Gene Modification in Mice
The 2007 Nobel Prize in physiology or medicine is awarded to Drs Mario R. Capecchi, Martin J. Evans and Oliver Smithies for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells. Their work has made it possible to modify specific genes in the germline of mammals and to raise offspring that carry and express the modified gene. The toolbox of experimental genetic methods developed by Capecchi, Evans and Smithies, commonly called the knockout technology, has permitted scientists to determine the role of specific genes in development, physiology, and pathology. It has revolutionized life science and plays a key role in the development of medical therapy.
Martin Evans identified and isolated the embryonic stem cell of the early embryo, the cell from which all cells of the adult organism are derived. He established it in cell culture, modified it genetically, and reintroduced it into foster mothers in order to generate a genetically modified offspring. Mario Capecchi and Oliver Smithies, independently of each other, discovered how homologous recombination between segments of DNA molecules can be used to target genes in the mammalian genome and developed methods to generate genetically modified mice. Such animals have become indispensable in medical research. Furthermore, the knowledge concerning stem cell biology and gene technology obtained during the research that led to the “knockout mouse” has changed our understanding of normal development and disease processes and identified new avenues for medical therapy. Fig. 1 shows the general strategy for gene targeting in mice.
|Figure 1. General strategy for gene targeting in mice.
A) Gene targeting of embryonic stem (ES) cells in culture is followed by cloning of an ES cell line containing the desired mutations. Positive-negative selection is used to enrich for ES cells containing the modified genes.
B) These ES cells are injected into blastocysts, which are injected into foster mothers to generate chimeric mice able to transmit the mutant gene to their progeny. To facilitate isolation of the desired progeny, the ES cells and recipient blastocysts are derived from mice with different coat colour alleles. In the figure, gene targeted ES cells and their progeny are shown in red and blastocysts in yellow.
Research that led to gene targeting
Embryonic stem cells
A stem cell is a cell that is capable of extensive proliferation, creating more stem cells (self-renewal) as well as more differentiated cellular progeny. Somatic stem cells are necessary for renewal of the tissues of the adult organism. For instance, hematopoietic stem cells in the bone marrow differentiate into blood cells, i.e. erythrocytes, megakaryocytes/platelets, and the different types of leukocytes. While each somatic stem cell of the adult organism is committed to a certain line of differentiation, the early embryo contains stem cells that are totipotent, i.e. they give rise to all cell types in the developing organism. Therefore, the thought that embryonic stem cells from the blastocyst could be used to create to a living mammalian organism has fascinated scientists for many years.
The concept that differentiated cells and tissues are derived from undifferentiated stem cells (“Stammzellen”) was already proposed a hundred years ago . However, their precise properties remained elusive for many decades. Studies of testicular teratomas showed that these tumours contain totipotent cells. In the 1950s, Leroy Stevens at the Jackson Laboratory found that mice of the 129Sv strain have a high frequency of such tumours. He showed that their cells could develop into embryoid bodies, i.e. aggregates of embryonic cells. When transplanted, such aggregates could induce solid tumours with many different cell types [2, 3]. A few years later, Kleinsmith and Pierce demonstrated that such tumours were derived from undifferentiated embryonal carcinoma cells .
The development of cell culture techniques permitted investigators to establish cultures of embryonal carcinoma cells (EC cells) from murine testicular teratocarcinomas. Several scientists including Martin Evans at the University of Cambridge reported on such cultures in the early 70s [5-7].
Evans obtained 129Sv mice from Stevens, established a colony of mice, and characterized the teratoma derived cells in culture [8, 9]. These embryonal carcinoma (EC) cells could be grown on feeder layers of irradiated fibroblasts. When the latter were withdrawn, extensive in vitro differentiation occurred. It proceeded through a primitive embryonic endoderm, which clumped into embryoid bodies. Attachment on a solid surface gave rise to all kinds of cell types, including skin, nerve, beating cardiac muscle, etc. This showed that the EC cells differentiated in the same way as the inner cell mass of the mouse embryo [8, 9].
Evans saw the potential in using these EC cells not only for cell culture studies but also for creating chimeric mice. In order to realise this vision, he established a collaboration with Richard Gardner in Oxford, who made injections of EC cells into blastocysts and reimplanted them into foster mice. The offspring was chimeric, with contributions from EC cells in nearly every tissue . Similar findings were made by several other groups at about the same time,  . However, chimeric mice carrying EC derived cells developed multiple tumours and could not contribute to the germ line due to karyotypic abnormalities.
It became obvious to Evans that an alternative strategy had to be used if one were to obtain germline transmission derived from cultured embryonic stem cells. With the use of monoclonal antibodies, he characterised cell surface macromolecules of EC cells and their normal counterparts, thus identifying molecular markers of early differentiation . The results suggested that normal cells with a similar phenotype as EC cells could be found and used for experiments. In 1980, Evans teamed up with the embryologist Matt Kaufman to combine cell culture and embryo manipulation. As described by Evans in a later review , he had intended to use haploid embryos for cell culture but prepared some diploid ones as controls. Evans writes :
“When I cultured these blastocysts as explants in tissue culture, using a medium that had been honed for optimum cloning efficiency of both mouse and human EC cells, I immediately noted an outgrowth of EC-like cells. These cells were clearly recognizeable as the sought-after pluripotential cells, and they passed every test: They formed teratomas in vivo, and they differentiated in vitro. They bore the cell surface antigens that we expected. They stained strongly positive for alkaline phosphatase, were karyotypically normal and, most importantly, made splendid chimeras.”
These cells were the embryonic stem cells (ES cells) that became critical for the success of gene targeting. Evans and Kaufman published their report on ES cells in a seminal paper in Nature in July, 1981 . Gail Martin, a former co-worker of Evans, reported similar findings half a year later . In their Nature paper, Evans and Kaufman pointed out the possibility of using ES cells for gene modification. They wrote :
“Their [i.e. ES cells] use as a vehicle for the transfer into the mouse genome of mutant alleles, either selected in cell culture or inserted into the cells via transformation with specific DNA fragments, has been presented as an attractive proposition. In many of these studies the use of pluripotential cells directly isolated from the embryos under study should have great advantages.”
Evans’ team set up blastocyst injection techniques to test whether indeed ES cells could contribute to functional germ cells and thus be used to create a chimeric mouse. They reported successful germline transmission in 1984, in another landmark paper in Nature .
The next step was to determine whether ES cells could be used to introduce genetic material into the germline. Evans and his co-workers infected ES cells with a recombinant retrovirus before injecting them into blastocysts . Retroviral DNA was identified in the founders and transmitted to the F1 offspring, demonstrating introduction of the foreign DNA into the mouse germline . In October, 1986, Evans et al. reported their findings in Nature and concluded that “cultured embryonic cells provide an efficient means for the production of transgenic animals” . In December of that year, another laboratory reported germline transmission of a neomycin resistance gene that they had introduced into ES cells by retroviral infection .
Evans now took the important step of introducing a mutant form of a specific, endogenous gene into the mouse genome. He and his co-workers transferred a mutant gene for hypoxanthine phosphoribosyltransferase (HPRT), which is defective in Lesch-Nyhan syndrome, an X-linked monogenic defect of purine metabolism . Several copies of the mutated HPRT gene were introduced into the genome of the ES cells by retroviral infection in culture. Mutated ES cells were injected into blastocysts and contributed to chimeras. The mutations were transmitted germline and identified in the male offspring as loss of HPRT activity. In a paper published in Nature back-to-back with the one from Evans’ lab, Hooper et al in Edinburgh reported germline transmission of another mutated HPRT gene, a spontaneous deletion mutation in ES cells . For the first time, models of human disease had been created by genetic manipulation of ES cells.
In their paper , Evans and his co-workers point out that their success “opens up the possibility of deriving strains carrying specifically induced alterations in other genes” and suggest that “it may also eventually be possible to produce specific alterations in endogenous genes through homologous recombination with cloned copies modified in vitro”, citing the work of Capecchi and Smithies [23, 24]. Indeed, the combination of the two technologies revolutionized experimental medicine, as we now know nearly 20 years later.
Gene targeting and transgenesis in mammals
The mouse has been a favourite animal for genetic studies for many decades and was an obvious choice for the first attempts to introduce new genes into the mammalian genome. Work in several laboratories had defined conditions for manipulating fertilized mouse eggs and blastocysts in culture. Using these culture techniques, SV40 virus DNA was introduced into blastocysts, which were subsequently implanted into pseudopregnant foster mothers. SV40 DNA could be detected in the offspring but it was impossible to demonstrate with certainty whether the DNA was integrated into the host genome, or remained as episomes . A few years later, the first transgenic mouse was created by infecting embryos with Moloney leukemia virus . A DNA copy of the viral RNA was present in the genome of the transgenic mice and was transferred to the offspring in a Mendelian fashion, therefore virus DNA had been introduced into the mouse germline. Subsequent development has made it possible to introduce and overexpress a large number of transgenes in mice and also other mammals . However, integration of the foreign DNA in the genome occurs at random and the number of copies varies. Although an important tool in life science, transgene technology of this kind lacks precision with regard to the inserted gene and cannot be used to manipulate endogenous genes in a predetermined manner. These inherent problems with the transgenic overexpression technique limit its usefulness.
The principle of recombination between homologous genes has been known for half a century and was recognized by a Nobel Prize to Joshua Lederberg in 1958 for his studies in bacteria. In the 70s, it became evident that eukaryotes employ a similar machinery to mediate exchange of genetic information between homologous chromosomes during meiosis. Early studies in yeast were followed by experiments demonstrating recombination between retroviral DNA sequences in the mammalian genome and introduced oligomeric retroviral DNA. Pioneering work by Richard Axel (2004 Nobel Prize for the discovery of odorant receptors) showed that cultured mammalian cells defective in thymidine kinase could be rescued by introduction of the herpes virus thymidine kinase (tk) gene . Mario Capecchi decided to improve the method and used a fine glass pipette to inject DNA directly into the nucleus . This improved the efficiency of gene transfer dramatically and Capecchi’s method was rapidly adopted by other investigators to introduce new genes into fertilized mouse embryos and produce transgenic mice . However, the transferred gene was still introduced at random in the host genome.
Capecchi now made a crucial observation: when the tk gene was injected, copies were integrated in only one or two loci of the host genome, with multiple copies forming head-to-tail concatemers. He reasoned that such concatemers could only be generated by two mechanisms: either by replication or by homologous recombination. A series of careful experiments were performed, which unequivocally demonstrated that head-to-tail concatemers were generated by homologous recombination . This, in turn, provided evidence that mammalian somatic cells possess an efficient enzymatic machinery for mediating homologous recombination. If this machinery could be harnessed to accomplish homologous recombination between a newly introduced DNA molecule and the same DNA sequence in the recipient cell’s genome, any cellular gene could be mutated.
Capecchi now submitted a grant proposal to the U.S. National Institutes of Health to test the feasilibity of gene targeting in mammalian cells. It was rejected since the reviewers considered it extremely unlikely that the introduced DNA would find its matching sequence within the host genome (cited by Capecchi in a later review )! At about the same time, Martin Evans et al in England proposed a similar strategy in a grant application to the UK Medical Research Council, which was also turned down for being over-ambitious!
Capecchi decided to continue working on homologous recombination in spite of being turned down by NIH. He generated recipient cell lines that carried a defective neomycin resistance gene (neor) and was able to repair it by introducing a functional neor gene . Correction occurred at a relatively high frequency (in one cell per 1,000 injected cells), making it likely that homologous recombination could be used to manipulate genes of the mammalian genome.
In parallel with Capecchi’s work, Oliver Smithies had developed the concept that homologous recombination might be used to repair mutated genes. As early as the 1960s he had already established that an allelic variant of haptoglobin had occurred through recombinatorial events . Later on, he cloned human fetal globin genes and concluded that Gγ and Aγ had arisen through a process involving homologous recombination . He devised a stepwise selection procedure for recovering targeted cells carrying modified genes. The strategy was successful and he reported in a landmark paper in the September 19, 1985 issue of Nature the successful integration by homologous recombination of a plasmid into the chromosomal β-globin gene of human erythroleukaemia cells .
By 1985, Capecchi had shown that homologous recombination occcurs with high frequency in mammalian cells and Smithies had used homologous recombination to insert a plasmid DNA sequence into a chromosomal gene of a human cell. However, all this work was carried out in cell culture. Could homologous recombination be used to target genes in the germline and obtain strains of genetically modified animals? Both Capecchi and Smithies had heard of Martin Evans’ ES cells and decided to give them a try. With the help of Evans, they both set up ES cell culture for use in homologous recombination experiments.
Smithies first used homologous recombination to correct a mutant HPRT gene in cultured ES cells . For this purpose, an ES cell line was used that carried a deletion mutation; this cell line had previously been used for production of mutant mice. The HPRT gene was repaired with a plasmid carrying the missing promoter and first 2 exons and Smithies showed that treated cells survived and grew in HAT selection medium, which requires HPRT enzyme activity. Smithies and his co-authors concluded that “This modification of a chosen gene in pluripotent ES cells demonstrates the feasibility of this route to manipulate mammalian genomes in predetermined ways” .
Capecchi’s team also chose the HPRT gene for their early studies. Standard methods were available for selectively growing cells with functional HPRT enzymes and had already been used for several years for selection of mutants, hybridoma cells in monoclonal antibody production etc. Thomas and Capecchi  introduced a neomycin resistance gene into an exon of the HPRT gene in ES cells and showed that clones of transfected cells had lost HPRT but gained neoR activity. They concluded in their Cell paper that “It is hoped that this combination of using ES cells as the recipient cell line and site-specific mutagenesis achieved by gene targeting will provide the means for generating mice of any desired genotype.”  They continued by outlining an experimental strategy:
“An advantage of this scenario is that the first generation chimera will usually be heterozygous for the targeted mutation and that subsequent breeding can be used to generate the homozygous animal. Thus, only one of the two loci need be inactivated, and recessive lethals can be maintained as heterozygotes. If successful, this technology will be used in the future to dissect the developmental pathway of the mouse as well as to generate mouse models for human disease.”
This vision has become reality and is now a cornerstone of experimental medicine.
It was important to proceed from the “model gene” HPRT to a general strategy that would allow targeting of genes whose function cannot be selected for in cell culture. Thomas and Capecchi  had pointed out that the frequency of homologous recombination vs random integration was 1/1,000, which should be high enough to permit targeting of non-selectable genes as well. This observation prompted work to develop the methods needed for such approaches. The following year, Capecchi’s positive-negative selection strategy for enriching ES cells containing a targeted disruption of any transfected gene was presented in Nature  (Fig 2). A neomycin resistance element (neoR) is introduced into an exon of the replacement vector, which also has a thymidine kinase (HSV-tk) element at its end. Homologous recombination of the targeted gene will result in neoR expression but the tk element will be lost since it was outside of the recombining DNA sequences. In contrast, random integration of the replacement vector will introduce tk as well as neoR into the gene. This strategy was successfully used to disrupt the int-2 gene, which is a member of the fibroblast growth factor (FGF) family .
All the components were now in place for producing gene-targeted mouse strains: the development of ES cell culture, the demonstration that gene modification in such cells can be transmitted to the germline and registered in the offspring, the observation that homologous recombination occurs with high frequency in the mammalian genome, the application of gene transfer methods to ES cells, and the invention of strategies for enriching transfected cells. Several laboratories joined the race and 1989 saw the birth of several different knockout mice [38-41]. Fig 3 shows the molecular evidence for correction of the mutant HPRT gene in the germline by Smithies and co-workers .
Further development of gene targeting technology
After the establishment of gene targeting technology, several important modifications and developments, in several laboratories, have extended its use in significant ways. An ingenious development of gene targeting has been made by introducing recognition sites for the enzyme Cre recombinase, so-called loxP sites, into existing genes. When mice carrying such “floxed” genes are mated with transgenic mice expressing Cre recombinase, the target gene of the offspring is modified through Cre action [42-44]. Another site-specific recombinase, Flp, is also frequently used to construct conditional targeting of genes in mice . The activity of the Cre, or Flp, gene can be controlled by placing it under a suitable promoter to achieve tissue-specific gene targeting . Expression of Cre and hence targeting of the floxed gene can be restricted to e.g. T cells (lck promoter), cardiac muscle (cardiac myosin promoter), neurons (enolase promoter) or epithelia (cytokeratin promoter).
Cre expression can also be controlled temporally, by introducing an element into the promoter which requires a ligand such as a drug for induction . Tetracyclin, type I-interferon and tamoxifen (which binds to an estrogen receptor-binding element) have all been used to obtain drug-inducible promoters. In this way, a desired gene can be targeted by administrating the drug. By introducing a tamoxifen site into a tissue-specific promoter, gene targeting can be obtained selectively in a certain tissue when the mouse is treated with the drug.
Cre-lox technology can also be used to replace an existing gene with another one . Such “knock-in” has been used e.g. to replace murine immunoglobulin or MHC genes with human ones in order to “humanize” the mouse with regard to immune function. It has also been used to replace an allele with another one, the latter for instance being an allele suspected to cause disease.
Significance of gene targeting for physiology and medicine
Gene targeting has transformed scientific medicine by permitting experimental testing of hypotheses regarding the function of specific genes. Prior to gene targeting, our understanding of the role of genes in higher organisms was deduced from observations of spontaneous mutations in patients and experimental animals, linkage and association studies, administration of gene products to animals and, to some extent, from cell culture experiments. However, cell culture is not helpful for understanding functions and diseases involving multicellular, integrative responses. Insights into organ systems such as the nervous system,the cardiovascular system, and the immune system, were fragmentary at best, as was knowledge of mammalian development. As the cardiovascular physiologist Heimo Ehmke put it, “cells don’t have blood pressure” . The possibility of observing the effects on the intact organism of destroying a candidate gene transformed these areas of research. For instance, cardiovascular physiologists switched from rats to mice as models, downscaling their instruments and techniques in order to study the genetic regulation of hemodynamics. A new era of genetic physiology was born.
The genomes of man and mouse contain about 22,400 genes. Several thousand of them have already been investigated by gene targeting. Collectively, these studies have provided a wealth of information about gene function in development and disease. They have helped fuse mechanistic molecular biology with integrative life sciences such as embryology, physiology and immunology and have prompted new technical developments in physiological sciences. For medicine, the modeling of human diseases by gene targeting in mice has been particularly informative.
At this stage, it may be helpful to recapitulate the criteria first proposed by Claude Bernard for the scientific method in medicine : Medical scientists use observations, hypotheses and deductions to propose explanations, theories, for natural phenomena. Predictions from these theories are tested by experiment. Any theory which is cogent enough to make predictions can be tested reproducibly in this way. Therefore, the scientific method is essentially a cautious means of building a supportable, evidence-based understandingof our natural world. Experiments are crucial in this process.
Prior to gene targeting, genetic medicine lacked the means for experimental testing. If we make an analogy with Robert Koch‘s approach to infectious diseases , genetic medicine could apply the first of Koch’s postulates (i.e. observe an association between microbe, or in this case, gene or allele, and disease) and with the advent of gene cloning, the second one (isolate the microbe/gene from the diseased individual and establish it in culture), but applying Koch’s third postulate (induce the disease by transferring the microbe/gene to a host organism) required gene targeting. By mutating a gene to destroy its function (knock-out) or switching it to a disease-associated allele (knock-in), disease is induced if the hypothesis is correct. Alternative approaches based on genetic epidemiology are currently being developed but currently available methods do not have the precision of hypothesis-based experiments. This digression into scientific theory may suffice to make the point that only by targeting candidate genes did it become possible to formally establish causality between gene and disease. Let us now look at some specific examples of the impact of gene targeting in medicine.
|Figure 3. Molecular genetic evidence for germline transmission of repaired HPRT gene. From ref . The Southern blot shows genomic DNA hybridized to a probe specific for a sequence in the targeting vector. ES cells were from agouti mice, blastocysts from black mice and the HPRT gene resides on the X chromosome. The tumor was from a chimeric mouse that carried the targeted gene, as did the targeted ES cell line. In the F1 offspring, female agouti mice derived from targeted ES cells carried the targeted HPRT gene, while it was present neither in black mice derived from recipient blastocysts nor in male agouti mice. The lower part shows the targeting strategy, with the deletion mutation in the HPRT gene (a), the targeting construct (b), the HPRT gene corrected by gene targeting (c), and the probe (19*) used for hybridization (d). Reprinted, with permission, from Proceedings of the National Academy of Sciences. Copyright 1989 National Academy of Sciences, U.S.A.|
The first area to which experimental geneticists turned their attention after the birth of gene targeting in mammals was monogenic diseases. The Lesch-Nyhan syndrome, a defective nucleotide metabolism caused by a mutation in the HPRT gene, actually served as the model condition during development of the technology, both in Evans’ and Smithies’ laboratories (see above). One of the reasons for chosing this particular medical condition was because selection conditions for isolating transduced cells were available for HPRT. The first examination of HPRT-/- mice was disappointing since neither neuropathological nor behavioural features of human disease could be observed [21, 39]. This prompted analysis of purine salvage pathways in mice and led to the findings that mice depend largely on adenine phosphoribosyltransferase (APRT) for purine salvage and are therefore not as sensitive to HPRT deficiency as humans. Administration of an APRT inhibitor to HPRT-/- mice induced persistent self-injurious behaviour resembling the clinical features in human disease . This is an illustration of the need for sophisticated analysis of integrative functions when characterising the phenotype of gene-targeted mice.
Cystic fibrosis is one of the most common monogenic disease and was chosen for gene-targeting studies by Smithies and his co-workers [53, 54]. The defective gene had been identified by linkage studies in patient families followed by molecular cloning. It turned out to be a cAMP-activated choride channel and was termed cystic fibrosis transmembrane conductance regulator (CFTR). By knocking out CFTR in mice, a condition was generated that reproduced many features of the human disease. Thus, CFTR-/- homozygotes displayed defective chloride transport in epithelia of airways and intestines, failure to thrive, meconium ileus, and pathological alterations of gastrointestinal glands. These studies were among the first to create a model of a human disease by gene targeting in mice. They have been followed by an avalanche of such knock-out models.
The pathogenesis of inherited heart diseaseshave been explored successfully by gene targeting approaches [55, 56]. For instance, targeting of genes encoding components of the contractile apparatus in cardiomyocytes leads to cardiomyopathy; targeted mutations in connexin proteins of gap junctions cause conduction defects; disrupted genes for transcription factors involved in heart development lead to congenital heart malformations; and targeting of genes controlling energy metabolism causes cardiomyopathy.
Complex diseases involving the action of more than one gene, and in addition, gene-environment interactions, represent a particular challenge for medical research. Inheritance, penetration and interactions are usually poorly understood, it has been difficult to dissect the contribution of an individual genetic factor, and the distinction between causation and correlation has been problematic. In order to prove causation in such a complex system, experiments must permit detection of the effects of changing only a single variable at one time. Gene targeting made such experiments possible and has permitted proof of causation in complex diseases.
Oliver Smithies has been the leader in this development. Together with Nobuyo Maeda, he focused on two important, complex diseases, hypertension and atherosclerosis (reviewed in ). Twin studies suggest that genetic factors may account for approx 70% of familial aggregation of essential hypertension. However, at least 10 genes have been shown to alter blood pressure and their gene products appear to interact in complex ways. In spite of the discovery that angiotensinogen (AGT) gene polymorphism is associated with essential hypertension, the genetics of this disease has remained poorly understood . Little is known about the number of genes actually involved in human essential hypertension, their quantitative effect on blood pressure, their mode of transmission, or their interaction with other genes and environmental components.
Smithies suspected that gene dose effects would impact on blood pressure levels and designed a new method for titrating gene dosage by producing mice with one, two or three functional copies of the AGT gene . “Conventional” gene targeting was used to produce the one- and two-copy mice and gap-repair gene targeting to produce mice with three copies of the AGT gene. This resulted in proportionally higher levels of gene products (i.e. plasma angiotensinogen protein) and, importantly, proportionally higher blood pressure with increasing gene copy number. When Smithies et al targeted another important gene for blood pressure regulation, the one coding for the angiotensin-converting enzyme (ACE), no such linear relationship was observed, in spite of the effectiveness of ACE inhibitors in reducing blood pressure. The investigators submitted their data to a computer simulation for complex interacting systems and could propose a model for blood pressure control through the renin-angiotensin system, which has proven to be useful for understanding essential hypertension . It shows that gene dosage, gene expression, and gene product clearance/catabolism must all be considered when evaluating the genetic regulation of blood pressure.
In 1992, Nobuyo Maeda, working in Smithies’ department at the University of North Carolina, developed a mouse model of atherosclerosis by targeting the gene for apolipoprotein E (Apoe) . The same gene was targeted independently by investigators at Rockefeller University . The Apoe-/- mouse develops spontaneous atherosclerosis which is remarkably similar to human disease. The following year, Michael Brown and Joseph Goldstein (1985 Nobel Prize for discoveries concerning cholesterol metabolism) and their co-workers targeted the gene for the low density lipoprotein (LDL) receptor (Ldlr) and obtained a mouse that develops atherosclerosis when fed a cholesterol-rich diet . The introduction of the two mouse models with defective Apoe and Ldlr genes have completely changed atherosclerosis research. By crossbreeding them with other gene-targeted mice, it has been possible to deduce the importance of genes regulating inflammation, lipid metabolism, blood pressure and other factors proposed to be involved in atherosclerotic cardiovascular disease . They are also used abundantly in the pharmaceutical industry for development and testing of new drugs against coronary artery disease.
Gene targeting has been exceptionally useful in cancer research. A large number of protooncogenes, tumor suppressor genes, angiogenetic factors etc have been targeted in different tissues in mice to shed light on the induction and spreading of tumours . Gene targeting of tumour suppressor genes have helped clarify their role in the formation of tumours. For instance, mice carrying a targeted p53 gene were predisposed to tumour development . Conditional targeting (using Cre-lox technology) of the adenomatous polyposis coli (APC) gene induces colorectal tumors in mice and APC-targeted mice have become useful models for research on solid tumours . Targeting of genes for endothelial growth factors and proteolytic enzymes have been essential for understanding mechanisms of neoangiogenesis and metastasis of solid tumours and are also used for developing therapeutic strategies to prevent spreading .
Contemporary research into most if not all major human diseases involves gene targeting in mice and there are “knockout models” for endocrine, metabolic, neurological, inflammatory and other disorders. Gene-targeted mouse models have also become increasingly important in studies of host defense against pathogens. Indeed, gene targeted mice have become indispensable in virtually all aspects of medical research.
Gene targeting has transformed physiology and medicine. Among the basic biomedical sciences, it is difficult to imagine contemporary medical research without the use of gene targeted models. The ability to generate predictable designer mutations in mouse genes has led to penetrating new insights into development, immunology, neurobiology, physiology, and metabolism. It has also allowed disease models of human pathologies to be generated in a tractable mammalian system and consequently enabled experimental dissection of disease states, identification of new therapy targets and the development of test systems for pharmacology. Finally, it is obvious that the development, in the future, of novel therapies to correct genetic defects in man will build on the experience of gene modification in mice that is based on the discoveries made by Mario Capecchi, Martin Evans and Oliver Smithies.
Göran K Hansson
Professor of cardiovascular research at Karolinska Institutet
Member of the Nobel Committee for Physiology or Medicine
Their work and discoveries range from how cells adapt to changes in levels of oxygen to our ability to fight global poverty.
See them all presented here.


What’s the best way to slow climate change? Governments have mostly wasted the last twenty years, conspicuously failing to effectively reduce the greenhouse gases in the Kyoto Protocol basket ( CO2, methane, nitrous oxide, hydrofluorocarbons, perfluorocarbons, and sulphur hexafluoride). Maybe we can do better on soot – also known as particulate air pollution, which is easier and cheaper to control.
Recent research shows that soot plays a significant role in global climate systems. It has been linked to changes in cloud cover, dust storms, and forest fires (especially in the boreal forest) as well as both global and regional climate effects including Arctic warming, Himalayan glacier melt, and changes in monsoon patterns. For a survey of this research, see, for example, V. Ramanathan, & G. Carmichael “Global and regional climate changes due to black carbon” (2008) 1 Nature Geoscience 221.
Another attribute of soot that has attracted attention is its short lifecycle and the corresponding opportunity to see much faster reductions in climate forcing following a reduction in soot emissions (as compared to reductions in the long-lived Kyoto basket gases). Soot is removed from the atmosphere by wet removal and direct deposition in as little as one week . This is in contrast to the gases in the Kyoto basket which have a lifetime of at least 2 years and carbon dioxide, which has a lifecycle of 100 years or more
Earlier this month, Canada joined the Global Alliance for Clean Cookstoves. The aim of the initiative is to provide efficient cookstoves to people who are reliant on biomass stoves and open fires for cooking and heating. They hope to support the adoption of clean cookstoves and fuels in 100 million households by 2020.
What’s missing from this announcement about soot reduction is what Canada will be doing at home. As illustrated in the charming video produced for Ecojustice’s Stop Soot Campaign, soot is the second leading cause of arctic warming and tighter standards on diesel fuel would help to reduce this impact.
by Meredith James and Dianne Saxe


About Arch of Constantine
The Arch of Constantine was a triumphal arch built by the Roman Emperor Constantine I, also known as Constantine the Great, in 315AD.
Erected to commemorate Constantine’s victory at the Battle of Milvian Bridge in 312AD, the Arch of Constantine contains an inscription dedicated to the emperor which can still be read today.
The Arch of Constantine is situated next to the Colosseum and near to the Palatine Hill and Roman Forum. It is free to visit and there are no opening hours. As well as the Arch of Constantine, there are two other triumphal arches in Rome, the Arch of Titus and the Arch of Severus.


When you hear of a disease named after a certain sporting activity – tennis elbow or runner’s knee for instance – you naturally tend to think that people who do those sports like crazy are the only ones who are affected. Of course, the truth of the matter is, these conditions are named this way for no particular reason. While runners knee is something that runners are often afflicted with, any kind of athlete who uses their legs – i.e. a cyclist, pole vaulter, boxer or even a weight lifter can be struck down by the ailment just as easily. In fact, runners knee isn’t actually one specific condition , it’s a term that doctors use loosely to describe any of a set of problems that occur in the kneecaps.
For instance, any kind of athletic activity that requires you to bend your knee and straighten it repeatedly can bring the condition on. Doing that can rub the nerves the wrong way. Sometimes, the tendons that connect your muscles to the bones around the knees can get injured with repeated bending. And about half the time, overworked and inflamed tendons act up in such a way that you experience quite a bit of pain. In some people, simply to fall hard on their knees can cause the kind of nerve or tendon injury that overuse can cause. Not good!
Runner’s knee can feel really painful. You experience a serious pain around the kneecap – most pointedly where the thigh bone meets the kneecap. You can also feel a grinding and popping sensation in the knee and there is usually a good bit of pain when you climb stairs or when you try to walk or sit. Your doctor can give you an MRI or an x-ray or CT scan to diagnose you. While all of this does sound kind of scary, the good news is that it can be relatively straightforward to treat runner’s knee. Most minor cases go away with a bit of the rest and icing your knee for a half hour every day for a week or using a tight knee support. If it hurts when you sit down, you could try elevating your knee on a pillow. Athletes often take NSAIDs – painkillers like Advil – to settle the swelling and pain.
Athletes often have aching joints and muscles and often pop acetaminophen in place of Advil to try to prevent the stomach problems that can be associated with some painkillers. If you have runners knee and you need to take a painkiller, doctors are usually of the opinion that Tylenol is the safest bet – providing you take no more than six tablets a day.
The military also has a huge problem with runner’s knee, especially in boot camp. Recruits who show up and go through the military’s grueling series of exercises often complain of this affliction and, in fact, runners knee happens to be one of the major reasons why military recruits drop out.
Researchers have found now that simple well thought-out stretching exercises can help a great deal with keeping running knee away. Exercises especially that strengthen thigh muscles and leg muscles making them stronger and more flexible make the occurrence of runner’s knee a lot less frequent.


Military professionalism is commonly grounded in several overriding principles: the subordination of the military to democratic civilian authority, allegiance to the state and a commitment to political neutrality, and an ethical institutional culture. These principles are enshrined in values that distinguish the actions of a professional soldier such as discipline, integrity, honor, commitment, service, sacrifice, and duty. Such values thrive in an organization with a purposeful mission, clear lines of authority, accountability, and protocol. Despite the disappointing track record, these same principles and values of professionalism resonate deeply with African military leaders and ordinary citizens alike. The problem has been that in too many African countries the adaptation and implementation of these concepts have been disrupted.
Democratic Sovereign Authority
A democratic political culture is typically the foundation of professional militaries. In Samuel E. Finer’s classic The Man on Horseback: The Role of the Military in Politics, the level of democratic political culture in a country is determined by the extent to which there exists broad approval within society for the procedures of succession of political power and a recognition that citizens represent the ultimate sovereign authority.5 Democratic processes also need to be protected by state institutions, such as the armed forces. The notion of military professionalism in democratic states, therefore, must embody basic values such as acceptance of the legitimacy of democratic institutions, nonpartisanship in the political process, and respect for and defense of individuals’ human rights. In a strong democratic political culture, legitimately elected civilian authorities are fully responsible for managing public and political affairs. The armed forces implement the defense and security policy developed by civilian authorities.
A majority of African states have duly adopted these democratic values and basic principles of military professionalism in their various constitutions and military doctrines. They are shared and accepted by the majority of African countries that have transitioned or are in the process of transitioning to a democracy. Moreover, many military leaders have been exposed to these values and principles through trainings in Western military academies and staff colleges. It is important, too, to note that these values are rooted in African culture. Protection of the kingdom, submission to the king, loyalty, and integrity vis-à-vis the community were core values of African ancestral warriors. It was only during the colonial and neo-colonial eras that this civil-military relationship foundered and these values eroded. The newly created African states set up armies to symbolize their nations’ independence, but these militaries essentially provided security just to the new regimes. Since then, we have witnessed an ongoing struggle to recapture the historical values of military professionalism.
Building professional militaries is dependent upon establishing a clear and balanced allegiance to the state and respect for civil society and not interfering in the oftentimes robust political discussion between the two. Democratization is a turbulent process, one that can be exploited by certain actors to create temporary domestic instability. Without a military’s steadfast support for neutrality and democratic sovereign authority, the process of democratic self-correction and consolidation will be difficult.6
One African military that has adhered to this principle is the Senegalese Armed Forces. Since independence, Senegal has never experienced a coup d’état. The Senegalese democratic culture has been periodically tested by political tensions, but the military has not challenged the constitutional order. Having passed these tests, Senegal’s democratic culture has grown stronger over the years.
In addition to Senegal, Botswana, Cabo Verde, Mozambique, Namibia, Zambia, and a few others are part of a small group of African countries whose governments have never been toppled by a military coup.7
Countries that have experienced a coup d’état pay a steep and longterm price for their militaries’ misplaced and sometimes cyclical interference in political discourse. Once the precedent of a coup has been established, the probability of subsequent coups rises dramatically. In fact, while 65 percent of Sub-Saharan countries have experienced a coup, 42 percent have experienced multiple coups.8 Most African coups were directed against an existing military regime that itself had come to power through a coup. Between 1960 and 2012, 9 of the attempted coups in Sudan were against military regimes as were 7 of the 10 in Ghana during the same time period. Once in place, such precedents become a burden that is difficult to throw off and have contributed to the collapse or destabilization of some states. Reflecting a degree of progress, while the threat of coups remains a real concern in Africa, the frequency of successful coups has diminished considerably (and has been concentrated in West and Central Africa) since the mid-2000s compared to previous decades (see Table 1).
Factors such as political and economic weakness, corruption, and a lack of institutionalized democratic structures create openings for military forces to justify overthrowing political leaders. Unsurprisingly, Sub-Saharan countries with low per capita gross domestic product (GDP) growth since independence have experienced more military coups than countries with higher per capita GDP growth rates.10 Yet, with very few exceptions, military coup leaders fail to restore stability and hand power back to civilians. Unfortunately, military-led governance is likely to be ruinous for a country’s economy. Thus, the vicious cycle is perpetuated. Guinea, Mali, Mauritania, and Nigeria have all seen their real GDP shrink more than 4.5 percent following military coups.11 Figure 1 highlights the negative correlation between multiple coups and a country’s long-term per capita GDP growth. Repeated coups have thrown some African countries, such as Burundi, the Central African Republic, Comoros, the Democratic Republic of the Congo, and Guinea-Bissau, into prolonged periods of economic contraction. Apparent exceptions like Equatorial Guinea, in fact, reflect natural resource-driven growth that has not translated into improved living conditions for the general population. Instability deters investment and development. In contrast, non-resource-rich states that have realized the highest levels of sustained growth are almost uniformly those with few or no coups.
Samuel Huntington argued that military interference in governmental affairs was more a political than a military problem, an observation that remains particularly relevant for most African countries.13 In the absence of well-established rules and strong institutions regulating political processes, labor unions, students, clergy, lobbies, and the military all engage in political competition for the control of state power. This characterized the post-independence political environment in many African countries. Given their size and inherent influence, militaries in Africa thus became major players on the political scene—and held onto this privilege.
With the emergence of alliances between top military, political, and economic leaders (including, at times, foreign partners) around shared financial interests, militaries’ intrusion into the economic sphere also became more diffuse and complex. In Angola, for example, members of the military participate in contract negotiations with foreign companies, sit on corporate boards, and are majority shareholders of telecom companies.14 The various military administrations that governed Nigeria also entangled themselves in the economic sphere. Appointments of military officers to company boards of directors and to top political posts, such as state governors, made these individuals immensely rich and politically powerful. Even in retirement, many of these officers remain powerful actors in Nigerian politics.
In 1999, when the Nigerian people elected the democratic government of Olusegun Obasanjo, a retired general, they ended 16 years of military rule. The new government understood what years of military involvement in commercial enterprise had done to its reputation and effectiveness. The government quickly swept many officers into retirement, revoked oil licenses, and reclaimed plots of land suspected to have been illegitimately allocated to senior military officers.15 Such efforts to improve security sector governance in Nigeria are ongoing.
The subversion of the profession of arms for financial enrichment distorts the incentives for public service required of an effective, professional military. It simultaneously undermines a military’s commitment to protect the country and its citizens. Plato noted some 2,400 years ago that the meddling of soldiers into other professions will “bring the city to ruin.”16
Egypt’s Complex Civil-Military Relations
“Egypt’s second republic will only come to life when the officers’ republic ceases to exist.”17 This somber warning reflects decades of complicated civil-military relations in Egypt. Egypt became a nominal republic in 1952 when the military deposed King Farouk. The country’s first president, Gamal Abdel Nasser, was a former military officer. So were successive leaders, Anwar Sadat and Hosni Mubarak, amassing 59 years of de facto military governance. The military was effectively the power behind the power in Egypt, intimately involved in policymaking and maintaining the status of the incumbent. The military took over state-owned enterprises,18 controlling many economic ventures—from running daycare centers and beach resorts to managing gas stations. And it reported none of this income to the government.19
The events of the Arab Spring in 2011 precipitated a break in this pattern. The massive scale of the protests led the military to conclude that stability of the state was threatened, and it orchestrated the arrest and deposal of Hosni Mubarak in February of that year. The military earned widespread plaudits from Egyptians and the international community during this time for the restraint it demonstrated in not responding with violence against the largely peaceful demonstrations. The military effectively took control of the government at that point, ruling under the auspices of the Supreme Council of the Armed Forces (SCAF).
Facing a spiraling economy and numerous domestic and international pressures for change, the SCAF organized a hasty process for drafting a new constitution and a timetable for parliamentary and presidential elections. Parliamentary elections were held in late 2011 with the Peace and Justice party, representing the Islamist Muslim Brotherhood, emerging as the dominant political actor.
Eager to protect the military’s many political and economic privileges from a new head of state, the SCAF issued a supplemental constitutional declaration just days before presidential elections in June 2012. In it, SCAF assumed control over all military affairs and gave itself a significant role in the committee charged with drafting the new constitution.
After Mohamed Morsi became Egypt’s first democratically elected leader, he purged the generals who had issued the constitutional addendum, but he was unable to roll back the powers and privileges the SCAF had bestowed upon itself. The military and the new civilian leaders appeared to have reached an uneasy governing arrangement with the military retreating from a public governance role and even acceding to the replacement of top generals by President Morsi.
The constitution that subsequently passed through referendum in December 2012 made the military’s autonomy official. It required that the minister of defense be a military officer. Parliament did not have oversight of the defense budget. Instead the budget was placed under the purview of the National Defence Council—a 15-member authority, 8 of whom were military20—leaving the public and government in the dark about how the military would spend its annual budget allocations and expenditures. It also meant that the military could continue to earn untold levels of revenue through its economic privileges. From television sets to olive oil, the military’s vast economic ventures pay no taxes, use soldiers as laborers, and buy public land on favorable terms.21
The exclusive governing style of President Morsi, his assertion of expansive and ill-defined powers, continuing economic turmoil, deepening divisiveness borne of fears that Islamists were seeking to dominate Egyptian society, and ongoing uncertainty over the legality of the 2012 constitution led to steadily growing tensions within Egyptian society. Massive protests in late June 2013 again created an opportunity for the military to intervene. On July 2, the military ousted the sitting president, again on the justification of maintaining national stability. In the subsequent protests by Morsi’s supporters the military opted to use force to disperse the protesters and arrest leaders of the Muslim Brotherhood. This crackdown claimed over 1,000 lives.
In the ensuing year, the military further expanded its control, arresting an estimated 20,000 people perceived as a threat to the military and clamping down on endent media and civil society.22 Through a new constitution passed by referendum in 2014, the military affirmed autonomy over its budget, immunity for its members from prosecution outside of a military court, and the power to arrest and try civilians in a military court. The military, moreover, not only reasserted but expanded its control of the economic sphere from basic items, such as bottled water and furniture, to larger infrastructure, energy, and technology projects.23 The military simultaneously paved the way for its commander, General Abdel Fatah al-Sisi, to run for and win one-sided presidential elections in 2014 (garnering 96 percent of the votes cast), formalizing his role as head of state.
While the military no doubt views these events as a return to the military’s rightful role as the dominant institution in Egyptian society, it is clear that the Egyptian military failed to uphold the principles of military professionalism: subordination to democratic civilian authority, allegiance to the rule of law, and an ethical institutional culture. As the events in Egypt reveal, when a military is unable to adhere to one of the pillars of professionalism, it finds itself in conflict with all three.
An Ethical Institutional Culture
In addition to democratic civilian control of the military and allegiance of the military to the nation, an ethical culture is a prerequisite for building a professional military. This entails values such as merit-based promotion, accountability of military leaders and soldiers for their actions, as well as demonstrating competent, impartial, and humane security enforcement. These institutional values do not come naturally. They must be taught. Soldiers must be inculcated with specific training in ethics, just as they learn discipline, law, and combat—all within the bigger picture of the military’s role in a democratic society.
A soldier’s ethos is an essential pillar of the institutional culture of the military and to the success of its mission. Along with proper training, soldiers need the courage to defend society’s interests before their own. The bravery, dedication, sacrifice, and sense of duty to protect and serve fellow countrymen should motivate recruits as much as any paycheck. Soldiers must have a calling to their profession. Being a professional soldier is not for everyone.
This ethos is important for building and maintaining a professional military. In Botswana, for example, citizens and members of the Botswana Defence Force (BDF) see the military as “the most capable of the country’s ‘disciplined services’…. Its members believe they are faithful stewards of resources entrusted by the nation to their care.”24 Thanks to this prestige, the BDF has the luxury of picking from a large pool of qualified candidates and has become increasingly selective in order to achieve the institutional culture it desires: highly disciplined, educated, and led by competent leadership. “In 2004 the BDF sought 80–100 new officers and received some 3,000 applications. It sought 500 enlisted recruits and received over 15,000 applications for these positions.”25
Significant steps have also been taken at the regional level to develop normative instruments to prevent military interventions in the political processes of African states. These include frameworks, such as the African Union’s (AU) “Common African Defence and Security Policy,” the “Framework for an African Union Response to Unconstitutional Changes,” the Southern African Development Community’s “Strategic Indicative Plan for the Organ on Politics, Defence and Security Cooperation,” and the Economic Community of West African States’ (ECOWAS) “Draft Code of Conduct for Armed and Security Forces.” The evolution of these explicit regional standards reflects a growing recognition of the need to improve the institutional culture of militaries on the continent. If upheld, these standards will have far-reaching implications for Africa’s political development.


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What Causes High Cholesterol?High cholesterol is a threat to many people of all ages around the world, and in the U.S. especially. More people die from cardiovascular disease every year than from any other cause, so it's essential that you do what you can to avoid heart problems. Keeping high cholesterol away is important, and knowing what can cause your high cholesterol is essential if you want to stay healthy.
Causes of High Cholesterol: Factors Out of Your ControlAge -- Many people have high cholesterol as a result of their age. The risk of developing high cholesterol increases once men pass the age of 45 or women pass the age of 55. Even those that have had healthy hearts their entire lives may find that their cholesterol levels get out of control once they pass these age markers. Race --Did you know that African Americans are more likely to develop high cholesterol than the average Caucasian? If you have high cholesterol, it may be the result of your race. Gender -- Men usually have higher cholesterol than women before the age of 50 or 55, but women that have gone through menopause will actually have higher cholesterol levels than men. Menopause can cause a drastic increase in cholesterol levels. Family History -- Those that have a history of cardiac disease are often more likely to develop high cholesterol, as they are genetically predisposed to having higher cholesterol levels. If anyone in your family has had high cholesterol or problems with their heart at a young age (younger than 55), it may mean that you are at a great risk of developing heart problems yourself.
Causes of High Cholesterol: Factors You Can ControlSmoking -- Smoking will suppress your body's production of HDL cholesterol, which will allow the bad LDL cholesterol to accumulate unchecked. Smoking will also make your arterial walls more conducive to buildups of cholesterol, and will weaken them the point that they are more likely to crack when the plaque builds up. Diet -- If you eat a lot of food that is rich in saturated and trans fats, you will find that your cholesterol levels will rise drastically. These fats cause your body to produce more cholesterol, and you will increase your total cholesterol levels. Seeing as your body already produces cholesterol naturally, you don't need to add more via your diet. Weight -- If you weigh more than you should, you are increasing your risk of high cholesterol. The extra weight on your body is stored fat, which is a form of cholesterol called triglycerides. High triglyceride levels means that your levels of good cholesterol are too low, which stops the good cholesterol from eliminating the bad cholesterol that can cause heart problems. Losing as little as 10% of your body weight can do wonders for your cholesterol levels. Activity -- Those that lead sedentary lifestyles always run a higher risk of health problems, and cholesterol is just one of the many problems that can set in if you don't do exercise. Exercise will increase the production of the good HDL cholesterol in your body, and increasing your HDL cholesterol just 3% can lower your bad cholesterol by as much as 8%. Doing exercise is the key to keeping cholesterol levels low, as burning fat means that you are lowering the levels of triglycerides in your body. Now you know what causes high cholesterol, you can take steps to lower your cholesterol levels by eating foods that lower cholesterol. It's in your best interest to get your high cholesterol under control as soon as possible!


March 2, 2008
For Building, Coop, Condo Owners
Own buildings, coops, and condos in NYC.
Dense, urban living is the most sustainable, resource and energy efficient, and environmentally responsible way to organize modern society. Hard to believe? Consider this: ever drive to work? live in a space with more than 1000 ft2 per person? It’s true that NYC buildings can be horribly energy inefficient (and there are a lot of low cost, easy things to do about it), but density by far makes up for it, so that on a per-capita basis, NYC is the most energy and resource efficient places in the country.
Use/Spec non-toxic materials and products.
Here’s a simple rule of thumb: If it’s poisonous, carcinogenic, triggers asthma, or wreaks havoc on your nervous system, you probably don’t want it in your building. And yet most of the products we use to build and maintain our buildings – including paints, cleaners, insulation, cabinetry, and carpets – are portable Superfund sites, making their way Trojan Horse-like, into our common and living spaces.
Use high quality, energy efficiency compact fluorescent lighting and Energy Star appliances.
Just because it saves energy doesn’t mean fluoresecent lighting looks good. Know what to look for when shopping for fluorescent lighting. See the following article: Understanding Lighting: The Good, The Bad, and the Environmental.
Use/Spec materials and products with post-consumer recycled content.
Search for products that state the percentage of post-consumer recycled content. Paper is a biggie. So are plastic and paper packaging (essentially, anything you can recycle should be made from recycled materials to keep the cycle going). And if you’re buying wood, tiles, countertops, carpet, or insulation there are options recycled and resource minimizing options for these as well.
Enact low-cost, fast payback, energy efficiency measures.
Schedule a building energy audit from a qualified firm. Auditors can usually quickly pinpoint places where energy is being wasted, and drastically reducing energy consumption and expenditures with a few simple adjustments, often costing very little money.
Install motion sensors for lighting in common areas.
Increase your comfort and reduce your energy consumption by controlling the the indoor temperature.
If you have a radiator, and control the heat in your apartment by getting up and adjusting the valves all winter long, or even worse, opening the windows, then having just the right temperature is probably a rare event. The same goes for cooling with AC — turning it off and on is a pretty crude way to control temperature. Erratic temperature is not only uncomfortable, but it wastes lots of energy, especially if the radiator’s kicking out heat or the AC’s keeping things nice and cool when nobody’s home. You can control the temperature in your living space by installing low cost, easy to use, thermostats and automatic radiator controls, simultaneously saving energy and increasing comfort.
Prepare for future building investments/capital management plan
by speccing energy efficient boilers, motors, and other equipment today. Otherwise you’ll be stuck with what’s available when the boiler goes down.
Switch to Green Power.
Two utility companies now offer “green power,” — electricity made from in-state wind and small, low impact hydro (no dams) — for utility customers in New York City. That means that city residents now have a low-cost, no-hassle renewable energy option. It costs a few bucks more a month, but that money helps grow the local renewable energy industry. The two companies are 1st Rochdale Cooperative
, and Consolidated Edison Solutions
(a subsidiary of the namesake parent). Read more about green power options for New York City
. Or, see the testimonial
of a recent Green Power switchee.
Create/enhance recycling and clean trash areas; create reuse opportunities.
The City has restored full recycling and your building is required to provide the appropriate bins. If you don’t have bins, ask your super or call your building management company. And check every now and again that the separated recyclables aren’t being tossed in with the trash when it’s all taken out to the curb.
Support Community Gardens.
New York needs more greenspace and vegetation. It filters the air and the noise, reduces the summer heat, and cleans the water.
Convert your rooftop – green roofs, solar PV, solar hot water.


Americans are rapidly coming to accept the reality of climate change. According to two studies released late last year, some 73 percent of the public now agrees climate change is happening, an increase of 10 percentage points since a similar survey in 2015. The number of Americans who say global warming matters to them personally jumped even higher — up 9 percentage points since March 2018 — to a new record high of 72 percent overall.
Despite growing acceptance, there is no clear path forward. Close to 70 percent of Americans say they wouldn't give even $10 a month to the government to fight climate change. Forty percent wouldn't even pay a dollar.
It's a sharp disconnect, and scientists say time is running out before we hit a tipping point. What would it take to get people to act on climate change? Guest host Tiffany Hanssen spoke to two climate scientists about the studies behind the shift in perception and the science that compels action.
Waleed Abdalati — Director at the Cooperative Institute for Research in Environmental Sciences, former chief scientist for NASA and professor in geography at the University of Colorado Boulder.
Heidi Roop — Lead scientist for science communications for the Climate Impacts Group at the University of Washington.
To listen to the full conversation you can use the audio player above.


Map Courtesy CIA World Factbook
The Kingdom of Morocco is a country in northwest Africa. It has a long coastline on the Atlantic Ocean that reaches past the Strait of Gibraltar into the Mediterranean Sea. It borders Algeria to the east, though the Algerian border is closed, Western Sahara to the south and Mauritania, and the Atlantic Ocean to its north and west. Morocco claims ownership of Western Sahara and has administered most of the territory since 1975. The status of Western Sahara is disputed, pending a United Nations referendum.
The full Arabic name of the country translates to The Western Kingdom. Al-Maghreb (meaning The West) is commonly used in Arabic. The name Morocco in most other languages originates from the name of the former capital, Marrakech (meaning "Country of God" in the Berber language).
Morocco became a French protectorate by the signing of the Treaty of Fez on March 30, 1912. It remained a Kingdom until it achieved independence in 1956. It then recovered Tangier, formerly an international city. The northern area of Morocco was under a Spanish protectorate concurrently. Morocco annexed Western Sahara in the 1970s, which had been a colony under the Spaniards since the 19th century. Previous to that it had been an area of Moroccan influence, but this annexation has not been recognized by any nation.
Morocco was the first nation to recognize the fledging United States in 1777 and has the oldest non-broken friendship treaty with the country, the Moroccan-American Treaty of Friendship, which has been in effect since 1783. John Adams and Thomas Jefferson were the American signatories. The United States legation (consulate) in Tangier, is the first property the U.S. owned abroad. It now houses the Tangier American Legation Museum. Morocco was granted Major Non-Nato Ally status in June 2004 and signed with the United States and the European Union free trade agreements.


Before anything else, I am sure you will have one question: What is The Mandela Effect? I will try to explain it to you and give you some examples but after, it is your turn to decide what is real and what is not.
An Explanation in Simple Words
The Mandela Effect is just a memory. It is a memory of something that you are very sure happened and you can even remember clearly the details, but actually it never happened. Not in this time -line.
Usually a group of people, strangers, remember the same thing and in reality, this thing is totally different. So it is not a dream. If you were the only one to remember this, ok, but there are a lot of people. So, may this be a memory from a parallel reality?
In psychiatry, it is a phenomenon in which many people have the same false memory of something which happened in the past.
When did this theory start?
This theory actually started in 2010 when Fiona Broome, a Blogger, and paranormal enthusiast said that she surely remembered Nelson Mandela’s death in a South African prison in the 80s. Along with her a big number of other people also remembered this event .They even claimed to have learned about it in school. Fiona had clear memories of Mandela’s funeral on a news channel.
Actually, Nelson Mandela died in 2013 from a respiratory infection, in Houghton, Johannesburg, in his home.
What was her explanation?
Broome, trying to explain this phenomenon claims that these false memories shared by many people with no evidence that they ever happened, may this be an occasional interaction of parallel words, parallel timelines.
We have evidence of these interactions found in our common false memories, and the death of Nelson Mandela is not the only example.
Mandela Effect Examples
I will give you a few classical Mandela effect examples and see if you are familiar with one of these. Tell me later 🙂
Of course, the first and the most important is the death of Nelson Mandela. As I said before, a large number of people remember him dying in the 80s in prison, and even remember even the ceremony on TV. The reality is that he died in 2013.
Another example is the spelling of the famous cartoon show ‘The Berenstain Bears’. Many people remember the name spelled The Berenstein Bears’. How do you remember it?
Of course, we have some other spelling examples as McDonald’s (real) or MacDonald’s, Looney Tunes (real) or Looney Toons. To be honest talking about the last one, I always remember it as Looney Toons.
Also, Fiona Broome discovered other facts in history where some people remember a different version. For example, the protester who in 1989 in Tiananmen Square defied the tanks, they remember he was killed by them. This group of people even claim that were taught about this historical moment in school.
How others explain this effect
Elizabeth Loftus, a famous psychologist explains in 1978 that memories can be changed. A psychological theory is that the memory is constructive not reproductive as we think.
So what this mean? Our brain builds our memories from small different pieces of information and they can be changed, destroyed, by some factors as imagination, association, and others.
What do we understand from this? Memories can be fallible and not pure at all. So can this be the explanation for the Mandela Effect? But now, my personal question…how can be the same mistake be made by many people, the same false memory?
Maybe a psychologist can give us a scientific explanation but again, for me, it is still a mystery. Ok, some facts can be misremembered from a person but how can you explain that a big group of people misremember the same thing?
Maybe because I am a time travel passionate, I believe in the multiple universes and parallel existence theory but I still believe that …that out there, there is something more …
I personally think that for a big number of people to misremember the same things maybe in another time line, that at some point interacted with ours, their memories were true.
Maybe Nelson Mandela really died in the 80s in another world line and maybe there Looney Tunes is written Looney Toons 🙂
What do you think? Do you have any memory that you are 100% sure that happened and you even remember details but it actually never happened?
I would like to tell you something that a friend of mine told me. He remembered going on a school camp with his sister when they were teens but later his sister didn’t remember anything about this, and he was also sure that they took some things from there, some stone collections and also she didn’t know anything about this.
So… when my friend asked me how I explain this …I just said…Mandela effect 🙂
Thanks for reading and I am waiting for your comments:)
==>for further reading ,scientific articles, viewing interesting videos and more TIME TRAVEL STUFF click below <==


See Saw Weather - Alaska comes to Cape May
Cape May, NJ - In mid-January we were asking, Is it really winter? The temperatures were balmy, spring flowers were blooming three months early, and fish that normally wouldn’t be active were swimming just off the beaches.
There was no ice, no snow, and the grass was still green.
We have no doubt today that winter's arrived. Frigid arctic air from Alaska is enveloping South Jersey.
How things have changed in only two weeks!
In January our gardens witnessed an unseasonal bloom by Daffodils, Flowering Quince, Wall Flower, Rosemary and Viburnum. They were producing flowers at a time when they had no business doing so.
No more. The flowers are encased in ice or are long gone.
Fish and Birds
Fish and birds also have been affected by the strange weather patterns.
This year, some fishermen were actually catching striped bass in mid-January in the back bays. This is virtually unheard of. In past decades, fish like striped bass and bluefish mostly disappeared in early December. The cold temperatures now have sent those fish packing.
Also this winter, we've witnessed unusual patterns among migrating birds. During a normal January, we would see fewer ducks and few gannets. However, this winter, hundreds of gannets were catching herring off of Cape May’s beachfront until the recent cold snap. There've been more ducks around this winter as well.
End of the Warm Winter
The winter of 2006-2007 started out looking like one of the warmest on record, thanks to a combination of global warming and El Nino.
Our warm spell has come to a crashing end as the jet stream has shifted, bringing us icy blasts of arctic air.
Why does it feel like Siberia all of a sudden? The jet stream is flowing directly from Alaska. The cold air it's bringing is the same polar air that makes Siberia and Russia shiver. In fact, Moscow is experiencing record cold temperatures.
El Nino Did It
Our winter has actually been classic for the El Nino pattern. In an El Nino year, winter is generally much milder, at least for the first half. Then the real cold of winter commences.
El Nino also ushers in cooler temperatures in early spring. So, brace yourself. We are likely to have cooler temperatures for a while.
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Public employees in New York state have the right to be represented by unions and to bargain collectively with their employers for salary, benefits and other terms and conditions of employment. This right should not be taken lightly. It did not exist in New York until the Legislature enacted the Taylor Law in 1967, and it does not exist today for school employees in 18 other states. Private-sector members are covered under the National Labor Relations Act, which likewise gives private-sector members the right to be organized, represented and to bargain collectively.
Before the Taylor Law was enacted, public employees in New York had no collective bargaining rights. Under the Condon-Wadlin Act, a 1947 law that the Taylor Law replaced, striking public employees were penalized by being fired. They could only be reinstated under a three-year pay freeze and five-year probation. The Public Employees’ Fair Employment Act (Taylor Law) was enacted in 1967 following a series of public-sector strikes, including the 12-day New York City transit strike a year earlier. The state Legislature granted amnesty to the striking employees, and Gov. Nelson Rockefeller appointed a committee to recommend legislation regarding public-sector employee rights. The result was the Taylor Law. Its provisions include:
- The right of public employees to organize and bargain collectively with their employers;
- The right to representation by employee organizations (unions) of their own choosing;
- The requirement that public employers (including school districts) negotiate with their employees and enter into written agreements (contracts) with their employees’ chosen representatives;
- Procedures for resolution of contract disputes (impasses);
- Prohibition of improper labor practices by either side;
- Creation of the Public Employment Relations Board (PERB) to administer the law; and
- The requirement that bargaining unit members who choose not to join a union pay an agency fee, and that use of the fee for political and ideological purposes only incidentally related to bargaining to which the agency fee payer objects is subject to a rebate procedure.
While the Taylor Law grants public employees the right to collective bargaining, it denies them the right to strike. The penalties for striking are loss of pay for each day the employee is on strike, plus a fine of an additional day’s pay for every day on strike and potential discipline for misconduct.
NYSUT is always working to win legislation to improve the provisions of the Taylor Law. For exam- ple, a 1982 amendment mandated that if a collec- tive bargaining agreement expires, its terms and con- ditions continue until a new agreement takes effect. That amendment has helped thousands of members avoid hardships when negotiations are impeded by harsh economic conditions, recalcitrant employers or both. An earlier improvement eliminated the “probation penalty” (probation for one year) against tenured teachers who went on strike.


Click an assembly to find out more.....
Being a good friend
Falling out and making up
Telling the truth
Good to be me
Trying your best
Being a Good Friend
This assembly includes-
- What does a good friend do?
- An example from, 'Britain's got talent' and shows what real friends are like.
- A fun game to get the children to make decisons about friends for themselves.
- A chance to look at the real challenges that we face in friendships.
Each slide makes a simple point so that you don't need to learn a 'script' and has slides that you can add you own text to or remove and add in slides of your own.


Aboriginal and Torres Strait Islander Peoples are entitled to the full realisation of their rights as articulated in relevant human rights treaties, including the Universal Declaration of Human Rights (UDHR) and specifically the Declaration on the Rights of Indigenous Peoples (UNDRIP).
The Australian Government indicated its support for the UN Declaration on the Rights of Indigenous Peoples on 3 April 2009.The Declaration is a comprehensive statement of Australia’s existing human rights obligations to Aboriginal and Torres Strait Islander peoples. Although it does not create new rights, it provides a comprehensive framework for action.
The UN Declaration on the Rights of Indigenous Peoples is a guarantee that Indigenous Peoples rights to self-determination, to lands and territories, to cultural identities, to self-representation and to their unique values and beliefs will be respected at the international level.
“As an international instrument, the Declaration provides a blueprint for Indigenous peoples and governments around the world, based on the principles of self-determination and participation, to respect the rights and roles of Indigenous peoples within society. It is the instrument that contains the minimum standards for the survival, dignity and well-being of Indigenous peoples all over the world” - Mick Gooda, Aboriginal and Torres Strait Islander Social Justice Commissioner.
ANTaR exists to work in partnership with Aboriginal and Torres Strait Islander people to advocate for rights, justice and reconciliation. We are committed to applying the principles of the UN Declaration on the Rights of Indigenous peoples in our planning and the delivery of our campaigns and programs. We will continue to give voice to these rights in our advocacy work.


Childhood tends to be carefree, yet as kids grow up, they must understand situations, make decisions, and come to conclusions. So, let’s find out the best yet simple problem solving activities for toddlers.
When understanding things around them and making such decisions, facing numerous challenges and problems is not unusual. This is where we all need problem-solving skills.
Where does this problem-solving ability come from? Do human beings inherit this from birth, or must they develop it? Problem-solving is an ability that one should develop through different mechanisms.
Most importantly, this is a skill that is needed to be practiced from early childhood. In this article, we will discuss teaching problem-solving activities to toddlers.
One can follow various activities to enhance problem-solving skills; however, ensuring their productivity and suitability is essential when introducing such activities to toddlers.
What activities can you use as an adult to help toddlers develop their problem-solving skills? Problem-solving requires a combination of skills, such as creativity, coping skills, imagination, critical thinking, communication, reasoning, etc.
Hence it is a must to follow activities that improve given abilities in toddlers, which will ultimately help them with problem-solving.
Some of the best activities that are effective in developing problem-solving ability in toddlers include puzzle games, building blocks/ model building, hide and seek, memory games, board games, treasure hunts, building a maze, and sorting games.
Simply put, problem-solving is identifying a problem and adapting to correct solutions. Problem-solving is a mandatory life skill that every individual need. Therefore it is essential to master this skill from the earliest possible age.
Using effective mechanisms from a young age will significantly help humans strengthen their critical thinking abilities. Keep reading this article to better understand how to teach toddlers problem-solving skills and what activities to use in this matter.
Table of Contents
Simple yet effective problem solving activities for toddlers
Thanks to their rapidly developing brains, toddlers have a fascinating ability to learn many new things. One of the essential skills that toddlers should practice is problem-solving because this plays a pivotal role in merging and surviving in society.
Plenty of skills are embedded within the term problem solving, including analytical thinking, logical reasoning, lateral thinking, creativity, persistence, negotiation, listening, and cognitive skills.
Teaching problem-solving to kids might sound like a complex thing to do. Nevertheless, plenty of accessible activities effectively contribute to evolving problem-solving skills in toddlers. Below you can find some.
- Puzzle and board games
Puzzle and board games are commonly used productive activities to uplift problem-solving skills in small children.
Puzzles contain pieces, and to place them in the correct order, toddlers must use their brains thoroughly, which will benefit them in developing their problem-solving abilities.
When using puzzle games for toddlers, remember to use ones specifically created for their age category. Similar to puzzles, board games are also crucial for developing problem-solving.
The same rule also applies to board games; they should be fun and engaging yet should not be too intricate. You can use board games such as tic-tac-toe or even be creative and make your own ones that suit toddlers.
- Building blocks and model making
Yet another ideal activity that aids toddlers in developing problem-solving skills is model-making activities. You can give them either building blocks or DIY model kits for this activity.
These activities allow toddlers to think differently and bring their imagination into real life. To make this activity more impactful, you can enable toddlers to do it in groups; that way, they develop various skills essential for problem-solving.
Storytelling is a fun and versatile activity for toddlers that improves problem-solving skills. Pick a story that sparks interest in toddlers, and present it interestingly.
You can also ask related questions meanwhile telling the story. Moreover, you can allow toddlers to narrate their own stories.
As much as you make storytelling exciting and diverse, it will help develop kids’ problem-solving abilities.
- Hide and seek
Hide and seek is an exciting outdoor activity helpful in developing problem-solving skills in toddlers. How can a simple game like hide and seek help improve toddlers’ problem-solving skills?
Surprisingly it does. Hide and seek is an activity that allows toddlers to develop visual tracking, working memory, and social-emotional skills. All, in return, are essential in problem-solving.
When introducing this activity for toddlers, start it easy, and gradually you can level up the complexity.
- Treasure hunt
This is one of the best activities for toddlers who love exploring the outdoors. This game can be played in groups, and toddlers have to think deeply when they find clues; therefore, it improves their problem-solving skills.
Role-playing is yet another enjoyable activity that aids toddlers in developing their problem-solving skills. You can present a certain scenario to toddlers, assign them a character and ask them to act accordingly.
This activity allows toddlers to be creative; moreover, it makes them think critically, so all these skills make them stronger at problem-solving.
- Building a maze
This is a game that you have to put a considerable amount of effort into executing. Nonetheless, it is highly beneficial in developing multiple skills toddlers need to be better at problem-solving.
You can use different materials to build the maze, such as cardboard, books, tables, or chairs. This game pushes small children to think critically to find the end and escape the maze.
- Sorting things
Sorting things is another practical activity you can allow toddlers to do, which will benefit their problem-solving skills.
For this activity, you can give them objects, for example, bottle lids of varying shapes and colors, and ask them to sort similar ones. Sorting activities are effective for cognitive development.
How to do problem-solving activities for toddlers?
In the previous section, we discussed some impactful activities that help toddlers to develop their problem-solving skills. No matter what activity you introduce to a toddler, there are things you should be aware of when helping them develop their problem-solving ability.
As mentioned earlier, toddlers are fast learners. Still, it is self-explanatory that you can’t teach something to a small child just the way you do it to a grown-up.
So how can you teach problem-solving activities to toddlers? What factors should you put into consideration? Let’s find out.
- Keep things simple, and ask questions
When introducing problem-solving activities to toddlers, it is essential to keep things simple. If you make things complex initially, toddlers will lose interest. Therefore always start small; gradually, you can make things more complicated.
In some instances, you do not even have to conduct specific activities to uplift the problem-solving skills of your toddler.
Particular scenarios in daily life provide chances for spontaneous lessons, so you can use them wisely to help your toddlers improve their problem-solving skills. Practical life examples are indeed helpful in developing problem-solving skills in toddlers.
- Ask questions
It is essential to ask questions from toddlers to develop their problem-solving skills. Along with the activities that you do, ask questions.
Asking questions allows toddlers to recall things and give better answers by analyzing the information they receive.
Toddlers are naturally curious; therefore, asking them questions gives them the extra boost of curiosity to ask more follow-up questions and learn things better. You must ask them questions; subsequently, you must encourage them to ask you questions.
- Be creative, and make it fun
Being creative and innovative is crucial when teaching toddlers problem-solving activities. Following the same activity will likely reduce productivity; hence, think out of the box and introduce diverse creative activities.
Introducing creative and varied activities to toddlers will thrive them understand things from various perspectives, which is a must to enhance problem-solving skills.
Toddlers love to have fun, so when deciding on problem-solving activities, ensure the activities are effective at the same time, fun.
If small children are not necessarily interested in something, it will be hard to convince them. Try to embed activities that help develop problem-solving in toddlers for their playtime. It makes playtime enjoyable at the same time, productive.
- Accept failure, and keep them encouraged to fix errors
Sometimes when your toddler attempts to solve a problem, he/she might fail. Failure is typical when learning problem-solving skills. In such cases, you should not demotivate the toddler.
Furthermore, they do not always provide answers to toddlers. If they are in a problem, as an adult, you have to help them, yet push them as much as possible to find solutions independently.
Allow failure, and keep encouraging them to adjust what went wrong. It is necessary to create a space for toddlers to learn from their errors and mistakes.
Problem-solving is one of the fundamental skills that humans require in their regular lives. Humans face various problem scenarios where they must think deeply and act correctly. In order to do it, problem-solving skills are mandatory.
One cannot become excellent at problem-solving overnight. It is a process that should be developed from a young age. This article discussed some effective activities that uplift toddlers’ problem-solving skills.
Also, how should you approach a toddler when introducing such activities? If you follow these mechanisms correctly, your toddler will eventually master problem-solving.
Stay with us for more interesting posts like this!


We recently passed an Internet milestone: April 12 marked the 20-year anniversary of commercialized spam. But even with two decades and trillions of unsolicited messages behind us, it seems there is still no end in sight.
There were certainly unsolicited messages sent before April 11, 1994, but that’s the day two lawyers pitched a green card lottery to the vast Usenet News audience. It’s been mostly downhill since.
You’ve probably been emailed by your fair share of African “royalty” or other foreign nationals seeking your assistance in transferring millions of dollars of wealth to the United States. Perhaps you’ve received email ads for Viagra or unsolicited pitches to for a low-interest mortgage or other loan.
Cloudmark wrote a blog post and created an infographic looking back at key dates in the 20-year history of spam. That first blast on Usenet News was nothing. Internet service providers, businesses, and individuals have invested millions and wasted countless hours in the battle against spam.
According to Kaspersky Lab, spam still makes up nearly 70 percent of all email sent. That means, roughly speaking, that if you were to receive 100 emails today, there’s a good chance that 70 of them will be unwanted solicitations.
The scourge of spam has pushed users to seek alternative forms of communication, and the spammers have followed along. Instant messaging, social networking, SMS text messages—if there are millions, or even tens of thousands, of people utilizing a given form of communication, you can bet there are spammers working to exploit it.
The good news is that things actually have improved dramatically on the email side. While 70 percent of all email might be spam, webmail services and email servers have greatly improved the ability to identify and block those messages before they get in the door. Most email client software also has very good junkmail filtering capabilities. The result is that only a handful of the spam messages intended for you ever actually make it to your inbox.
Unfortunately, the tech world is still trying to catch up to newer spam attacks on social networks and text messaging. Depending on the platform or service, there are some controls to prevent unsolicited messages, but the onus is on the user to be aware of and configure those security features in most cases.
But the best defense against spam is the human using the technology. The only reason spam continues to be an epidemic is because it works. It costs virtually nothing to put a spam message in front of millions of users, so any response at all is profitable.
You are your own best protection: don’t open file attachments, and don’t click on links. Better yet, just delete unsolicited messages without even opening them. If enough of us do this, maybe we can stick a fork in spam once and for all.
Tony is principal analyst with the Bradley Strategy Group, providing analysis and insight on tech trends. He is a prolific writer on a range of technology topics, has authored a number of books, and is a frequent speaker at industry events.


Data helps school districts see whether they are meeting their goals. With data, we can measure student progress, measure program effectiveness, guide curriculum development, allocate resources effectively, and promote accountability to the community.
Some common sources of data for California Schools:
- California School Dashboard - In the past few years, California has shifted away from a single way to measure schools' performance (test scores) to a set of measures displayed in an interactive, online format.
- The Dashboard aims to display how schools are doing in key areas, like graduation rates, parent engagement, and students' mastering of state standards. The Dashboard is centered on equity, identifying student groups that can be better served. Rather than finding fault, it is meant to help schools and districts identify areas where they need to grow and build on strengths.
- To learn some basics about the Dashboard, watch this video, which was kindly shared by the Alameda County Office of Education: Dashboard Video - English | Dashboard Video - Spanish
- DataQuest - Use the level and subject drop down menus to see a variety of state data about our schools.
- EdData - Find fiscal, demographic, and performance data on our schools.