimed to evaluate the effects of drug combinations on CRCs organoids with WT and mutant RAS pathways, as well as on non-tumorigenic normal organoids. In all seven independent CRC patients with a mutant RAS pathway, we observed synergistic activity when combining EGFRi and MEKi (Sun et al., 2014) or EGFRi and ERKi (Figure 6B). Nevertheless, RAS WT organoids, either of CRC or normal origin, reveal higher sensitivities to combination therapies that include EGFR inhibition than RAS mutant CRCs. In contrast, therapies that do not include EGFR inhibition, but target MEK (selumetinib) and ERK (SCH772984), were similarly effective in all organoid lines, regardless of the mutational status of the RAS pathway or cellular state (Figure 6B and Figure 6—figure supplement 1).
Another important observation after comparative analysis on this wide CRC panel concerns patient-to-patient variability in the response to anti-EGFR monotherapy, even within the RAS WT and mutant subgroups (Figure 6B, upper left). Combining EGFRi with either MEK or ERKi results in a more consistent inhibitory effect over this set of CRCs (Figure 6B, lower panels), thereby not only improving individual responses but also augmenting success rates on a population scale. These findings are supportive to the previously published concept that proposes to combine EGFR with MEK inhibition directly at the start of therapy in patients with WT RAS tumors with the rationale of preventing sub-clones with acquired resistance to anti-EGFR monotherapy from reigniting tumor growth (Misale et al., 2015).
Besides the direct effects of therapeutic treatments on tumor mass, the ability of cancer cells to recover from the treatments and restart tumor growth is of utmost relevance. We therefore studied the recovery of CRC organoids after release from (i.e. washout of) targeted inhibitors. More precisely, we monitored cellular viability, proliferation and cell death induction by quantifying viable nuclei (marked green) and dead nuclei (marked red) in 3D confocal tiled-scans at multiple time-points before and after treatment of targeted inhibitors (Figure 7A and Materials and methods).
In P18T, MEK or ERK inhibition (selumetinib or SCH772984 resp.) did not induce significant cell death after three days of monotherapy. Afatinib (EGFR/HER2i), either alone or in combination with selumetinib (MEKi), induced significant degrees of cell death, in line with our 72 hr monitoring of drug response. Similar effects were obtained by combined inhibition of MEK and ERK (Figure 7B, left panel). Additionally, this experiment shows that the described effects persisted for seven days after drug washout. As expected for P18T-KRASG12D, suppressing EGFR/HER2 activity upstream of mutant KRAS using afatinib proved ineffective, while monotherapy of MEKi or ERKi inhibited proliferation only to a minor extent (Figure 7B, right panel). Only the inhibitor combinations EGFRi/MEKi and MEKi/ERKi induced complete proliferative stagnation, albeit with minor induction of cell death. Importantly, independent of a therapeutic strategy, the CRC organoids quickly restored proliferative activity after drug release. Comparable results were obtained in CRC organoids P8T (KRAS WT) and KRAS mutant P26T, with the exception that cell death induction in P8T was less pronounced than in P18T (Figure 7C). In summary, these data indicate that inhibition of the EGFR-RAS-ERK pathway, independent of inhibitor combination, predominantly inhibits cell-cycle progression in KRAS mutant CRC organoids.
To characterize the induced cell cycle arrest in RAS mutant tumor cells in more detail, we performed cell cycle analysis by flow cytometry using a 3 hr EdU pulse in combination with DNA staining to discriminate between the different cell cycle phases (G1, S and G2 respectively) in P18T-KRASG12D and P26T. Indicative of a G1 arrest, we observed a sharp decline in the amount of RAS mutant tumor cells in S-phase using inhibitor combinations EGFRi/MEKi and MEKi/ERKi, while a similar fraction of cells accumulated in G1 (Figure 8A).
To further characterize drug-induced arrest, we investigated whether the regrowth after drugs washout involves all therapy-surviving tumor cells or only a minor subpopulation that fuels tumor relapse. For this, we performed two functional assays.
First, we performed EdU incorporations at various time points during the drug response and during organoid recovery after drug withdrawal. In agreement with the previous cell cycle analysis, almost no EdU incorporation was detected in the presence of inhibitor combinations EGFRi/MEKi and MEKi/ERKi (no cells in S-phase) (Figure 8B). However, during the first three days after drug withdrawal, the vast majority of growth-arrested tumor cells incorporated EdU again, suggesting renewed proliferative activity in virtually all tumor cells, thereby excluding the presence of senescence or minor subpopulations being responsible for restored growth (Figure 8C).
In addition, we performed live-cell imaging on tumor organoids after drug withdrawal and quantified the number of mitotic and apoptotic events over time (see Materials and methods). Indeed, in line with a G1 arrest, we observed a delay of about 20–24 hr after withdrawal of the drugs (EGFRi/MEKi) before observing numerous mitotic events again in all regions of the arrested organoids (Figure 8D and Figure 8—figure supplement 1 and Video 3). (Similar results were obtained for MEKi/ERKi, data not shown).
As described above, none of the therapies targeting the EGFR-RAS-ERK pathway could induce the desired degree of cell death in RAS mutant CRC organoids. As opposed to this, it has been reported that combined inhibition of anti-apoptotic BCL2 family members and effectors of the RAS pathway can effectively induce cell death in KRAS mutant cancers (Corcoran et al., 2013; Hata et al., 2014, 2015; Tan et al., 2013). These results prompted clinical trials to evaluate combined targeting of MEK and BCL2/BCLXL in KRAS mutant solid tumors (NCT02079740). Unfortunately, clinical application of BCL2/BCLXL inhibitors is hindered by on-target toxicity of BCLXL inhibition in blood platelets (Hata et al., 2015) and might therefore strongly benefit from strategies that allow minimized doses.
Here, we explored the use of navitoclax, a clinically tested BCL2/BCLXL inhibitor, in targeted therapies against RAS mutant CRC organoids. Indeed, straightforward ATP-based screening confirmed that navitoclax, when combined with afatinib (Figure 9A, left panel), selumetinib (Figure 9A, middle panel), or both (Figure 9A, right panel) is far more efficient in antagonizing tumor organoid growth than any of the related monotherapies. Importantly, regarding the dose-limiting effects of navitoclax, we show that robust dual inhibition of the EGFR-RAS-ERK pathway (1 µM afatinib/1 µM selumetinib) is exceptionally potent in sensitizing navitoclax-induced effects (Figure 9A, right panel). Such strong sensitization could not be achieved by afatinib (1 µM) alone or selumetinib (1 µM) alone. Similar results were obtained in other patient-derived CRC organoids that harbor a KRAS mutation (Figure 9—figure supplement 1) or, alternatively, with different combinations of inhibitors targeting the EGFR-RAS-ERK pathway (Figure 9—figure supplement 2).
To ensure that the observed effects represent cell death rather than cell-cycle arrest, we performed qualitative and quantitative microscopic analyses (Figure 9B). Indeed, cell death could be induced by low concentrations of navitoclax when combined with high concentrations of afatinib and selumetinib.
Next, we designed a medium-throughput assay to monitor the persistence of drug response after wash-out of the above-mentioned inhibitor combinations (Figure 9C). As expected, even dual inhibition with afatinib (EGFR/HER2i) and selumetinib (MEKi) at high concentrations does not induce sufficient cell death, since these cultures can recover to the levels of untreated controls within six days (Figure 9C). In contrast, the addition of low concentrations navitoclax to high concentrations of inhibitor combination afatinib/selumetinib potently induces cell death, as shown by the sustained inhibitory effects on culture growth (Figure 9C, black bars).
In light of the dose-limiting toxicity of navitoclax in blood platelets, we performed full matrix-screens to explore optimal combinations of drug concentrations (Figure 9D). In agreement with previous results, the more efficient inhibition of the RAS pathway, i.e. high concentrations and/or dual targeting (Figure 9—figure supplement 3) the lower the concentration of navitoclax that is necessary to affect cellular viability. Furthermore, venetoclax, a BCL2-specific inhibitor, is unable to reproduce the effects of navitoclax (BCL2/BCLXLi) (Figure 9D), suggesting that in agreement with the reported findings in lung cancer (Corcoran et al., 2013), it is BCLXL that protects against apoptosis upon targeted inhibition of a mutant RAS pathway in CRC organoids (Figure 9D).
Patient-derived CRC organoids were recently introduced as a model system in cancer research that is complementary to cell lines and PDX models (van de Wetering et al., 2015). We assembled a panel of normal and CRC organoids with either WT or mutant RAS that had been derived from different patients (van de Wetering et al., 2015). Moreover, we included normal (Drost et al., 2015) and tumor organoids in which the oncogenic G12D mutation was introduced in the endogenous KRAS locus by CRISPR-Cas9-induced homologous recombination. These engineered organoid lines, in combination with patient-derived CRC organoids of different genetic backgrounds, allowed us to study the effect of mutant KRAS on drug response to targeted inhibition of the EGFR-RAS-ERK pathway. Moreover, real-time imaging allows the monitoring of exact cellular fates in drug-challenged CRC organoids with spatial (3D) and temporal resolution.
Using this panel, we show that the presence of mutant RAS is sufficient to confer resistance to EGFR inhibitors. Moreover, we confirmed the synergistic effect of the clinically tested combination of pan-HER and MEK inhibition on mutant RAS organoids. However, we found that RAS mutant organoids remained largely resistant to apoptosis and became largely arrested in proliferation. Importantly, for KRAS mutant tumor organoid P26T we observed similar drug sensitivities in vitro as in vivo upon xenotransplantation. Moreover, similar growth arrested responses were reported in PDX mouse models of KRAS mutant CRC cancers ( Sun et al., 2014), underscoring the notion that tumor organoids are a reliable model system to test drug responses.
We report, for the first time, how normal tissue organoids respond to inhibitors targeting the EGFR-RAS-ERK pathway. Intriguingly, drug effects were almost identical in normal organoids and patient-derived CRC organoids when WT for RAS. This may imply that the sensitivity of RAS WT colon cancer for EGFRi is not an acquired oncogene-addiction, but merely represents the dependency of normal colon (stem) cells on EGFR signaling activity (Wong et al., 2012). Indeed, normal organoids from the human colon consist predominantly of proliferative stem and progenitor cells due to the WNT ligands in the culture medium. Therefore, the observed toxicity in the normal organoids may very well be most representative for direct effects on the stem cell compartments of the normal colon. Indeed, one of the direct side effects of anti-EGFR monotherapy is diarrhea (Miroddi et al., 2015). In analogy to drug responses with WT RAS, normal organoids harboring a CRISPR-introduced oncogenic KRAS mutation showed resistance profiles towards targeted therapies that closely resemble those of RAS mutant CRC organoids, again underscoring the dominance of the RAS mutational status on drug response.
Anti-EGFR therapy in patients with KRAS WT colon tumors is standard of care, whereas patients with RAS mutant tumors are excluded. Our results confirm the drug sensitivity profile of colorectal cancers with and without mutant RAS, as has been established both in other model systems as well as in the clinic (Sun et al., 2014; Karapetis et al., 2008; Amado et al., 2008). For RAS mutant tumors, a number of different drugs and drug combinations have been tested, but thus far this has been without significant effect (Ryan et al., 2015). Our analyses confirm that drug treatments targeting the EGFR-RAS-ERK and the PI-3K/AKT cascades, including combinations thereof, are largely ineffective in RAS mutant CRC organoids.
In contrast, for CRC patients with RAS WT tumors combination therapies that target the EGFR-MEK-ERK pathway may be an improved alternative over anti-EGFR monotherapy. First, we observed that this combination treatment induced cell death more systematically over a panel of individual patient-derived CRC organoids with WT RAS (i.e. with decreased variability) than monotherapy with a pan-HER inhibitor. Furthermore, combination treatments may decrease the potency of low-abundant RAS mutant subclones to initiate tumor-relapse during therapy against a predominantly KRAS WT tumor (Misale et al., 2015).
The combined inhibition of pan-HER and MEK is currently tested in patients with RAS mutant cancers in several clinical trials (e.g. NCT02450656, NCT02230553 and NCT02039336). Also in our hands, this combination showed a clear synergistic effect in suppressing growth of RAS mutant CRC organoids, as determined by a straightforward ATP-based assay. Importantly however, our data revealed that this inhibitor combination induced a cell cycle arrest in mutant RAS organoids but no cell death. As a result, the cells rapidly restored proliferative activity after withdrawal of these drugs. The inability to induce cell death likely affects the long-term effectiveness of this combination in CRC patients with mutant RAS.
An alternative combination that is currently in clinical trials is the combined inhibition of MEK and ERK (NCT02457793). The rationale behind this combination is the notion that resistance to targeted inhibition of RAF and MEK often involves reactivation of ERK (Ryan et al., 2015), while suboptimal suppression of ERK activity in RAF mutant cancers may underlie the limited efficacy (Bollag et al., 2010; Corcoran et al., 2015). Although our RAS mutant CRC organoids showed sensitivity to dual inhibition of MEK and ERK, also this drug combination induced cell-cycle arrest rather than cell death, questioning whether it will be sufficient for the treatment of RAS mutant CRC.
With respect to clinical applications, we here report that effective inhibition of the EGFR-MEK-ERK pathway through combinatorial targeting does significantly prime the cytostatic RAS mutant cancer cells for apoptosis. This can be utilized by low concentrations of navitoclax, one of the most clinically advanced inhibitors of anti-apoptotic BCL family members. Minimizing navitoclax concentrations would be beneficial due to its on-target effects on BCLXL in circulating platelets, thereby causing thrombocytopenia (Hata et al., 2015). However, triple combination therapy with low concentrations of navitoclax (50 mg/kg; five days on, two days off) proved to be too toxic for the mice (data not shown). Nevertheless, we consider the navitoclax-induced apoptosis as a proof-of-principle that EGFR-MEK-ERK pathway inhibition in combination with alternative signaling nodes holds great promise in identifying therapeutic drug combinations that kill RAS mutant tumor cells while being tolerated by the patient.
In summary, we show drug responses of a wide panel of patient-derived CRC organoids to multiple clinically advanced targeted inhibitors, either alone or in combinations, against the EGFR-RAS-ERK pathway. Importantly, the drug phenotypes that we observe in the organoids appear representative for previous reported responses in vivo. We believe that organoid collections will facilitate the identification and optimization of effective targeted therapies, since drug screens can be performed at a scale that is currently unprecedented when using resource-intensive PDX models. Due to the reliability and scalability of tumor organoids as a model system, we advocate that novel drugs should be tested on a panel of tumor organoids before their use in clinical trials.
The patient-derived organoids used in this study were previously established and characterized (van de Wetering et al., 2015). Human CRC and healthy colon organoids were cultured as described previously (van de Wetering et al., 2015). In short, organoids were cultured in drops of Basement Membrane Extract (BME; Amsbio) and medium was refreshed every two days. The CRC culture medium contained advanced DMEM/F12 (Invitrogen) with 1% Penicillin/Streptomycin (P/S, Lonza), 1% Hepes buffer (Invitrogen) and 1% Glutamax (Invitrogen), 20% R-spondin conditioned medium, 10% Noggin conditioned medium, 1X B27 (Invitrogen), 1.25 mM n-Acetyl Cysteine (Sigma-Aldrich), 10 mM Nicotinamide (Sigma-Aldrich), 50 ng/ml EGF (Invitrogen), 500 nM A83-01 (Tocris), 10 μM SB202190 (ApexBio) and 100 µg/ml Primocin (Invitrogen). The medium of healthy colon organoids had additional Wnt conditioned media. Organoids were splitted through shear stress (pipetting) and/or Trypsin-EDTA (Sigma-Aldrich) treatment. Culture medium after splitting was supplemented with 10 µM Y-27632 dihydrochloride. Organoid cultures have repeatedly been tested negative for Mycoplasma. Western blots are performed as described previously (Drost et al., 2015). Antibodies used: ERK (RRID:AB_390779), pERK (RRID:AB_331646) and GAPDH (RRID:AB_2107445).
CRISPR guide RNAs (sgRNAs) were generated as described by Drost et al. (2015). The KRAS target sequences used were: 5′-GAATATAAACTTGTGGTAGTTGG-3′ and 5′-GTAGTTGGAGCTGGTGGCGTAGG-3′. Transfections of p18T and p26N organoids with sgRNAs and subsequent selections by withdrawing EGF and adding the EGFR inhibitor gefitinib to the culture medium were performed as previously described (Drost et al., 2015). The presence of KRAS G12D mutation was verified by sequencing the PCR product obtained using the primers KRAS_for, 5′-TGGACCCTGACATACTCCCA-3′ and KRAS_rev, 5′-AAGCGTCGATGGAGGAGTTT-3′ (Drost et al., 2015).
Organoids were infected with lentivirus encoding histone2B fused to mNeonGreen (bright monomeric GFP variant) linked to a puromycin-resistance gene (pLV-H2B-mNeonGreen-ires-Puro) (Shaner et al., 2013) to visualize and track nuclei. Infected organoids were selected using 2 µg/ml puromycin.
Five days after organoid typsinization, 1 mg/ml dispase II (Invitrogen) was added to the medium of the organoids and these were incubated for 15 min at 37°C to digest the BME. Subsequently, organoids were mechanically dissociated by pipetting, filtrated using a 40 μm nylon cell strainer (Falcon), resuspended in 2% BME/growth medium (15–20,000 organoids/ml) prior plating of 30 µl (MultidropTM Combi Reagent Dispenser) on BME pre-coated 384-well plates. The drugs and their combinations were added 3 hr after plating the organoids using the Tecan D300e Digital Dispenser. Drugs were dispensed in a randomized manner and DMSO end concentration was 0.4% in all wells. 72 hr after adding the drugs, ATP levels were measured using the Cell-Titer Glo2.0 (Promega BV) according to the manufacturer's instructions. Results were normalized to vehicle (DMSO = 100%) and baseline levels (multi drug ATP plateau at high concentrations = 0%) that were manually determined per organoid type and screening day. Multiple identical drug combinations were averaged. Heatmaps were smoothened using a moving average. Bliss scores were calculated as described previously (Tan et al., 2013).
Afatinib, Dacomitinib, Lapatinib, Selumetinib, Trametinib, BYL719, MK2206 and GDC-0994, navitoclax and venetoclax were purchased from Selleck Chemicals. SCH772984 was obtained from MedChem Express and Cobimetinib from Active Biochem. These compounds were dissolved in dimethylsulfoxide (DMSO, Sigma-Aldrich) and stored as 10 mM aliquots.
Data analyses were performed using GraphPad software by applying the nonlinear regression (curve fit) and the equation log(inhibitor) vs. normalized response (variable slope).
Five days after trypsinization, H2B-mNeonGreen-expressing organoids were plated in matrigel in glass-bottom 96-well plates and mounted on an inverted confocal laser scanning microscope (Leica SP8X) under controlled conditions (37°C, 6% CO2). Drugs were added to the organoids just prior to imaging. For 72 hr, organoids were imaged every 15 min in XYZT-mode using a 40x objective (1.1NA) and a 506 nm laser excitation light from a tunable white light laser for 72 hr. The images were converted using ImageJ/Fiji software into manageable and maximally informative videos, combining z-projection, depth color-coding and merging with transmitted light images (see source code files, ‘Organoid Movie Macro’).
Five days after trypsinization, H2B-mNeon-expressing organoids were plated in glass-bottom 96-well plates. Prior to drug addition (day −3), the organoids were imaged on a Leica SP8X. One 3D tile scan (merging 3×3 images, ~175 µm in Z in total, 5 μm Z-step) was acquired per well, allowing the visualization of 10–20 organoids per well. On day 0, 3 and 7, exactly the same fields of organoids were imaged again and medium was refreshed. A custom-made ImageJ/Fiji macro (see source code files, ‘Macro Drug&Release’) was developed to analyze 12–15 organoids per well/condition in a paired manner, i.e. individual organoids were tracked over the entire experiment (>10 days). Per organoid, a pseudo-quantitative measure for absolute numbers of living and dead cells was established as follows: (1) Thresholding on H2B-Neon fluorescence to select H2B-positive pixels (total). (2) Marking of ‘dead’ pixels in each slice (initially automated, based on particle size and eventually manually by selection). (3) Dividing pixels in dead and alive (total minus dead). (4) Integrating the pixel areas representing alive and dead H2B, respectively, from the slices that make up for the lowest 50% of the organoid volume. This was done to avoid analysis on the upper 50% of the volume, which is inevitably of lower image quality.
The analysis was performed in 12 to 15 organoids per well/condition. No biological replicates.
Of note, this method is independent of non-permeable DNA dyes such as PI to avoid their potential long-term effect on organoid growth. In order to validate the current method, a single time point data set was acquired with the use of PI (see Figure 7—figure supplement 1), validating the robustness of the strategy.
For the analysis of organoid recovery after drug withdrawal, organoids were treated with the indicated drugs for 72 hr as described. Subsequently, drugs were washed out through aspiration of the drug-containing medium, followed by a washing step (10’ incubation at 37°C with basal DMEM/F12 (+++)). After washing, organoids were incubated with CRC-medium containing 2%BME for recovery. After 48 hr, the medium was replaced by CRC-2%BME combined with 10% AlamarBlue (AB) cell viability reagent (ThermoFisher Scientific) according to the manufacturer's instructions. The increase of AB fluorescence (excitation 544 nm, emission 590 nm) was monitored over a time course of 2 hr (with measurements at 15’ intervals) at 37°C, using a SpectraMax M5 microplate reader (Molecular Devices). Fluorescence kinetics were plotted over time (as relative fluorescent units (RFU) per hour) to define the linear range of the assay. Cell viability was then defined as the maximum AB fluorescence (RFUmax, within the linear range of the assay), corrected for background fluorescence (RFU at time point 0). Viability data from drug treated organoids were normalized to vehicle (DMSO) treated controls. Upon the AB time course, organoids were washed two times with basal DMEM/F12 and incubated with CRC-2%BME for another 96 hr, after which the AB assay was repeated.
H2B-mNeon-expressing organoids were plated in 384-well plates and provided with drugs as described for the viability assay. To selectively stain dead cells, propidium iodide (PI) was added 2 hr before starting imaging (2 µM). 3D stacks of 150 μm (7,5 μm per plane) were acquired on a Leica SP8 scanning confocal microscope, using a 10x dry lens for large field-of-view (3000×3000 pixels). Being an endpoint assay, high laser intensities could be applied for optimal imaging quality (and hence analysis). Green (mNeon) and red (PI) signals were sequentially acquired to avoid spectral mixing. Signals were pooled for 15–20 organoids before ratio calculation, hence no error bars.
Custom-made software (ImageJ/Fiji, see source code files, 'Macro PI versus H2B') was designed to determine surfaces (i.e. numbers of pixels) representing H2B-mNeon and PI, respectively; the ratio of these surfaces (PI/H2B) is the quantitative measure for cell death in the drug-challenged organoids. Central to the unambiguous determination of these surfaces is setting of the threshold. Our algorithm initially measures surfaces and corresponding mean intensities with ramping threshold, and from these data mathematically derives the threshold-optimum by combining the highest mean signal and most confined surface area.
Depth-coded projection movies were analyzed for life and death in time: mitotic and apoptotic events were marked with help of custom-made ImageJ/Fiji macro (see source code files, ‘ScoreEvents’). Indicated events were automatically drawn in the movie (essential when aiming to mark all events) and data were automatically sorted into Excel-files containing a (chronologically sorted) list of events.
3 hr prior trypsinization (TriplE, 5 min at 37C) to a single cell, organoids were incubated with 500 nM EdU. Single cells were fixed with ethanol (5%). EdU click-it reaction was performed according to manufacturer's protocol. DNA was stained using 1 µg/ml DAPI. Cells were analysed using a FACSCanto II (BD).
Approval for this study was obtained by the local animal experimental committee at The Netherlands Cancer Institute (DEC-NKI; OZP = 12012 and WP5727 and WP5689). P26T patient-derived organoids were trypsinized and 200,000 cells were resuspended in 50 µl medium/Matrigel (BD Biosciences) mixture at a 1:1 ratio and injected subcutaneously into NSG mice (JAX stock no: 005557). Mice with established tumors (average volume of 300 mm3) were treated with afatinib (12.5 mg/kg; five days on, two days off), selumetinib (20 mg/kg; same schedule) or with a combination of both drugs for four weeks. After three weeks recovery from the drug treatment, mice were sacrificed.
For the second in vivo experiment, P26T organoids (~300.000 cells) were resuspended in 50% matrigel/medium with 10% collagen type I (BD Biosciences) and injected subcutaneously into NSG mice (JAX stock no: 005557). Mice with established tumors (average volume of 200 mm3) were treated with afatinib (20 mg/kg; five days on, two days off), selumetinib (25 mg/kg; same schedule) or with a combination of both drugs for four weeks.
Tumor volumes were evaluated three times per week by caliper and the approximate volume of the mass was calculated using the formula Dxd2/2, where D is the major tumor axis and d is the minor tumor axis. For in vivo dosing, afatinib was dissolved in 1.8% hydroxypropyl-b-cyclodextrin (Sigma), 5% of a 10% acetic acid stock and aqueous natrosol (0,5%). Selumetinib was resuspended in 0,5% hydroxypropylmethylcellulose (Sigma) and 0.4% Tween-80 in distilled water. All agents were administered via oral gavage.
The results presented are representative of three independent experiments run in triplicate, unless otherwise indicated. Student’s t test and two-way ANOVA were carried out using GraphPad Prism to calculate significance. Differences were considered significant at p<0.05. Results are expressed as mean ± standard error (S.D.).
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Jonathan A CooperReviewing Editor; Fred Hutchinson Cancer Research Center, United States
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Thank you for submitting your article "Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Ivan Dikic as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Michael Yaffe (Reviewer #3).
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
This paper makes an important contribution to the emerging concept that CRC organoids are useful as a middle ground between 2-D cultures and mouse models. The authors use organoids with wildtype KRAS, organoids with mutant KRAS, and normal organoids engineered to express mutant KRAS, and find that KRAS mutational status determines the sensitivity to HER-RAS-ERK axis targeted therapies. While these observations had been previously described in vitro or in clinical trials, one new result is that MEKi/HERi or MEKi/ERKi combinations induce cell cycle arrest rather than cell death. Importantly, addition of BCLi during HERi/MEKi removal induces death. This could have clinical relevance given the platelet toxicity of BCLi. The paper demonstrates the potential of using patient-derived CRC organoids in testing and evaluating combinatorial drug responses.
The reviewers agree that the strength of the paper lies in the extensive data validation and use of patient-derived (and engineered) organoids. The chance of artifacts has been reduced by the use of varied inhibitors against common targets and the different approaches to measure cell death. However, the use of organoids for drug screens is not new, CRISPR-mediated mutagenesis of KRAS has been reported, and the efficacy of combinations of EGFRi and MEK/ERKi is well-established. Moreover, the reviewers were concerned that there is limited new biology or molecular mechanisms in the paper. For this reason, the reviewers are only willing to consider the paper for eLife if the new findings (of cell stasis rather than death following MEK/EGFRi and of the efficacy of BCLi) are better substantiated.
The reviewers have discussed their findings and agree that the following points must be addressed in a revised submission:
1) Organoids do not completely reflect the biology of cancer in vivo. The response of CRC organoids to combination therapy should be confirmed in in vivo models such as mouse flank xenograft of kidney capsule (e.g., Fujii et al. 2016 Cell Stem Cell 18:827).
2) The argument that EGFRi/MEKi and MEKi/ERKi cause cell cycle arrest rather than senescence or death is based on H2B fluorescence and a limited set of other assays. This evidence needs to be bolstered by independent assays, such as caspase-3, annexin V, cell cycle (p21), checkpoint, proliferation and senescence markers. The authors could sort for dead cells and then separate arrested cells by DNA content and identify arrested cells.
In addition, the authors should consider the other, less major, comments that are listed below.
1) Figure 1, Panel B. A color key should be provided to understand the colored organoids, in order to easily identify a given z-stack depth with a particular color.
2) Figure 2, Panel B. The authors claim this full-matrix screen as good illustrator of synergy. Given that the heatmaps have been smoothened with walking (i.e. moving?) averages, it would be good to clarify better the concept.
3) Figure 2, Panel C. It looks like the introduction of mutant KRAS in P18T has an effect in cell proliferation even in absence of treatment (organoid size at 65h is clearly smaller that P18T one), which is not commented in the text. Is the data normalized to the starting number of cells?
4) Also, the authors claim the cells stop proliferating, but no additional validation than cell size is used (i.e. checkpoint activation, p21 expression).
5) Figure 4. The authors try to show that the response to targeted therapies is only dependent on KRAS status independently of cellular mutational status, as the results in normal organoids are comparable to cancer ones. I would be more cautious in this respect, as the culture of normal organoids requires high Wnt activity media, growth factors and inhibitors, which may not reflect the normal mucosa in the intestine. Additionally, I find kind of surprising that the introduction of an oncogenic KRAS does not result in oncogenic senescence in a normal organoid. It would be nice to see some organoid pictures in this set of experiments and/or a comparison of WT vs. KRAS-mutant organoids growth curves in absence or treatment.
6) Figure 5. I find this figure as the major strength of the work, as it validates the organoid lines screening as a real tool to assess drug/drug combination sensitivity. The data here shows a pretty wide range of sensitivities to afatinib alone for both the wt and mutant K-Ras organoids, as the authors note in the subsection “Therapy-surviving cancer cells quickly restart tumor growth after release of targeted inhibition”, second paragraph. Can the authors use the genotypic information they have on the organoids to elucidate any other contributors to the relative monotherapy resistance?
7) Figure 6. The authors describe here a novel and smart strategy to track organoid viability in real-time cultures, and developed ImageJ macros to quantify the phenotypes, that might be really useful for other researchers performing 3D cultures (organoids, spheroids, etc.). Nevertheless, they should clarify in the figure legend that cell dead/alive status is identified by nuclear size (red colored nuclei may be associated to π staining, also described in the Methods section and used in following experiments in the manuscript). Also, given the novelty of the method, I would recommend further validation of nuclear size as a proxy of dead/alive status (co-stain with apoptotic markers and/or viability dyes).
8) The authors conclude that the inhibition of the EGFR/MEK/ERK axis induces mostly a cell cycle arrest that, in absence of drug, can be reverted and cells proliferate again. I would be more cautious in this affirmation, given the data presented. It is not clear from the data presented whether there is a global cell cycle stop (no assessment of G1/S checkpoint activation nor cell cycle progression markers) and the data does not rule out the possibility of a small population of pre-resistant cells within the organoid, which are then able to repopulate in absence of treatment.
9) Figure 6, Panel B. – Please add a color code legend.
10) Figure 7, Panel A. I would suggest splitting this graph in at least three different ones, in order to better point out the synergy between targeted drugs and navitoclax. Panel B. PI final concentration is not provided in the methods section, which may impact the threshold applied for the analysis (i.e. π unspecific staining/background). Panel C. The authors claim they need a robust inhibition of both MEK and ERK in order to Navitoclax to be effective at low concentrations. It would be very helpful, to show some western blots of p-EGFR, p-ERK and downstream signaling to demonstrate such inhibition at 1uM of afatinib and selumetinib vs. lower concentrations of them (i.e. 0.065uM).
11) Figure 3—figure supplement 1 – what is the rectangle code for the inhibitors?
[Editors' note: further revisions were requested prior to acceptance, as described below.]
Thank you for resubmitting your work entitled "Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening" for further consideration at eLife. Your revised article has been favorably evaluated by Ivan Dikic (Senior editor), a Reviewing editor, and two reviewers.
The reviewers are now very positive about the paper, but there are some remaining concerns. New experiments are not required, but you may have the data and could add them or discuss. Points 1-3 address whether growth rate differences between WT and mutant KRAS organoids might be detectable in low serum/glucose conditions and whether EGFRi and MEKi are really inhibiting as expected in vivo. Point 4 suggests adding in vivo histology to complement the in vitro histology of the organoids. Point 5 concerns whether the in vivo resistance is due to selection. Point 6 requests statistics for the growth curves and cell cycle and this point must be addressed. Point 7 suggests adding comments about Bcl2 vs. BclXL drugs.
1) The authors write that "Upon addition of oncogenic KRAS, no overall differences in morphology or growth rates were observed." (Results). It has been established that Kras mutant cell lines exhibit greater proliferation than Kras wild-type cell lines in low serum or low glucose conditions, and that tumors in Kras genetically engineered mouse models also grow faster than Kras wild-type GEMMs (Yun et al. Glucose deprivation contributes to the development of KRAS pathway mutations in tumor cells Science 2009 and Martin et al. Development of a colon cancer GEMM-derived orthotopic transplant model for drug discovery and validation Clinical Cancer Research 2013). Therefore, it will be of interest to the readership if the authors could assess using CRISPR engineered and/or human CRC organoids how KRAS mutant vs. wild-type organoids grow in vitro under low serum or low glucose conditions, and/or in vivo upon transplantation.
2) Figure 3—figure supplement 3. The authors performed western blots on Kras mut and WT organoids in response to drug treatment, as requested. The lower P-ERK level in control KRAS MUT organoids vs. control WT organoids may an artifact of the media conditions. The authors should assess P-ERK and/or P-MEK in Low serum / low glucose conditions.
3) Figure 2 and Figure 2—figure supplement 1. Western blots for P-ERK and/or P-MEK should be provided for in vivo experiments to demonstrate that tumor growth / stasis is due to the mechanism of action of the drugs. This would easily address the authors' point that they "speculate that the in vivo drug concentrations were insufficient to effectively block the EGFR-MEK-ERK pathway" for the low dose combination treatment.
4) Please provide in vivo histology of the tumors compared to the in vitro organoids, with or without drug treatment.
5) It would have been more interesting to me to see secondary in vitro passage of the residual in vivo tumors after HIGH dose treatment. This would answer the question of whether a therapeutically relevant drug dose reveals primary resistance or induces secondary resistance in the tumor organoid tissue. This is a comment rather than a request for more experiments – may be more of a topic for future study.
6) The drug dose response curves and cell cycle data do not indicate significant differences or appropriate statistics in the figures nor in the legends. This should be addressed.
7) Regarding the naviticlax combination. AbVie has drugs that now target either Bcl2 or BclXL so future experiments could investigate the effects of separate drugs or knockdown and then co-treatment with RAS pathway targeting drugs.
Figure 3—figure supplement 3: phosphorylation is misspelled.https://doi.org/10.7554/eLife.18489.040
- Jarno Drost
- Hans Clevers
- Johannes L Bos
- Hugo J Snippert
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank our colleagues for continuous support and discussions. CSV, IVK, and BP were supported by a ‘Sta op tegen Kanker’ International Translational Cancer Research Grant to HC and JLB. Stand Up to Cancer is a program of the Entertainment Industry Foundation administered by the AACR. HJS is supported by a KWF fellowship from the Dutch Cancer Society (UU 2013–6070) and JD by a VENI grant from the Netherlands Organization for Scientific Research (NWO-ZonMw) (91614138). The Mouse Clinic for Cancer and Aging (BvG, MvdV) is supported by National Roadmap grant for Large-Scale Research Facilities of the Netherlands Organization for Scientific Research. General support came from the Netherlands Organization for Scientific Research gravitation program Cancer Genomics Netherlands and the Josephine Nefkens Foundation.
Animal experimentation: Approval for this study was obtained by the local animal experimental committee at The Netherlands Cancer Institute (DEC-NKI). (DEC-NKI; OZP=12012 and WP5727 and WP5689). All of the animals were handled according to approved institutional animal care and use committee.
- Jonathan A Cooper, Reviewing Editor, Fred Hutchinson Cancer Research Center, United States
© 2016, Verissimo et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

domain: medical


text:
Where the Water Tastes Like Wine Lyrics Sheet
A downloadable lyrics booklet
Please enjoy this gorgeous book of lyrics for the Where The Water Tastes Like Wine soundtrack. Lyrics written by Ryan Ike, translated to Chinese by Ryan Zhangquan and team, and book designed by Lysandra Nelson.
Click download now to get access to the following files:
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domain: artistic


text:
Swimming Australia is mourning the death of long-serving swimming administrator Greg Lalor who passed away on Saturday 26 October, in Brisbane.
Tag Archives: training swimwear
Australia to take on the world at the BHP Billiton Aquatic Super Series in Perth
Today marks ?100 Days to Go? until the start of the BHP Billiton Aquatic Super Series at VenuesWest Challenge Stadium, with two new international teams set to compete in Perth this summer.
Seven swimmers to send off Go Swim Month
As the end of October quickly approaches so too does the end of Swimming Australia?s GO Swim Month for 2013.
To and Hurley bring home 16-medal haul from Middle East
After the final night of competition in Doha, Australia?s Kenneth To and Bobby Hurley have finished the Middle East legs of the FINA World Cup with a total of 16 medals between them.
To sets Australian standard at short course world cup
Short course specialist Kenneth To has set a new Australian record in the men?s 100m individual medley overnight, hitting the wall in a time of 51.19, to take gold at the FINA World Cup in Doha.
Hurley heats up with dual gold in Dubai
On the second night of competition at the Hamdan Sports Complex in Dubai, Robert Hurley has added two more medals to his haul, picking up gold in both the 200m freestyle and 50m backstroke.
Hurley and To back on top in Dubai, Seebohm secures silvers
Australian swimmers Robert Hurley and Kenneth To have added to their medal haul from the Moscow leg of the FINA World Cup Series with two more medals each overnight in Dubai, while Emily Seebohm has won three individual silver.
Swimming Australia announce Jacco Verhaeren as new National Head Coach
Swimming Australia President John Bertrand AM and CEO Mark Anderson have today announced Jacco Verhaeren as the new National Head Coach to work under Director of High Performance Michael Scott.
Next generation inspired to GO Swim
The 2013 GO Swim Month is in full swing as clubs across the country jump on board the Swimming Australia initiative to educate communities on the benefits of club swimming.
Golden night for Australian trio in Moscow
The Australian trio of Ashley Delaney, Robert Hurley and Kenneth To have each picked up a gold medal on the final night of competition at the FINA World Cup in Moscow.

domain: sports


text:
Silent Hill: Betrayal (Vanilla Edition) : Shaun Jooste
A5, 385p. Vanilla Edition is the raw story with the single, dogmatic ending. No maps or images have been included.
Trevor travels through the haunted, abandoned town of Silent Hill, in search of his lover Kathy and best friend Jay. What he finds is echoes of his deceased wife and her murdered brother, intent on exacting vengeance for the crimes committed against them.

domain: literature


We don’t know what she’s saying, but it sounds INCREDIBLE!
We’ve thought highly of Jennie Lena before, but this is another level!
On the most recent episode of The Voice Of Holland tonight, she performed a song called Vraag Jezelf Eens Af, which translates to “Ask Yourself.”
We are asking ourselves how this woman can’t be the next champion of The Voice in the Netherlands!!
Ch-ch-check out the incredible video (above) on PerezTV!!!
What’d U think??


By BILL HUTCHINSON, ABC News (NEW YORK) -- A flight instructor and a student pilot were rescued by a U.S. Coast Guard crew Saturday night after their plane experienced engine trouble, forcing them to ditch the aircraft in the ocean off the coast... Read More.
By AMANDA MAILE, MINA KAJI and ALEX STONE, ABC News (NEW YORK) -- Federal investigators on Tuesday said the helicopter carrying Kobe Bryant, his daughter and seven others, including the pilot, crashed last January when the pilot became disoriented after flying in thick... Read More.
(LOS ANGELES) -- While some Star Wars fans could have anticipated at least some of the upcoming projects that were announced as part of the Disney Investor Day presentation Thursday, a major surprise was a movie called Rogue Squadron. Wonder Woman's Patty Jenkins has been tapped... Read More.
By ROSA SANCHEZ, ABC News (NEW YORK) -- The F-16 pilot who crashed in Michigan Tuesday has been pronounced dead.The Wisconsin Air National Guard's 115th Fighter Wing announced his death Friday morning, stating that, per the Department of Defense's policy, they... Read More.
By LUIS MARTINEZ, ABC News(CENTRAL VALLEY, Calif.) -- A Navy F/A-18E fighter jet crashed in California on Tuesday, but the pilot was able to eject safely, according to a Navy spokesman."An F/A-18E Super Hornet from Naval Air Station... Read More.
By MINA KAJI, ABC News (WASHINGTON) -- The head of the Federal Aviation Administration is set to pilot the 737 Max Wednesday in Seattle -- a key step in the aircraft's eventual re-certification."I'm not going to sign off... Read More.
BY: JULIA JACOBO, ABC News(NEW YORK) -- A rescue team in Australia is attempting to save as many pilot whales as possible after more than 270 became stranded in waters off the coast of Tasmania.The whales are currently... Read More.
By HALEY YAMADA, ABC News(PORTLAND) -- A helicopter pilot died Monday while battling the White River Fire in Mount Hood National Forest, officials said Tuesday.About 20 miles east of Portland, Oregon, the pilot was flying a Type 1... Read More.
By HALEY YAMADAVIA, GMA(WASHINGTON) -- One woman is soaring to new heights as the first Black female tactical jet pilot for the U.S. Navy."I don't think the goal in my life is to necessarily be the first at... Read More.
By MORGAN WINSOR, ABC News(LONDON) -- An American fighter jet crashed into the North Sea off the coast of England on Monday morning, killing the only pilot on board, officials said.The F-15C Eagle, from the U.S. Air Force's... Read More.


text:
Charlottenburg is an affluent area in Berlin known for the Charlottenburg Palace, shopping centric Kurfürstendamm avenue and is home to many theaters and museums. Originally an independent city, Charlottenburg was incorporated into Greater Berlin in 1920.
Must See In Charlottenburg
The Kaiser Wilhelm Memorial Church is centrally located in Charlottenburg near a popular shopping area. The church was nearly destroyed by bombing raids during World War II. After the war, the decision was made that the church would be left standing as a memorial and reminder of the consequences of war.

domain: geography


We've all heard of Raspberry Pi, which is a pretty small computer board that can be used to create virtually anything. The only limit is what your mind can come up with. Now we have another one, that's much cheaper and that's the VoCore. It's a Mini Linux Computer, and you can make it into a router, build a motherboard and even create an entirely new device. Not to mention repurposing old appliances into smart wireless appliances. Like your old speakers into a smart speaker. With VoCore you are able to use it as a standalone device which would run on OpenWrt, or you can use it as an embedded component of a larger system. With some electronics knowledge, you can pretty much build anything.
VoCore does also work on open-source hardware, it also provides up to 20 GPIO lines and includes an on-board WiFi adapter. This way you won't need to use an external one. VoCore can easily connect with other peripheral devices as well. It can extend ethernet and USB interfaces using the dock. It integrates a 802.11n MAC, baseband, radio, FEM & 5-port 10/100Mbps Ethernet Switch. Now the VoCore Mini Linux Computer is on sale right now over on the Android Headlines Store for just $30.


text:
Easy. Affordable. LIVE!
Take an online cooking class with me and learn how to cook delicious Japanese Sweets. Learn how fun and simple it is to make Wagashi in this exclusive cooking class.
Wagashi are quite unusual to the western palate due to their unique texture. I have a curious passion for them, and own many books in Japanese about them. To the best of my knowledge, I am the only one running wagashi cooking classes in Europe. I choose a selection of seasonal sweets for each class, and serve them with matcha tea.
Wagashi are traditional Japanese sweets, usually made from rice, adzuki beans and fruit. They evolved as an accompaniment to the tea ceremony, to balance the bitterness of the matcha tea, mainly in areas such as Kyoto and Kanazawa. Japanese sweets are highly seasonal, and beautifully decorated. The oiran culture in the Edo period was known for popularising wagashi sweets to the broader population in Japan. Learn how to make different types of traditional sweets in this class, that make an elegant gift for someone special. I usually include daifuku mochi, yokan, namagashi, and nama chocolates in the classes.
NOTE: Classes can be booked between 1pm and 10pm Central European Time (Paris/Monaco). I do make exceptions if you live in a different time zone, so feel free to ask.
If you would like to book an online class for a company teambuilding session, then you can be up to 40 people. See the team building page for details.
Kimura's Kitchen has been pioneering live online cooking classes since 2015.
But what is an online cooking class? Simply put, it's the most affordable, most comfortable, and easiest form of cooking class there is.
Forget the days of poor quality video streaming, those years are behind us. In a live online class with me, I will broadcast HD video from my kitchen to your kitchen via a secure private session. You can follow my steps and cook along with me, and I can see what you are doing from your side and help you to get the best results.
Before the class, I send you a list of ingredients to buy, and we agree a time to go live. Simple as that. Classes last 90 minutes.
5 simple steps:
1. Choose your class: select your class from the cooking classes page. You can also choose a subscription, where you can take several classes over the course of a year. *Remember to select how many people will be in the class.
2. Request your time and date: write your preferred date and time in the box when booking (or email me at email@example.com) and I will get back to you to confirm the date within 24 hours.
3. I send you a list of ingredients: a link will be sent to you with the ingredients to buy for the class and list of online shops.
4. You download the app: I usually use Zoom for my classes.
5. We go live on video and cook! I stream HD video from my kitchen so you can cook along, and I can see what you are doing from the other side so you get the best result.
In order to be eligible to reschedule a cooking class (if you are unable to do the class on that day, or if you are less people than originally booked) you must notify me 3 days before the date of your booking. If you notify me with less than 3 days before your booking, you will not be eligible for an exchange or reschedule. Refunds are not possible at Kimura's Kitchen regardless if this is due to the customer or in the unlikely event that Kimura's kitchen has had to cancel your session. However this item may be rescheduled or exchanged for another product or service instead. We are always happy to help you reschedule or offer an alternative.
If you have more questions feel free to contact us at firstname.lastname@example.org
By making a purchase on this website you accept the above terms and conditions.

domain: hobby


This hoodie is insulated with ThermoBall powered through PrimaLoft, our artificial choice to down that achieves extraordinary heat in chilly and rainy climate. As compressible as down, the ThermoBall clusters lure and maintain heat inside of small air wallet to supply efficient insulation, even if rainy.
Ultralight, packable, hooded insulated jacket keeps heat even if rainy
ThermoBall powered through PrimaLoft gives the light-weight loft, heat and compressibility of down with the rainy-climate insulating efficiency of synthetics
15D nylon ripstop with ThermoBall insulation
Hooked up totally adjustable hood
Uncovered molded-teeth entrance zip


text:
“We influence, engage and develop a skilled workforce to deliver high quality health & social care research.”
– Mission statement
If you are a new member of staff, please visit the Induction website, where you can find more information about your role, the role of your colleagues and the CRN.
The NIHR Academy is responsible for the development and coordination of NIHR academic training, career development and research capacity development.
The academy plays a pivotal role in attracting, training and supporting the best health and care researchers to tackle the complex challenges of the future. The training and career development awards provided complement the additional NIHR investment in world-class research facilities and a skilled research workforce.


Wolf Shuts the Door15th Jan 2020
Following the news of Restaurant MCR's closure, there's more bad news for foodies in Manchester, as recent Northern Quarter opening, Wolf at the Door, has temporarily closed with a completely different concept being planned for its reopening.
Originally opened as Wilderness Bar + Kitchen in August 2019 in the former Odd bar and Folk & Soul unit, they had to rebrand in October due to a potential name clash with a Birmingham cafe.
~ Advertisement ~
Serving up some of the finest cocktails in the city, the menu was shortlisted for a prestigious Imbibe Award, with the talented Adam Day (formerly of Wood & Co.) originally heading the bar, before leaving unexpectedly in early December.
The Scandi-inspired fine dining food menu was equally impressive, as former Manchester House / Restaurant MCR chef James Lord produced some of the best dishes to be found anywhere in town. His technically perfect creations were priced at a surprisingly accessible level, aiming at the traditional Northern Quarter crowd, with the venue's relaxed cafe-style design inspired by similar venues in Copenhagen.
Despite the fact Wolf at the Door's owners are some of the most experienced operators in town, (Junkyard Golf Club, Bunny Jacksons, Liar's Club, Crazy Pedro's, Cain & Grain, Bay Horse, Cottonopolis, and Trof) it was the first time they had ventured into the incredibly challenging fine dining game.
Owner Lyndon Higginson confirmed the news of the venue's temporary closure: "We had a simple plan, to open a really cool neighbourhood bar with amazing drinks and brilliant food.
"We somehow ended up with Michelin Star worthy food and spectacular award winning cocktails.
"We got a little too excited about the food, the drinks and the design and we just went with it.
"I really do believe we were knocking out some of the best food in our city and I’m eternally grateful to all of our staff who made this happen.
"The issue has always been that we never planned to open so much of a restaurant.
"So... We’re taking a couple of weeks to change our offer slightly and we’ll be back open before the end of the month with a new food offering and some more amazing drinks.
"We’re gonna give people what they want. We’ll still have the same ethos. We still want the best bar. And we will.
"We’re gonna have a big party at the end of the month and you’re all invited."
What's On in Liverpool


text:
AMERICAN HISTORY: A Visual Encyclopedia
Connect with a representative to create a custom curriculum for your district.
Created in collaboration with the Smithsonian Institution, this abundantly illustrated reference features 18 chronological chapters with more than 750 photographs and artworks plus extensive maps, biographical portraits, sidebar stories, and more.
Each chapter opens with a timeline of important events, and each two-page spread focuses on a different topic. Appendixes contain an illustrated table of U.S. presidents, facts about states, the Declaration of Independence, the Constitution, and the Gettysburg Address.
8½" x 11"

domain: library


text:
Smartphones and tablets are no longer emergent technologies: they are dominant
forces in driving e-commerce around the world. These ubiquitous little devices
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this presents a profitable opportunity. Don't let countless bookings slip away:
tap this ever-growing source of revenue with a creative and effective BookPro™ mobile
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Google's mobile research shows that over 30% of mobile users in countries across the world
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. If your site isn't optimized for these devices,
mobile shoppers will encounter clunky, awkward versions of your pages, with limited functionality
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Mobile is here to stay, so get started building your BookPro™ mobile site today
Booking Engine Compatibility
Good mobile sites for hotels will build brand awareness and aid visitors in planning
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From iPads® to iPods®, Blackberry® to Android®, there are a range of devices and operating systems
that mobile shoppers will use to access your site. BookPro™ mobile sites ensure complete cross-device
compatibility, so no matter how your mobile customers are reaching your site, they will be able to
view it with full functionality and easy navigation.
Dynamic Content Updates
With our BookPro™ hotel Content Management Systems
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make or information you add will seamlessly integrate with your mobile site, giving mobile
users the same up-to-date experience as visitors to your desktop site.
BookPro™ Website GPS Integration
By integrating your BookPro™ website with the GPS functions of smartphones and tablets, our BookPro™ mobile
sites can use a visitor's mobile location to direct them to points of interest around your hotel. By
engaging with interactive maps and specific directions on their device, users can then quickly and
easily find their way to attractions that you publicize on your site.

domain: travel