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Today we held a whole school assembly to celebrate Robert Burns Day. The pupils performed a range of poems, dances and songs. It was wonderful to have everyone together sharing their learning.
Eco Group
This term we started the year by getting outside to explore our environment. We were excited to get outside and look at our playground to decide what we needed to do. The first thing we noticed was the amount of Read More
Adventure Aberdeen Easter Holiday Programme
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Rights group
Our new rights group have met 4 times this school year. We have been working on posters to tell people about the rights. We will soon put them up around the school but they might not be there if you Read More
Sport Aberdeen Oldmachar ASG Primary Schedule
Oldmachar ASG – Primary Schedule Term 2 – 2018.19
Is your child ready for Primary School?
If your child is ready to start Primary 1 in August 2019,you will need to go online to enrol them in January. Enrol your child for school at: www.aberdeencity.gov.uk/schoolapplications You can also: • Get help filling in the online form Read More
Primary 4 and Primary 4/5 October – December 2018
The children in P4 and P4/5 have been learning all about food and then they created their own Mandarin Menu!
Primary 3 October – December 2018
The Primary 3 pupils have been learning how to talk about their families in Mandarin.
Primary 1 + Primary 1/2 October – December 2018
The pupils in P1 + P1/2 have been working very hard with Mrs Sayer this term. They have been learning about colours and how to write numbers to 10.
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Today we had a wonderful day of performances! The pupils from P1-5 put on a Musical performance for their parents and friends. It was fantastic to hear all the amazing singing. We have uploaded some clips from today for you Read More


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Cells 2018, 7(12), 225; https://doi.org/10.3390/cells7120225
2D- and 3D-Based Intestinal Stem Cell Cultures for Personalized Medicine
The State Key Laboratory of Membrane Biology, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China
Author to whom correspondence should be addressed.
Received: 4 October 2018 / Accepted: 16 November 2018 / Published: 22 November 2018
Colorectal cancer (CRC) is one of the most common cancers that have high occurrence and death in both males and females. As various factors have been found to contribute to CRC development, personalized therapies are critical for efficient treatment. To achieve this purpose, the establishment of patient-derived tumor models is critical for diagnosis and drug test. The establishment of three-dimensional (3D) organoid cultures and two-dimensional (2D) monolayer cultures of patient-derived epithelial tissues is a breakthrough for expanding living materials for later use. This review provides an overview of the different types of 2D- and 3D-based intestinal stem cell cultures, their potential benefits, and the drawbacks in personalized medicine in treatment of the intestinal disorders.
Keywords:intestinal stem cells; organoids; monolayer; cell culture; personalized medicine
1. Introduction
The intestine is a key organ that is responsible for food digestion, nutrients absorption, and waste excretion [1,2,3], with the capacity to sustain microbial and defense pathogens, as well as to regenerate following damage by inflammation or irradiation [4]. The intestinal epithelium, which is composed of a single layer of cells and forms the crypt–villus structure, is rapidly renewed, with a complete turnover every 4–5 days [3]. The self-renewal is driven by intestinal stem cells (ISCs), which are marked by Leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) and localized at the base of the crypts and intermingled with Paneth cells that provide signals to support stem cell self-renewal and maintenance [5,6,7,8,9]. Along the villus, the epithelium consists of terminally differentiated functional cells, including enterocytes, enteroendocrine cells, goblet cells, tuft cells, and other cell types. These ISCs-derived cells move up toward the tip of villus during differentiation, and eventually die at the tip of villus and are shed off into the intestinal lumen [10,11]. Although there are no villus structures in the large intestine, the architecture of the epithelium is similar to the one of the small intestine, with the ISCs at the bottom of the crypts [3]. The self-renewal and differentiation of the ISCs are tightly controlled by the coordination of multiple niche factors [2,12], such as epidermal growth factor (EGF), Wnt ligands [13,14], bone morphogenetic proteins (BMPs) [15,16,17], Notch [18,19], and others [20], as well as Hippo/YAP signaling [21].
Colorectal cancer (CRC) progresses sequentially from adenomatous polyps to advanced adenomas, carcinomas, and adenocarcinomas, and gene mutations in multiple pathways have been reported in different stages of CRC development [20,22,23]. For instance, gain-of-function mutations have been observed in the various components of the Wnt/β-catenin pathway, such as APC, CTNNB1, RNF43 and, while loss-of-function mutations are found to be associated with transforming growth factor β (TGF-β)/bone morphogenetic protein (BMP) signaling components such as SMAD2, SMAD4, TGFBR2, BMPR2, and BMPR1A. Gene mutations have also been reported in KRAS, BRAF, TP53, and DNA repair machinery. Different mutations cast complexity upon CRC treatment. In addition, ulcerative colitis (UC) and Crohn's disease have abnormal immune responses in the colon and rectum, but whether these immune responses are a cause or a result of the disease is still under debate [24,25,26]. Although dextran sodium sulfate (DSS)-induced intestinal colitis in mice is a well-established and characterized model for acute intestinal injury and inflammation, this model, as well as the immune cells and epithelium co-culture system, have not yielded to effective therapies to treat inflammatory bowel colitis [27,28]. Furthermore, there are no specific treatments for enteric pathogen-caused illness [29,30].
Although chemotherapy and irradiation therapies are developing rapidly, gastroenterological disorders are still a major public health problem in the world due to the disease complexity. At the moment, most patients receive similar one-size-fits-all treatments. However, certain therapies work well for some patients, but they do not display promising results in others [31,32]. Therefore, personalized medicine or individualized treatments are in urgent demand to revolutionize future healthcare. To achieve this, precise patient-derived disease models are essential. Several patient-derived preclinical models have been established for intestinal disorders, including patient-derived tumor xenografts (PDX), culture systems of cancer cell lines, spheroids, and organoids and monolayers of intestinal epithelium and tumor tissues. These models have proven to be extremely valuable, not only for drug discovery and clinical trials, but also for a basic understanding of the fundamental mechanisms underlying tumorigenesis, inflammation, and interactions between the host–pathogen. This review summarizes the current 2D- or 3D-based intestinal epithelium culture systems, and discusses the advantages, caveats, potentials, and challenges, highlighting their applications in personalized medicine.
2. 3D-Based Organoid Culture System
Cell-based assays have been widely adopted in drug discovery for past decades. Historically, colon cancer cell lines, for instance HCT-116, HT-29 and SW480, have been used as the only practical option to discover drug candidates [31,32]. However, it has been demonstrated that the traditional culture system does not have sufficient clinical predictive power, due to the loss of tissue-specific architecture, the existence of gene mutations, and the lack of cell diversity in the intestine, therefore poorly recapturing tumor or inflammation conditions in patients [33]. Moreover, the necessity of adding three dimensions to the cell culture system that more closely resembles the natural environment, and its therapeutic potential, is being increasingly recognized for the future.
Scaffold-free and scaffold-based cultures are two major 3D culture systems. The scaffold-free system, like the sphere culture of the tumor cell lines in a low-attachment culture dish, suffers from similar defects as mentioned above [34]. Using droplets of a hanging drop plate, neural stem cells [35], mammary stem cells [36], corneal stem cells [37], and the embryoid bodies (EB) cultured from embryonic stem cells (ES) [38] could be cultivated in a spheroid, while it cannot be applied to the intestine. The other scaffold-based system, PDX models generated by transplanting the patient's tumor cells to an immune-deficient mouse, could recapture some aspects of niche effects, such as the interactions of tumor cells with the surrounding stromal cells and immune cells; however, the nonmetastatic colorectal cancers fail to establish engraftments in mice [39]. A significant breakthrough was achieved by the establishment of three-dimensional (3D) organoid cultures of intestinal epithelium [40]. This culture system can rebuild the morphological, functional, and certain microenvironmental features of the intestine in vitro. The 3D organoid culture can mimic the in vivo physiology of the intestinal epithelium and it can therefore be used as a new tool for drug discovery and drug testing (Table 1).
The development of the murine intestinal organoid culture system by Clevers' group in 2009 opened up a new era for the investigation of organ regeneration and homeostasis [41]. The successful establishment of the organoid culture system was based on the identification of the leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) as a feasible marker of intestinal stem cells [42]. The intestinal epithelial organoids, also named "mini-gut", can be cultured from the crypt epithelium, or even from single Lgr5+ cells [40]. Briefly, the isolated crypt epithelium or intestinal stem cells are embedded in Matrigel, a collagen- and laminin-rich extracellular matrix extract, and allowed to self-organize into a gut-like epithelial structure. The culture medium was supplemented with three growth factors: the Wnt agonist R-spondin1, the bone morphogenetic protein (BMP) antagonist Noggin, and epidermal growth factor (EGF) [40]. The 3D organotypic structures are very similar to that of normal intestinal epithelium in vivo, comprising of multiple crypt-like domains containing basal Lgr5+ cells interspersed with Paneth cells, and proliferative transient amplifying (TA) cells, enterocytes, goblet cells, and enteroendocrine cells [3,41,43]. Importantly, these cultured organoids retain their self-renewal and differentiation abilities, and their phenotype, karyotype, and gene expression profile are similar to the gut epithelium in vivo.
After the 3D culture system for murine intestinal organoids was set up, more efforts have been made to adapt this system for human tissues, especially for patient-derived colon cancers [44] (Figure 1). In addition to the three growth factors R-spondin, Noggin, and EGF, the generation of patient-derived organoids requires other factors such as Wnt3a, gastrin, prostaglandin E2, nicotinamide, the TGF-β receptor inhibitor A83-01, and the p38 inhibitor SB202190 [43].
3. 2D-Based Culture System
Although 3D-based organoid culture technology has had a great impact on the in vitro study of gut epithelium biology and its therapeutic potential, the culture system faces significant challenges. Firstly, the self-renewing organoids embedded in Matrigel could mimic some of the in vivo epithelium features, but these organoids cultured as pure epithelium lack niche cells such as stromal cells, immune cells, and vasculature [45,46,47], and are unable to fully mimic the in vivo microenvironment. Secondly, some key cell types were not found in organoids; for example, secretin+ enteroendocrine cells were missing in the 3D culture [16,48]. Thirdly, the spheroidal structure of organoids restricts luminal access of exogenous compounds and pathogens, therefore restricting physiologically relevant studies on apical transporters, metabolic enzymes, and microbiota [49,50,51,52,53,54]. They could not be used for drug screening to treat UC or Crohn's disease. Lastly, as the Matrigel is derived from animals with undefined hydrogel matrix compositions, it has limited broad applications for the 3D organoid culture system in mechanistic and clinic-related studies. To overcome some of these limitations, therefore, researchers have focused back on 2D monolayer culture systems.
Previous works had suggested that long-term culture of primary intestinal epithelium as a planar monolayer was impossible because of rapid stem cell loss and accelerated apoptosis. Recently, several groups have successfully established 2D monolayer culture systems of intestinal epithelia that can promote ISC maintenance [51,55,56,57,58,59,60].
Although different approaches have been used to culture intestinal epithelial monolayers (Table 1), a common method involves culturing cells on an extracellular matrix-coated surface. Use of a thin coating of type I collagen and recombinant human laminin is the most common choice, which has been shown to support the rapid expansion of proliferating monolayers of mice and human intestinal epithelium containing ISCs [57,60]. Although these approaches are appealing, the technical complexity associated with matrix neutralization of the acidic collagen solution with sodium hydroxide [60] or with intestinal subepithelial myofibroblast conditioned medium [57] may restrict the broad application of such systems. Based on the transwell system, several groups have described robust monolayer culture systems, which generate cells with the apical face of epithelium towards the culture medium [51,56,59]. Furthermore, a combination of the epithelial monolayer culture on the transwell insert and the niche cell culture on the transwell plate provides an effective tool to assess the effect of niche factors from immune cells or stromal cells in the intestine (Figure 2).
However, the Bolstering Lgr5 Transformational (BLT) sandwich culture system, consisting of a bottom collagen IV coating and a collagen I gel overlay, with intestinal epithelial cells between them [59], is still complex, and this may limit its application. Furthermore, mature secretory cell markers (Lyz1, Muc2, and Chga) were absent in the system established by Tong and colleagues. Similarly, the Paneth cell marker Lyz1 was not detected in the monolayer culture described by Puzan and colleagues [56]. A lack of important cell types raises the concern of whether these systems can recapture the pathophysiological features of the intestine in normal people and patients.
Most recently, we and Thorne et al. independently established simple and economical 2D monolayer culture systems that can recapture most of the features of 3D-cultured organoids and in vivo tissue [55,58]. The intestinal crypt epithelium is placed on a thin Matrigel layer on the culture dish. To maximize the expansion of Lgr5+ ISCs, the small molecules CHIR-99021 (inhibitor of GSK3 to activate Wnt/β-catenin signaling) and LDN-193189 (BMP type I receptor inhibitor) are added in the culture medium [55,58,59]. However, different from the system by Thorne et al., our system uses blebbistatin, which could improve the survival of cultured intestinal epithelium [61], and places Matrigel with uniform thickness, which could maintain the uniform growth of epithelial cells and reduce the cost of Matrigel.
Using a transwell-based 2D system, Wang and colleagues were able to establish a columnar epithelium with villus-like structures [62]. In their culture system, irradiated 3T3-J2 cells are seeded to transwell inserts coated with 20% Matrigel as a feeder layer. Then, human ISCs are placed onto the feeder layer in a medium containing R-spondin1, Jagged-1, Noggin, and other small molecules. More importantly, by removing the apical medium, the system allowed for columnar epithelium differentiation toward villus-like structures at the air–liquid interface. They further reported the cloning and propagation of ''ground state'' human intestinal stem cells (ISCGS) that had higher clonogenicity compared with organoids.
A 2D-based monolayer culture allows for better imaging observation of the dynamics of Lgr5+ stem cells and the interplay between stem cells and differentiated cells. Moreover, in contrast to the inaccessibility of the apical domain of the epithelium in organoids, exposure of the surface in the monolayer provides a suitable system to investigate the pathogen–epithelial cell interaction [63]. However, one vital limitation of the all monolayer culture systems that needs to be solved is that it cannot be easily passaged and propagated [64]. Although efficient passaging between 2D and 3D can be achieved, 2D-to-2D passaging can be accomplished with a limited number of iterations, and long-term passaging needs further optimization [16,55,57,60].
4. Applications in Personalized Therapy
Development of stem-cell-derived culture technologies raises an unprecedented opportunity to reform the health care system by transforming disease modeling to the assessment of efficacy and safety profiles, including potential drug development, the cytotoxicity evaluation of new therapeutic compounds, personalized medicine, and transplantation-based therapies for end-stage diseases [44,65,66].
As 3D organoid culture is based on patient-specific tissues and can recapture in vivo-like complexity and architecture, it has been used to investigate the mechanisms of tissue homeostasis and disease development, including colorectal cancer and ulcerative colitis [67]. Many genetic gastroenterological disorders can be modeled with patient-derived or gene-edited organoids. For instance, Jarno et al. and Mami et al. have genetically edited the genome of normal human intestinal stem cells isolated from the patients by CRISPR/Cas9 technology to target the most highly mutated colorectal cancer hotspot (APC, TP53, KRAS, and SMAD4) [68,69]. Moreover, Verissimo and colleagues reported that patient-derived tumor organoids with a CRISPR/Cas9-introduced oncogenic KRAS mutation could be used to evaluate drug efficacy in a preclinical setting [70]. Therefore, these gene-edited organoids yield a new avenue by which to screen anticancer candidates in different stages of tumorigenesis.
Another important application of intestinal organoids is to investigate the pathogenesis of various infectious diseases, and to understand the dynamics of the host–microbe interaction [29,30]. By microinjection into the organoid lumen (Figure 2), the interaction of enteric pathogens with the human intestinal epithelia have already been demonstrated, such as Vibrio cholera [71], enteroaggregative Escherichia coli (EAEC), enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC) [72], and rotavirus [73,74]. Similarly or even better, the 2D models can be used to explore the interaction between gut microbes and the intestinal epithelium, which could not only uncover the impact of microbes on epithelium homeostasis, but also help develop an effective and personalized treatment of the intestinal disorders.
In addition to the establishment of several specific disease models, organoids can be used for high-throughput drug screens [75]. A variety of organoid-based high-throughput screen systems have been established, such as organoid-on-chip [76,77]. As there is a strong correlation between gene mutation status and the therapeutic response, known as mutation-based drug sensitivity [78], the adoption of the tumor organoids has been used in precise cancer therapy. A good example was reported by van de Wetering and colleagues, who revealed that the organoids with TP53 loss-of-function mutations displayed resistance to the MDM2 inhibitor Nutlin-3a using 20 colon tumor organoids [65]. Interestingly, an investigation of drug sensitivities of tumor-derived organoids against the library of 85 therapeutic compounds including chemotherapy and targeted therapy agents resulted in the identification of an effective treatment for each individual patient. Together, modeling specific and rare subtypes of cancer by the means of genetically engineered organoids could help to identify effective and personalized treatments [75,79].
Although much work has been done with 3D cultured organoids as disease models, drug screening, and personalized therapy, the 2D monolayer culture represents a transformative technology for personalized medicine applications that depend on drug and compound screening related to dietary and microbial metabolites. Parasites (Cryptosporidium) and microbes (Shigella, E. coli, Clostridium difficile and Salmonella typhi) can be introduced into the medium of 2D cultures directly. Using a 2D monolayer system, Wang et al. found that tannic acid could significantly inhibit intestinal epithelium growth in 2D monolayers, but not in 3D organoids, which may be due to exposure of the compound to different cell surfaces (apical side in 2D vs basolateral side in 3D) [60]. Moreover, the monolayer also provides a system for characterizing ion transport across the intestinal barrier [63]. Indeed, Kozuka et al. identified an inhibitor of potassium absorption in the murine distal colon using an epithelial monolayer culture [51]. Furthermore, the transwell-based monolayer culture is an adequate system for investigating the crosstalk between intestinal epithelial cells and niche cells (mesenchymal cells, immune cells, and myofibroblasts) as well as the enteric nervous system [56,80].
5. Challenges, Limitations, and Perspectives
As patient-derived organoids and monolayers are faithful replicas of the patient's intestine epithelial tissues, these systems are great models that will undoubtedly facilitate diagnosis, molecule screening, drug testing, and transplantation as personalized approaches to treating intestine disorders. However, many challenges remain to meet the demands in quantity, quality, and processing robustness for commercialization and clinical trials.
For regeneration medicine, successful and efficient FDA-approved transplantation needs further improvement under culture conditions, including the animal-derived Matrigel and the high-cost of the growth factors. Several groups have used polyethylene glycol (PEG) and collagen to replace Matrigel as the supporting matrix in the culture [49,57,59,63], but the technical tediousness for handling PEG should not be neglected. Another big challenge is that niche reconstitution as the current culture system is designed for epithelial tissues. Considering the multi-functional and structurally complexity of the in vivo environment, niche cells including immune cells, stromal cells and other cell types, and vasculature, should be taken into account to better reflect the pathophysiological conditions. It is extremely important to understand the mechanism of inflammatory intestinal disorders, and ultimately to design efficient therapies to treat these untreatable diseases. For instance, the inclusion of niche cells would facilitate the screening of immunotherapy drugs and/or stromal-targeted agents. In this regard, the transwell-based 2D monolayer culture and microfluidics technology may provide some advantages of co-culture of epithelial cells and other types of cells to reconstitute the microenvironment [56,81]. Co-culture with niche cells has also been attempted in 3D systems [45], but the complex nature and high cost of 3D cultures limit their application, especially for a large-scale expansion. Recently, we have successfully established a growth factor-free culture system of murine intestinal organoids with two small molecules, which lights hope in this direction [82]. Furthermore, safe application in the clinic requires the long-term maintenance of the genome, without mutations or epigenetic changes.
With a number of major hurdles to be overcome, personalized medicine, taking advantage of the great promising potential of 3D- and 2D-based culture system, is rapidly evolving. Recently, a first-in-human trial of intestinal organoids for the treatment of inflammatory bowel disease is about to be carried out [83]. More excitingly, the patient-derived organoids have been used to test and select effective drugs for cystic fibrosis [71,84]. The 2D-based monolayer culture also holds great promises in personalized medicine. There is no doubt that 3D organoid and 2D monolayer cultures provide complementary, efficient, and clinically relevant models for personalized medicine.
Author Contributions
Y.L., and Y.-G.C. wrote the manuscript.
Research in the Y. G. Chen Lab is supported by grants from the National Key Research and Development Program of China (2017YFA0103601), and the National Natural Science Foundation of China (31330049) to YGC.
Conflicts of Interest
The authors declare no conflict of interest.
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Figure 1. Applications of organoid technology for drug screening and therapy in personalized medicine. Gene mutations in patients' tissues can be identified by genome or transcriptome sequencing. Meanwhile, the biopsy materials can be cultured in Matrigel to form organoids, which can be used to establish disease models via gene editing and co-culturing with niche cells and pathogens. The modified tumor organoids can be utilized for drug screening and testing. Gene mutations in organoids can be corrected by clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated gene editing for transplantation.
Figure 2. Models for intestinal epithelium-niche cells and epithelium–microbes/pathogen interactions. Both the 3D organoid culture and the transwell-based 2D monolayer culture can be used for the investigation of intestinal epithelium–niche cell interactions. To study the epithelium–microbe/pathogen interactions, microbes and pathogens can be introduced directly into the culture medium of 2D monolayer systems, while for the 3D cultures, pathogens need to be injected into the lumen of organoids to allow them to come into contact with the apical side of the epithelium.
|3D-Based Culture System
|2D-Based Culture System
|Air-Liquid Interface Culture
|Culture Technique
|Low-melting Agrose
|Transplanted into immune-deficient mouse
|Embended in Matrigel
|Cell lines
|Plate coated with Collagen I
|Transwell coated with Collagen I
|Plate coated with Matrigel
|Seeded onto 3T3-J2 cells which cultured on transwell coated with 20% Matrigel
|Cell lines
|Patient-derived tumors
|mIEC; hIEC; Patient-derived tumors
|Cell lines
|mIEC; hIEC
|mIEC; hIEC
|mIEC; hIEC; Tumors
|hIEC; Tumors
|Key Features
|In vivo-like complexity
|Lack of mature secretory lineage
|Lack of mature secretory lineage
|Stemness and multipotency
|Easily passaged
|Cancer subtype modeling (gene manipulation)
|Host-pathogen interaction
|High-throughput screening
|High-throughput imaging
|Time consumption for modeling
|Cost benefits
© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


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By Isabel L. Beck,Margaret G. McKeown,Linda Kucan
New to This Edition
*Reflects over a decade of advances in research-based vocabulary instruction.
*Chapters on vocabulary and writing; evaluation; and differentiating guideline for suffering readers and English language newbies, together with assurance of reaction to intervention (RTI).
*Expanded discussions of content-area vocabulary and multiple-meaning words.
*Many extra examples exhibiting what powerful guideline feels like in action.
*Appendix with an invaluable menu of educational activities.
See additionally the authors' Creating powerful Vocabulary: commonly asked Questions and prolonged Examples, inclusive of particular educational sequences for various grade levels, in addition to Making feel of Phonics, moment variation: The Hows and Whys, through Isabel L. Beck and Mark E. Beck, a useful source for K-3.
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LibreOffice x64 is the free power-packed Open Source personal productivity ... created to help users process their text documents, spreadsheets, presentations, drawings, databases and formulas. This software solution ... edit PowerPoint, Word or Excel files. LibreOffice x64 is a comprehensive, professional-quality productivity suite that you ...
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MedCalc 64-bit 16.8
MedCalc x64 is a complete statistical program for Windows designed ... It is fast, user-friendly and reliable. MedCalc x64 is the most user-friendly software for Receiver Operating ... method comparison studies. Data management Integrated spreadsheet with 16384 columns and up to 100000 rows. ...
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Matrex x64 2.2
Matrex x64 is an easy to use vectorial spreadsheet. It is better than usual spreadsheets when working with big data amounts coming from ...
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ANKHOR FlowSheet x64 184.108.40.20620
ANKHOR FlowSheet x64 takes table-based data processing to a new level ... of data graphs with the simplicity of common spreadsheets. FEATURES: Added: · PDF as an export format. · Header input to spreadsheet import. · Operator to extract a sub table ...
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Cytoscape x64 3.6.1
Cytoscape x64 is an open source bioinformatics software platform for ... expression profiles and other state data. Although Cytoscape x64 was originally designed for biological research, now it ... platform for complex network analysis and visualization. Cytoscape x64 core distribution provides a basic set of features ...
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WebIssues 64-bit 1.0.5
... other information, with the same flexibility as a spreadsheet, where columns can be freely added and modified. However, WebIssues allows many people to work simultaneously, maintains the full history of ...
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Newspaper article The Canadian Press
Editorial Exchange: Let Debate on Parliamentary Reform Act Begin
Article excerpt
Editorial Exchange: Let debate on parliamentary Reform Act begin
An editorial from the Toronto Star, published Dec. 8:
To call Canada's Parliament dysfunctional would be an understatement. It fluctuates between two extremes: all scandal all the time, and rote debates with MPs not daring to say what they believe or vote independently.
For four decades, parliamentarians have been wringing their hands ineffectually as a succession of prime ministers and their unelected officials have appropriated power that once belonged to the people's representatives.
Last week, in a welcome move, Michael Chong, a thoughtful Conservative backbencher, introduced a private member's bill designed to redress the imbalance between party leaders and their MPs and make parliamentarians more accountable to their constituents.
His timing was astute. Prime Minister Stephen Harper, who has imposed a rigid code of conformity on his Conservative colleagues, has been weakened in recent months by an embarrassing expense scandal in the Senate and an erosion of the unquestioning loyalty he commanded from his caucus for seven years.
Even so, the four-term MP faces plenty of opposition. Backroom organizers, cabinet ministers, pundits and political scientists immediately jumped on his bill, warning that his plan would destabilize Parliament, allow undesirable candidates to get elected and disrupt the business of government.
Chong reacted calmly. "I expect that many MPs will rightfully want to review the bill, understand it and make a determination," he said.
The proposed Reform Act would make three principal changes:
It would give the parliamentary caucuses of all political parties the power to trigger a leadership review, as they can do in Britain, Australia and New Zealand. Such a review would be triggered by a written request from 15 per cent of caucus members.
It would restore local control over party nominations by lifting the requirement that the party leader approve a prospective candidate. …


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Thursday, August 9, 2001
Code Red Nails Microsoft -- 10:16 am CST, Update by A.T. HunMicrosoft has confirmed that their Hotmail servers were hit by Code Red. I think I speak for everyone in the whole world when I say HAHAHAHAHAHAHA! Doesn't it say something when the company that makes the software doesn't patch their own servers running that software? Should anybody trust .NET to keep our data safe and secure? Hello?!? MS proves time and time again that they are utterly incapable of keeping servers running and secure for relatively mundane things like Hotmail and instant messaging. How will they possibly do it right with .NET? The answer: they won't and it will be .NET users who take it in the shorts. Thanks Slashdot.
J.t.Qbe comments: And let's not miss the irony here: just a short while ago Microsoft made a big deal about the fact that it had (allegedly) migrated all of Hotmail to Win2K, from its previous FreeBSD-based servers--where Code Red wouldn't have touched it at all! I'm with you, A.T.: HAHAHAHAHA!
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Alexa Moutevelis Coombs
Alexa is MRC Culture's On TV blog editor. She has previously worked for Students for Life for America and Citizens United Productions.
Alexa is MRC Culture's On TV blog editor. She has previously worked for Students for Life for America and Citizens United Productions. Alexa graduated in 2006 from Washington & Lee University.
Latest from Alexa Moutevelis Coombs
In its newest episode, "Got Milk?" Fox's post-apocalyptic comedy Last Man On Earth gave viewers a flashback to the time when the pandemic that has wiped out most of earth was just starting – and the show seemingly takes great delight in the current cabinet of Republicans dying in office.
Lena Dunham is a huge Planned Parenthood and abortion supporter. She was a Planned Parenthood abortionist for Halloween one year – and not in a bloody horror way, but in a "newsy, sexy, and cool" way. How much does Lena love abortion? Just in December she said, "I still haven't had an abortion, but I wish I had." Well, now she might get her wish, via her character Hannah on the HBO series Girls.
The slander of President Ronald Reagan's legacy by liberal gay activists continues. After ignoring his influence in stopping California's Prop 6 as governor, President Reagan is painted as a homophobe unwilling to acknowledge or help gays during the height of the AIDS crisis. This characterization of a conservative legend has been debunked but that still hasn't stopped the liberal gay activists who choose to re-write history in the ABC gay propaganda miniseries When We Rise.
On Wednesday night's episode of ABC's gay propaganda miniseries When We Rise, "Night II," the lesbians attempt to prove that so-called "male interference" is not required to have a child, while the gays think there's government interference on AIDS, with attacks on conservatives and President Reagan thrown in for good measure.
On a very special Black Lives Matter episode of Freeform's Switched at Birth, the show switches its focus from its main white and Latino characters to feature the stories of 3 black students at University of Missouri – Kansas City. It's about as preachy, melodramatic, and self-important as you'd expect, but it really strains credulity with an unintentionally comedic lecture on media bias.
ABC's eight hour gay propaganda event When When Rise isn't afraid to go low. Monday night's episode immediately gets into the leftist activism with a montage bashing Republicans and comparing gay rights to the fight against Nazism and the Civil Rights movement - and it all goes down hill from there.
The new CBS legal drama Doubt premiered on Wednesday night, and while it stars Katherine Heigl, Laverne Cox is getting all the attention for being the first transgender to be cast as a series regular on a network TV show - i.e. liberals are forcing the radical LGBQT agenda on American TV viewers again.
Lena Dunham's HBO show Girls was back for its sixth and final (thank God!) season on Sunday night and the notoriously liberal show shockingly took aim at New York Times columnist Paul Krugman and not-so-shockingly subjected viewers to the eye crime of seeing Dunham naked.
Tuesday night's episode of the TV Land comedy Teachers hilariously tackled Common Core and how much it "sucks." In the episode "Held Back," Ms. Feldman is minding her own business, creatively teaching her third grade class math when Principal Pearson comes in and tells her she must teach Common Core. Ms. Feldman balks, saying Common Core "sucks" and that she tailors her teaching to the kids' individual needs.
The fifth and final season of Freeform's Switched at Birth has the fingerprints of Social Justice Warriors all over it. The second episode, which aired on Tuesday night, "This Has to Do With Me," provided a "ripped from the headlines" storyline based on the Black Lives Matter campus movement that began sweeping schools and chilling free thought at universities in the fall of 2005.
We are now two episodes into the Jack Bauer-less re-make of Fox's 24, now titled 24: Legacy, and a nasty little trend has begun of showing our military heroes as men unable to function back in normal society.
If you just crawled out from under a rock and turned on Fox last night, you might have thought you were witnessing a presentation sponsored by the United Nations, instead of the Super Bowl. We weren't treated to "We are the World," but there was no shortage of advertisements pressing Americans to go the way of the world.
After a long delay, ABC's Scandal returned for its sixth season premiere on Thursday night, fast-forwarding to election night after ending season five in the middle of a presidential campaign between "Republican" First Lady Mellie Grant (Bellamy Young) and Democrat Francisco "Frankie" Vargas (Ricardo Chavira).
There is so much liberal propaganda to be appalled at from the third season of the Canadian teen drama Degrassi: Next Class, out now on Netflix. I suppose it's to be expected when the creator bemoans "some of the scary right-wing thinking that's out there." But all the teen sex – both hetero and homo, lectures on Syrian refugees, and talk of "safe spaces" pales in comparison to how the series handles abortion.
During the 74th Golden Globe Awards on NBC Sunday night, far left actress Meryl Streep delivered a dramatic, sanctimonious diatribe to the viewing audience and her fellow leftists in the audience. For almost six minutes, listeners were subjected to a laundry list of perceived wrongs that must be righted by liberal Hollywood. She may single-handedly assure Donald Trump a second term.
The new Fox "comedy" The Mick is receiving some much-deserved outrage for its depravity and sexualization of children. The show revolves around foul-mouthed, irresponsible grifter Mackenzie "Mick" Murphy (Kaitlin Olson) who is forced to look after her intolerable niece and nephews after her rich sister goes to jail.
Syfy's new futuristic anti-business series Incorporated continued on Wednesday night to push its global warming alarmist fantasies.
The new original anti-business drama Incorporated premiered this week on the Syfy network. Produced by Hollywood lefties Matt Damon and Ben Affleck, it's a liberal polemic preaching about the dangers of eeeeevil multinational corporations, capitalism, and global warming disguised as trendy, futuristic, post-apocalyptic entertainment.
ABC seemed intent on taking down organized religion on Tuesday night. In the Fresh Off the Boat episode "WWJD: What Would Jessica Do?" Jessica (Constance Wu) is terrified she will lose her youngest son Evan (Ian Chen) to God.
After last week's episode on the plight of the illegal immigrant, Tuesday night's episode of NCIS on CBS gives us the plight of the poor, innocent Guantanamo Bay detainee.


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After seeing how vindictive Angelina Jolie was to him throughout their custody struggle, Brad Pitt is now extra relieved than ever that he and the actress are divorcing. We've acquired the small print on why he's 'assured' together with his determination.
It was Angelina Jolie who filed for divorce from Brad Pitt approach again in Sept. of 2016. However after a bitter two-year custody struggle over their six children the place her authorized group tried to maintain Brad away from his kids, the 54-year-old actor looks like divorcing Angie is for the very best. "Brad feels that Angelina and her divorce techniques proceed to be hypocritical, vindictive, vengeful and controlling. The lengthy, arduous means of separating their lives and the custody struggle over their kids has solely strengthened Brad's resolve. He feels extra assured than ever that divorcing Angelina has been the precise factor to do for him," a supply near the star tells HollywoodLife.com EXCLUSIVELY.
Issues acquired so dangerous in June that the personal choose of their custody struggle admonished Angelina, 43, for inflicting potential hurt to their children by making an attempt to restrict their entry to their dad. He informed her she risked dropping main custody of Pax, 15, Zahara, 12, Shiloh, 12 and 10-year-old twins Knox and Vivienne. Eldest son Maddox, 17, was deemed sufficiently old to make his personal choices about his relationship with Brad. In court docket docs the choose informed Angie that "not having a relationship with their father is dangerous to them," including that "it's important that every of them have a wholesome and robust relationship with their father and mom."
"If the minor kids stay closed all the way down to their father and relying on the circumstances surrounding this situation, it could end in a discount of the time they spend with [Jolie] and should outcome within the Court docket ordering main bodily custody to [Pitt]," the paperwork continued. Brad ended up getting extra time with the kids and the previous couple lastly got here to a brief custody settlement on Dec. four to keep away from what may have been an unsightly trial.
"Whereas he was heartbroken over the lack of Angelina for some time, seeing the uglier facet of Angelina throughout this divorce battle has given Brad some peace and affirmation. He is aware of in his coronary heart that he's doing the precise factor and what's finest for his household. He's trying ahead to extra peaceable days forward, when the divorce is finalized and life post-Angie," our insider provides. Whereas the custody settlement is out of the way in which for now, the exes nonetheless have to financially divide up their property and resolve on any spousal help earlier than Brad and Angelina might be legally divorced.