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Staying frosty in plague times
The director wanted to show things as they were – badly-led, no one wanting to fight or knowing what they were fighting for, says Christopher Silvester
For a folk hero this Ned has zero contact with the ordinary Irish folk who will supposedly come to love him, says Christopher Silvester
The most successful modern actor-director, Clint Eastwood, rejects “the auteur crap”
As usual, the Academy flagellates itself over insufficient African American Oscar contenders
Bad jokes, no chemisty, lame action: the snoozathon that’s not woke enough
Some of the greatest movies ever made were incredibly long. But is a film lasting 13 hours just too much?
Martin Scorcese’s new Mafia movie is the culmination of a great career

domain: entertainment


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Cops Caught Kicking Man Fight To Get Jobs Back
2 APD Officers File Appeals To Be Reinstated
Two Albuquerque police officers fired for allegedly beating up a suspect are fighting to get their jobs back.
Officers John Doyle and Robert Woolever were caught on camera attacking a man, police said, and the police chief called their behavior criminal, but their lawyer has a much different story to tell.
Lawyer John D'Amato said the man was out to kill police, and the two officers were warned about it right before the incident.
"It does look crazy. It appears to be out of control," D'Amato asid.
The lawyer said the two officers tackled Nicholas Blume after he led them on a chase in a stolen car before crashing into a building. D'Amato said the officers thought Blume was reaching for a gun the whole time he was on the ground, and Doyle had to kick him to get his hands free.
"As soon as Officer Woolever handcuffs Blume, he stopped," D'Amato said.
Albuquerque police said they were warned about Blume, a violent gang member, not long after an officer killed his friend in the Aryan Brotherhood, who was threatening officers with a gun. The Albuquerque Police Department said it was worried Blume would retaliate and shoot an officer.
"Every encounter this man has had with APD has been violent. (He is) resistant and has always been armed," D'Amato said.
D'Amato said Blume, who has dozens of felony arrests, is wanted in connection with an Albuquerque homicide case and a police death in Colorado, but police said Blume hasn't been charged in connection with both cases.
Police said Blume didn't have a gun during the attack, but officers found a gun in the stolen car.
Doyle and Woolever have filed appeals to get their jobs back, and D'Amato said they will have a hearing by the end of the year.

domain: news


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Airway neuropeptides, in particular calcitonin gene-related peptide (CGRP), are likely to be important in the pathogenesis of chronic cough. The present authors evaluated the following: 1) the relationship between cough sensitivity and bronchoalveolar lavage (BAL) neuropeptides; and 2) the effect of reflux oesophagitis (RO) on cough, cough sensitivity and BAL neuropeptides in children not selected for cough. It was hypothesised that CGRP would be increased in children with chronic cough and would relate to cough sensitivity. Capsaicin cough sensitivity was performed in children undergoing gastro-duodenal endoscopy. CGRP, substance P and neurokinin A were measured in BAL obtained nonbronchoscopically. Children were defined as "coughers" if chronic cough was present. Coughers (n=21) had significantly reduced cough sensitivity but were just as likely as noncoughers (n=19) to have RO. The median CGRP was significantly higher in coughers with oesophagitis than in noncoughers with oesophagitis. CGRP significantly negatively correlated to cough sensitivity in coughers but not in noncoughers. Elevated calcitonin gene-related peptide, but not substance P or neurokinin A, is only associated with chronic cough in children when oesophagitis coexists. Calcitonin gene-related peptide in bronchoalveolar lavage relates to cough sensitivity and is likely to be important in the pathophysiology of chronic cough. Copyright � ERS Journals Ltd 2007.
|Number of pages||7|
|Journal||European Respiratory Journal|
|Publication status||Published - 2007|

domain: literature


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Molecular-Targeted Therapies for Hematologic MalignanciesView this Special Issue
Mutations in Epigenetic Modifiers in Myeloid Malignancies and the Prospect of Novel Epigenetic-Targeted Therapy
In the recent years, the discovery of a series of mutations in patients with myeloid malignancies has provided insight into the pathogenesis of myelodysplastic syndromes (MDSs), myeloproliferative neoplasms (MPNs), and acute myeloid leukemia (AML). Among these alterations have been mutations in genes, such as IDH1/2, TET2, DNMT3A, and EZH2, which appear to affect DNA and/or histone lysine methylation. Large clinical correlative studies are beginning to decipher the clinical importance, prevalence, and potential prognostic significance of these mutations. Additionally, burgeoning insight into the role of epigenetics in the pathogenesis of myeloid malignancies has prompted increased interest in development of novel therapies which target DNA and histone posttranslational modifications. DNA demethylating agents have been demonstrated to be clinically active in a subset of patients with MDS and AML and are used extensively. However, newer, more specific agents which alter DNA and histone modification are under preclinical study and development and are likely to expand our therapeutic options for these diseases in the near future. Here, we review the current understanding of the clinical importance of these newly discovered mutations in AML and MDS patients. We also discuss exciting developments in DNA methyltransferase inhibitor strategies and the prospect of novel histone lysine methyltransferase inhibitors.
The increasing use of systematic genome-wide discovery efforts in patients with a variety of myeloid malignancies has led to the rapid discovery of a series of recurrent genetic abnormalities underlying these disorders. Remarkably, a large number of these alterations appear to be in genes whose function is known, or suspected, to be involved in epigenetic regulation of gene transcription. In the last 3 years, alone mutations in the genes TET2, IDH1, IDH2, DNMT3a, and EZH2 have all been found in patients with myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDSs), and/or acute myeloid leukemia (AML). Although the functional implications of these mutations and how precisely they contribute to abnormal hematopoiesis and leukemogenesis is being heavily investigated and not yet clarified, a number of potentially clinically important implications of these mutations may already be apparent. First, mutations in several of these genes likely hold prognostic importance for patients, and these genetic alterations, thereby, may serve as prognostic markers for risk stratification and aid in therapeutic decision making. Secondly, mutations in several of these genes may specifically impact DNA methylation and/or histone posttranslational modifications in such a manner that is therapeutically targetable. Mutations in several of these genes, such as IDH1 and IDH2, have been proven to result in a gain-of-enzymatic function which holds the prospect for development of novel targeted therapeutics. This review focuses on the clinical relevance of recently discovered epigenetic alterations in patients with myeloid malignancies and the prospect for novel targeted therapeutics against aberrant epigenomic characteristics in patients with MDS and AML.
2. Recently Identified Mutations in Epigenetic Modifiers in Myeloid Malignancies
2.1. IDH1 and IDH2 Mutations
Genome sequencing of AML has recently led to the discovery of mutations in the genes encoding isocitrate dehydrogenase (IDH1 and IDH2). IDH1 is a key cytosolic enzyme in the Krebs cycle. It catalyzes the decarboxylation of isocitrate to α keto-glutarate (α-KG), leading to the production of nicotinamide adenine dinucleotide phosphate (NAD-P). The isocitrate dehydrogenase 2 (IDH2) gene encodes a homologous protein which catalyzes the same reaction in mitochondria. IDH mutations have been extensively studied and are frequently found alterations in low-grade gliomas. They have also been discovered in a small subset of the highly aggressive glioblastomas, where they confer a more favorable prognosis [1–6].
IDH mutations were subsequently identified in AML and other myeloid malignancies, including MDS and MPNs [7, 8]. All discovered IDH mutations reside in the active site of the enzyme and participate in isocitrate binding . They are missense alterations affecting arginine-132 (R132) in IDH1, and either the analogous arginine residue (R172), or the arginine-140 (R140) residue in the IDH2 protein [7, 10–15]. The common recurrence of IDH mutations in AML suggests an importance in leukemogenesis. All mutations are missense and heterozygous, suggesting that the alterations lead to a “gain of function” . It has been shown that the mutant forms of IDH cannot catalyze the conversion of isocitrate to α-KG . Dang et al. reported that the mutated R132H IDH1, in place of the normal process of isocitrate decarboxylation, catalyzes an NADPH-dependent reduction of α-KG to 2-hydroxyglutarate (2-HG). 2-HG is a metabolite which is normally present at very low levels in cells , 2-HG levels have been found to be elevated in IDH-mutant glioma samples, and Gross et al. reported that IDH1 R132 mutations also lead to production and accumulation of 2-HG in AML blasts, greater than 50-fold higher than their nonmutant counterparts. Elevated 2-HG levels in IDH-WT samples led to the first discovery of IDH2 mutations, which accounted for elevated 2-HG levels in these AML cells. The elevation in 2-HG levels has also been noted in sera of patients with IDH-mutant AML .
Studies of IDH mutations in gliomas have suggested that they are an early event in the pathogenic process . Their exact mechanism in leukemogenesis of AML is uncertain. Normal IDH function appears essential for normal cell growth and proliferation. IDH1 is one of only three cytosolic proteins which contribute to NADPH production which is essential for nucleotide and lipid synthesis. Ward et al. demonstrated that siRNA silencing of the IDH1 and IDH2 proteins led to a significantly reduced proliferative capacity . Some investigators have suggested that accumulation of 2-HG plays an important role in this process, the “gain of function” neomorphic enzyme activity that promotes cancer [9, 18, 19]. Patients with a rare inherited condition called 2-hydroxyglutaric aciduria have elevated levels of 2-HG with an increased propensity for brain tumors. 2-HG has indeed been shown to increase reactive oxygen species in these patients [20, 21]. Additionally, 2-HG is homologous to α-KG in structure and thus may bind and interfere with essential enzymes that are activated by α-KG. Among these are prolyl hydroxylases which control the stability of and downregulate HIF-1α transcription factors, implicated in the pathogenesis of multiple malignancies [9, 19, 22].
The association of IDH mutations with aberrant hypermethylation has only recently been discovered. By studying samples from 398 AML patients in an Easter Cooperative Group (ECOG) E1900 trial, we found that IDH-mutant AML is associated with consistent and aberrant hypermethylation of various promoter sites involved in myeloid differentiation and leukemogenesis  (Figure 1). Promoter CpG sites are extremely important in the regulation of gene expression, specifically those of genes which mediate tumor suppression and differentiation, and DNA methylation can lead to transcriptional inactivation or chromosomal instability [24, 25]. Aberrant hypermethylation has been extensive described as a pathogenic process in forms of MDS and AML [26–29]. The discovery of aberrant hypermethylation and transcriptional inactivation of loci in relation to IDH-mutant AML is intriguing and significant. The potential mechanism for the hypermethylation and leukemogenesis in IDH-mutant disease may be related to downregulation of normal α-KG levels. Multiple enzymes are dependent on α-KG for their function, including the TET enzymes, which appear to play an important role in the differentiation of myeloid cells and promote demethylation by hydroxylating methylcytosine groups [30, 31].
Another recent study reported that altered 2-HG/α-KG levels present in IDH1/2 mutant cells additionally results in the inhibition of a different set of α-KG-dependent enzymes, the Jumonji family of histone lysine demethylases (JHDM) . There are 3 classes of enzymes which are known to antagonize histone methylation: (1) peptidylarginine deiminase, which removes methylarginine modifications to produce citrulline , (2) lysine specific demethylase 1 which removes H3K4me1/H3K9me1 marks in a reaction requiring flavin is a cofactor , and the Jumonji C domain family of histone demethylases which require iron Fe(II) and α-KG as cofactors. Unlike LSD1, which can only remove mono- and dimethyl lysine modifications, the JHDMs can remove methyl groups from all three histone methylation states. So far, JHDM family members have been shown to reverse the following lysine methyl marks: H3K36me1/2 (JHDM1) , H3K9me1/2 (JHDM2) , H3K9me2/3 (JHDM3) , and H3K36me3 (JHDM3) , H3K4me2/3 (JARID1) , and H3K27me2/3 (UTX/JMJD3) . In addition, JMJD6 has been shown to encode an arginine-specific histone demethylase which demethylates H3R2me1/2 and H4R3me1/2 . All of these marks may, therefore, be affected by the presence of IDH1/2 mutations, and Xu et al. indeed demonstrated hypermethylation of many of these marks following introduction of IDH1/2 mutations into cells  (Figure 1). These findings lend significant credence to the theory that hypermethylation of DNA and histone lysine/arginine modifications play a key role in the pathogenesis of AML in this subgroup of patients. Further work to delineate the complex epigenetic alterations to the transcriptional changes which promote leukemogenesis will be very enlightening.
Currently, our clinical use of genetics in AML for prognostication relies on (1) the use of cytogenetics to delineate patients into favorable, intermediate, or adverse cytogenetic categories and (2) molecular genotype of the genes FLT3, NPM1 (nucleophosmin 1), and CEBPA (CCAAT/enhancer-binding protein alpha) for those patients with a normal karyotype. Multiple studies have demonstrated that patients with a normal karyotype but with an internal tandem duplication of FLT3 (FLT3-ITD) have an inferior outcome compared to those without a FLT3-ITD mutation [40, 41]. In several additional studies, the presence of an NPM1 mutation with or without the presence of a FLT3-ITD mutation was associated with higher complete response and event-free survival [42, 43]. Normal karyotype AML with mutations in CEBPA also appear to represent a subset of AML with more favorable outcome [44, 45]. Given the identification of multiple new molecular genetic abnormalities in patients with AML, an increasing number of studies have been performed to delineate the mutational frequency and prognostic implications of IDH1/2, TET2, and DNMT3A mutations in AML patients. The initial report of IDH1 mutations in AML found that 8.5% of AML samples contained a mutation at amino acid R132. All mutations were associated with samples that displayed intermediate risk cytogenetics, with the large majority having normal cytogenetics. With this relatively small group of 187 samples, the investigators noted no independent prognostic value on overall survival (OS), but subgroup analysis suggested adverse effects in those patients with no NPM1 mutations . Ward et al. also assessed for mutations in a series of 87 AML samples and found that 7.7% had IDH1 mutations. Additionally, they found that even a larger subset of samples (15.4%) displayed mutations in the IDH2 gene. All samples with IDH mutations were found to have normal karyotypes, without an effect on overall survival .
Larger studies, based in North America and Europe, have attempted to further investigate the prevalence and prognostic implications of these mutations. A CALGB study assessed bone marrow and peripheral blood marrow samples from 358 patients with cytogenetically normal AML. The investigators reported IDH mutations in the third of their patients, with 14% having IDH1 mutations and 19% displaying IDH2 mutations. AML patients with IDH mutations in this study tended to be younger with lower-risk disease as defined by a higher frequency of NPM1 mutations without the presence of FLT3-ITD mutations. Specifically, IDH1 mutations adversely affected disease-free survival (DFS) in this particular subgroup of patients with a favorable molecular profile. Interestingly, those patients with IDH2 mutations achieved a lower rate of complete remission with induction therapy .
A study from The Netherlands also found a significant prevalence of IDH mutations in 893 samples from patients with newly diagnosed AML, with 6% and 11% having IDH1 and IDH2 mutations, respectively. They also reported an association with normal karyotype AML and the NPM1 mutation. As in previous studies, no effect on OS was noted for the entire cohort of AML samples, but interestingly, in those samples without an NPM1 mutation, IDH mutations were associated with an inferior event-free survival (EFS) . A subsequent study from the UK found IDH1 mutations in 8% of patient samples, using data on 1333 adult patients from the UK MRC AML10 and AML12 trials. Similar to the Dutch study, they reported a strong association with intermediate cytogenetics and the NPM1 mutation. However, in contrast to the previous study but similar to results of the CALGB trial by Marcucci et al. , the investigators found that an IDH1 mutation preferentially reduced survival in the cohort of patients with a coexistent NPM1 mutation . These findings were supported by large studies from France and Germany, which again found that 12–16% of patients had IDH mutations, with the mutations adversely affecting rates of CR and survival in the subgroup of patients with CN-AML and with NPM1 mutations [11, 13].
However, the prognostic significance of the copresence of IDH and NPM1 mutations in CN-AML continues to be uncertain. A recent large German study reported shorter EFS and rates of CR in all AML patients with an IDH1 mutation, and specifically within the population of patients without NPM1 mutations . The same authors have recently also presented their data at the annual American Society of Hematology (ASH) 2010 meeting on 526 patients with cytogenetically normal AML and found that 28.7% of patients had IDH mutations. 12.9% of patients were found to have IDH1 mutations and 15.8% had IDH2 mutations. In their survival analysis, no effect was found on OS or EFS. However, in this presentation, a trend for shorter EFS was reported in all IDH mutant patients with NPM1+/FLT3-ITD genotype, and, interestingly, a significant adverse effect on EFS in those with specifically IDH2 mutations in the NPM1+/FLT3-ITD-group . The leukemia group from M.D. Anderson in Houston, Tex, USA, also presented their data at the ASH 2010 meeting on 358 AML patients treated with induction chemotherapy. They found that IDH1 and IDH2 mutations were associated with normal karyotype and NPM1 mutations and were present in 12 (7%) and 24 (14%) patients and also found an IDH1G105 single-nucleotide polymorphism (SNP) alteration in 24 (14%) patients. Overall, they found that 30% of patients had IDH alterations, but there was no association with achievement of CR, remission duration, OS or EFS, and IDH aberrations . Caramazza et al. from the Mayo Clinic examined IDH mutations in 157 patients with hematologic malignancies and suggested an association with isolated trisomy 8. Eighteen IDH mutations were identified, with the majority (15) being IDH2 mutations. Seventeen of the 18 IDH mutations occurred in myeloid malignancies, among which the large majority were MDS or AML. IDH mutations were fewer among 64 additional patients with AML or MDS without isolated trisomy 8. In MDS patients with trisomy 8 alteration, prognosis was similar between those with IDH mutations and those without .
2.2. TET2 Mutations
TET2, the ten-eleven-translocation gene 2, is located in chromosome 4 at band 4q24. Delhommeau and colleagues first described the presence of TET2 mutations in myeloid malignancies, by evaluating bone marrow samples from 320 patients with MDS, MPNs, and AML. TET2 defects, either mutations or deletions, were discovered in 17 of 90 patients with MDS (19%), in 24 of 198 patients with MPN (12%), and in 5 of 21 patients with secondary AML (24%) . Other groups have since reported a similar prevalence of TET2 mutations in myelodysplastic syndromes and other myeloid diseases [51–53]. TET2 mutations were also found in the large majority (median of 96%) of the precursor cells in the bone marrow of patients with mutations, including CD34+ progenitor cells . In addition, there has also been much interest in the role of TET2 as a transformative mutation in myelodysplasia and MPNs. By analyzing the DNA of paired samples from patients with transformed, secondary AML, it was found that TET2 mutations were detected most frequently after the transformation of MPNs to AML .
The pathogenic mechanism of TET2 mutations in myeloid diseases has been an area of intense investigation. Strong evidence supports the integral role of TET2 in demethylation of DNA and epigenetic regulation, specifically in the conversion of 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) [30, 31]. A recent intriguing study convincingly demonstrated that 5-hmC is an intermediary in the process of DNA demethylation, and that TET hydroxylases are essential mediators of this process . Ko et al. further demonstrated that mutations of TET2 lead to alteration and suppression of catalytic activity of the enzyme. They reported low DNA levels of 5-hmC in bone marrow samples taken from patients with TET2 mutations. In addition, the depletion of TET2 in murine models led to a suppression of differentiation of hematopoietic precursors in culture . This interruption in myelopoiesis may be a necessary “hit” or alteration in the process of leukemogenesis in patients with TET-2 mutant AML.
Most recently, a variety of groups have studied the prognostic impact of TET2 mutations in MDS and AML. They appear to have a particularly high prevalence in chronic myelomonocytic leukemias, in which they have been found to be associated with significant monocytosis and poor outcomes . Data on prognosis has also been presented in patients with MDS and AML, where there has been some controversy. Nibourel et al. sequenced sample DNA from 111 patients who had achieved CR after induction chemotherapy for de novo AML. They reported an overall incidence of 17% of TET2 mutations, which were associated with NPM1 mutations but did not appear to have an impact on survival. This lack of effect on outcomes was also reported by other investigators [59, 60]. Another group from France reported that TET2 mutations were actually independently associated with significantly improved overall and progression-free survival in patients with MDS. Only a small percentage of patients (7%) in this study were reported to have secondary AML . In contrast, other investigators have reported a significant adverse effect on overall survival in patients with AML , and a recent CALGB report of 427 patients with cytogenetically normal AML found that TET2 mutations were associated with a lower rate of CR and shorter disease-free and overall survival, with outcomes particularly worse for patients with favorable risk (CEPBA and NPM1 mutant) disease .
Perhaps, more intriguing has been the consistent finding in recent studies that TET2 mutations rarely cooccur with mutations affecting isocitrate dehydrogenase (IDH1 and IDH2) [60, 62]. As mentioned, the possible mechanism for aberrant methylation and leukemogenesis in IDH-mutant AML may be related to the downregulation of α-KG levels, on which TET2 enzymes depend for their activity . Others have reported that the metabolite 2-HG, markedly elevated in samples of patients with IDH mutant AML, can also directly inhibit TET2 function . Therefore, TET2 and IDH mutations may be leukemogenic through a common mechanism, that of suppression of TET2 function. They, thus, likely act as a distinct mutational class in AML with overlapping effects on DNA methylation and leukemogenesis (Figure 1).
2.3. DNMT3A Mutations
DNA methyltransferases catalyze the methylation of cytosine residues of CpG dinucleotides in DNA and are encoded by the human genes DNMT1, DNMT3A, and DNMT3B. DNMT3A mutations in AML have only been recently described [63–65] and found to be present in approximately 20–22% of patients with de novo AML. Interestingly, these mutations seem to be associated with intermediate-risk AML, a finding also noted with TET2- and IDH-mutant disease. In contrast, no cases of favorable risk AML contained these mutations . Unlike the exclusivity of TET2 and IDH mutations in recent studies of AML, DNMT3A mutations often cooccurred with IDH mutations, suggesting that these latter two mutations may not have overlapping functions in leukemogenesis.
Somatic mutations in DNMT3A have been reported as nonsense, frameshift, and missense mutations throughout the open-reading frame. However, a notable recurrent mutation in DNMT3A has been repeatedly reported in AML  and MDS  patients as a somatic missense mutation at amino acid R882. Although one study reported a decrease in DNA methylation activity of >50% with the DNMT3A R882 mutant  in an in vitro methyltransferase assay, AML patient samples with DNMT3a mutations were not found to have altered total 5-methylcytosine content or altered patterns of methylation  (Figure 1). Equally important is the fact that the DNMT3A R882A mutation appears to occur exclusively as a heterozygous mutation suggesting a potential gain of function which may or may not require a wildtype copy of DNMT3a for altered function. Future studies examining the function of the DNMT3A R882 mutation in vitro and in vivo in the presence of wildtype DNMT3A will hopefully shed further light on a pathogenic mechanism of DNMT3a mutations in AML.
DNMT3A mutations were subsequently noted in patients with MDS and secondary AML. Walter et al., sequencing samples from 150 patients, found that 12 harbored DNMT3A mutations. Similar to the trend noted in AML, the majority of mutations were at amino acid R882. These mutations were associated with worse overall survival and rapid progression to AML, although sample size was small and transplantation status was not considered . The adverse impact on survival has also been reported in patients with AML. Ley and colleagues found, in their cohort, that those with DNMT3A mutations experienced a significantly worse median overall survival of 12.3 months as compared to 41.1 months for those without mutations. Interestingly, it appeared that DNMT3A mutations accounted for the majority of the adverse effect on survival seen even in patients with FLT3-ITD alterations . A more recent study from China, studying patients with acute monocytic leukemias, also reported decreased overall survival and worse outcomes in those with DNMT3A mutations . Lastly, DNMT3a mutations have also recently been reported in patients with primary myelofibrosis . Larger studies of DNMT3a mutations in patients with additional MPNs will be needed to further understand the clinical and/or prognostic importance of DNMT3a mutations in MPNs.
2.4. EZH2 Mutations
EZH2 is a highly conserved enzyme which serves as a histone H3 lysine 27 (H3K27) methyltransferase. Although EZH2 has been known to be overexpressed in several epithelial malignancies for some time, only in 2010 was it discovered that EZH2 may be mutated in hematopoietic malignancies. Curiously, a recurrent mono-allelic activating mutation has been identified in EZH2 at tyrosine 641 in patients with lymphomas  while a series of apparent loss-of-function mutations have been found in patients with MDS and primary myelofibrosis [69–71].
The fact that EZH2 is altered by overexpression/increased activity in epithelial cancers and lymphomas, yet inactivated in myeloid malignancy, argues that the biologic consequences of alterations in H3K27me3 may be tissue specific. At the same time, rigorous assessment of whether EZH2 mutations affect the abundance and/or the distribution of H3K27me3 in the chromatin of malignant versus paired normal nonmutated cells has not yet been published (Figure 1). In addition, investigation of the effects of EZH2 mutations on alterations in DNA methylation may be particularly important given that EZH2 physically interacts with DNA methyltransferases 1–3 , and data suggests that H3K27 methylation is a necessary prerequisite for DNA promoter methylation .
From a clinical standpoint, inactivation of EZH2 by loss or mutation in MDS is enlightening as cytogenetic abnormalities of chromosome 7 which have been long recognized in MDS and AML and linked to adverse outcome [73, 74]. In fact, several studies suggest that MDS patients with EZH2 mutations have worsened overall survival compared to those without EZH2 mutations, regardless of gross cytogenetic findings [70, 71]. Larger studies incorporating EZH2 mutations in light of additional genetic abnormalities will be needed to further clarify the prognostic importance of EZH2 mutations in MDS and MPNs and are ongoing.
3. The Prospect of Novel Therapeutic Agents Targeting Epigenetic Modifiers in Myeloid Malignancies
3.1. DNA Methyltransferase Inhibitors (DNMTIs)
The first three epigenetic targeted therapeutics which have been FDA approved for use in the United States include 2 drugs targeting DNMTs (azacitidine (AZA) and decitabine (5-aza-2′-deoxycytidine)) as well as one histone deacetylase inhibitor (vorinostat). Although the use of DNMTIs has proven useful in the therapy of high-risk MDS as well as in AML, there are several questions which have lingered regarding the use of these therapies: (1) what is the ideal dose and schedule of DNMTIs? (2) what is the true mechanism of action of the nucleoside DNMTIs? (3) are there biomarkers which can be used for predicting response and/or resistance to DNMTIs? and (4) can we develop nonnucleoside direct inhibitors of DNMTs?
Despite questions regarding use and schedule of DNMTIs, there have been several interesting new developments in this class of therapeutics. Within the original category of nucleoside analog DNMTIs, an oral formulation of AZA has recently been developed . Parenteral azacitidine is approved for administration at 75 mg/m2 for 7 days every 28 days, and this dose is believed to result in DNA hypomethylation as well as cytotoxicity. Using several assays for DNA methylation, this dose of 5-aza has been shown to result in maximal DNA hypomethylation at approximately day 15 with gradual return of DNA methylation back to baseline around the time of next cycle . Given this, use of orally administered AZA on a more frequent schedule may result in altered effects on DNA methylation and cellular cytotoxicity and holds the potential for greater therapeutic efficacy. The initial phase I trial of oral AZA on a 7 days schedule revealed that the drug is bioavailable, safe, and clinically active in patients with MDS and AML. At the 2010 ASH meeting, results of the multicenter phase I study of extended oral AZA schedules revealed that oral azacitidine on a 14- or 21-day schedule is well tolerated, with no AZA accumulation, and promising clinical responses were observed .
The direct cytotoxic effects of the clinically utilizing DNMTIs as well as their chemical instability have prompted continuous rationale for developing additional DNMTIs. A third nucleoside DNMTI which has been under development for some time is the cytidine analogue zebularine. Although zebularine has a similar mechanism of action as decitabine and azacitidine, resulting in the depletion of DNMTs through covalent bonding with DNMTs, zebularine has a much longer half-life making oral administration of the drug possible [77, 78]. Additionally, zebularine appears to be selectively incorporated into malignant and not normal cells admixed with tumor in at least one setting, a property not seen with decitabine or AZA . Despite these properties, one limitation to the development of zebularine for clinical use has been the fact that higher concentrations of zebularine are needed to obtain similar levels of demethylation in cells in comparison with azacitidine and decitabine . Further preclinical works addressing the practicality of the drug as a clinical therapeutic agent are ongoing.
In addition to the nucleoside DNMTIs, there has been considerable efforts at developing nonnucleoside targeted molecules to directly inhibit individual DNMTs. One approach has been the development of antisense oligonucleotides targeting DNMT1 for in vivo use. One such molecule, the phosphorothioate antisense oligonucleotide MG98, was developed based on its ability to knockdown DNMT1 expression in various model systems [80, 81]. However, phase I clinical trials of MG98 in solid and hematopoietic tumors was disappointing with very little consistent knock down of DNMT1 mRNA in patients [82, 83]. This likely resulted from inefficient intracellular uptake of MG98.
Rational design of small molecules targeting DNMTs through noncovalent interactions with the catalytic sites of these enzymes has resulted in the development and characterization of several test compounds. The first rationally designed DNMT1 inhibitor is RG108 which was designed utilizing a three-dimensional model of the human DNMT1 catalytic pocket . RG108 has comparable demethylating activity to zebularine but appears to be less active than azacitidine and decitabine. Several additional small-molecular inhibitors of DNMTs have more recently been found within the NCI open database of compounds through a similar screening approach which led to the discovery of RG108 . Further preclinical characterization of all of these compounds is underway.
The use of DNMTIs in patients with high-risk MDS and AML has proven that while durable complete remissions are possible with these drugs, responses can be quite variable with no current routinely used clinical parameter known to predict likelihood of response to therapy. With the discovery of mutations in TET2 in these patients and the postulated pathogenic mechanism of TET2 mutations in MDS/AML, it has been hypothesized that TET2-mutated patients may have higher rates of response to DNMTIs. This has recently been suggested by a small French study of 86 patients with MDS and secondary AML. The investigators reported that those with TET2 mutations experienced a response rate (RR) of 82% to AZA in comparison to the TET2-wt group, which had a significantly lower RR of 45%. However, there was no effect on survival parameters, and the study group was quite heterogeneous with few additional genetic parameters studied . In contrast, other groups have found that TET2 alterations in a similar cohort of patients may actually predict for decreased responsiveness to demethylating therapies . The small number of patients included in these studies and the limited genetic characterization of the patients must be considered. Larger studies with more comprehensive genetic evaluation will be critical in determining if mutations in genetic factors suspected to be important in regulating DNA methylation (TET2, IDH1/2, and DNMT3a mutations, amongst others) affect response to DNMTIs.
4. The Prospect for Novel Histone Methyltransferase Inhibitors in Myeloid Leukemias: DOT1L Inhibition and Rational Design of Protein Methyltransferase Inhibitors
Recent discovery of the potential importance of aberrant hypermethylation of histone lysines in the pathogenesis of myeloid leukemias driven by MLL-translocations , NUP98-NSD1 translocations , and possibly IDH1/2-mutant disorders  suggests the possibly of targeting histone lysine methyltransferases in myeloid leukemias.
One novel and exciting prospect utilizing this therapeutic rationale is the study of DOT1L-targeted therapy for the selective treatment of MLL-translocated leukemias. In addition to the recently identified mutations in epigenetic modifiers in MDS and AML, a number of translocations disrupting the normal activity of epigenetic modifiers in myeloid malignancies have been recognized for a longer period of time (Table 1). Key amongst the frequent translocations altering the activity of an epigenetic modifiers in AML includes translocations involving mixed lineage leukemia 1 (MLL1) which occur in at least 10% of adult AML patients and >70% of infant acute leukemias. MLL1 normally serves as an histone H3 lysine 4 (H3K4) methyltransferase. MLL1 translocations result in fusion of the N terminus of MLL1 to one of >60 different translocation partners . In a recent landmark survey of MLL rearrangements, 760 MLL-rearranged biopsy samples were reviewed and 104 different MLL rearrangements were found in adult and pediatric acute leukemia patients . However, amongst AML patients with MLL rearrangements, 77% were accounted for by one of 7 translocations: MLL-AF9 (30.4%), MLL-AF10 (14.5%), MLL-ELL (10.9%), MLL-AF6 (10.1%), MLL-ENL (5.4%), MLL-AF17 (2.9%), and MLL-SEPT6 (2.5%). Many of these same MLL rearrangements are also common in acute lymphoblastic leukemia (ALL) along with MLL-AF4 which is the most common MLL rearrangement in ALL (accounting for 66% of MLL-rearranged ALL cases) .
Importantly, the four most frequent MLL translocations (MLL-AF4, MLL-AF9, MLL-AF10, and MLL-ENL) result in recruitment of DOT1L (disruptor of telomeric silencing 1-like) to the fusion protein and acquisition of histone 3 lysine 79 (H3K79) methyltransferase activity (Figure 1). A number of studies using both shRNA for DOT1L and conditional deletion of DOT1L have recently shown that the H3K79 methyltransferase activity is critical for leukemogenesis induced by MLL-fusion proteins [91, 92]. This has led to the concept of developing targeted therapy for DOT1L inhibition in the therapy of MLL-translocated leukemias. At the 2010 ASH meeting, investigators at Epizyme Inc. presented the initial results from in vitro studies of the first DOT1L inhibitor, EPZ01 . EPZ01 acts as a competitive inhibitor of the cofactor S-adenosylmethionine (SAM), the universal methyl donor for all enzymatic methyltransferase reactions. Despite the ubiquity of SAM in protein methyltransferase reactions, EPZ01 is reported to have a 500-fold selectivity for DOT1L over other lysine histone and arginine methyltransferases. Consistent with this, the investigators revealed selective killing of EPZ01 for leukemia cell lines bearing MLL1 translocations over non-MLL rearranged cell lines. DOT1L-inhibition appeared to downregulate H3K79me3 abundance globally and at critical loci, serving both as proof of concept of the mechanism of activity and potentially as a biomarker of response .
As mentioned earlier, MLL rearrangements are also frequent in ALL with MLL-AF4 translocation being the most frequent MLL rearrangement in ALL . Although two initial transgenic mouse models of MLL-AF4 fusion gene overexpression did not result in the development of acute leukemia, a conditional knockin model of MLL-AF4  as well as a retroviral transplantation model of MLL-AF4  did result in the development of ALL. Moreover, mice with overexpression of MLL-AF4 in the conditional knockin model by Krivstov et al. were clearly distinguished by increases in H3K79me3 indicating a clear link between the presence of MLL-AF4 fusion oncoprotein and acquisition of increased H3K79 methyltransferase activity. In addition, recent purification of the MLL-AF4 complex has clearly indicated binding of DOT1L to this complex . Equally important, use of an shRNA against DOT1L inhibited the expression of several genes critical for MLL-AF4-mediated oncogenesis in the MLL-AF4-conditional knockin model underscoring the potential importance of DOT1L inhibition in the therapy of ALL with MLL-AF4 rearrangement .
One key question which must be further addressed in the preclinical development of DOT1L-targeted therapy is the question of potential adverse ramifications of DOT1L inhibition. Jo et al. recently reported that mice bearing conditional disruption of DOT1L-developed pancytopenia and failure of hematopoietic homeostasis revealing a critical role of DOT1L in normal hematopoiesis . At the same meeting, Bernt et al. also developed a conditional deletion model of DOT1L in vivo using Vav-Cre technology for DOT1L deletion in the adult hematopoietic and endothelial cells but also in the germline . DOT1L-/- mice in this system were born at Mendelian rations and with blood counts at lower border of normal range. In addition, cardiac-specific deletion of DOT1L resulted in increased mortality in mice due to cardiac dysfunction which closely resembled human dilated cardiomyopathy . Interestingly, Nguyen et al. further discovered that DOT1L is downregulated in patients with idiopathic dilated cardiomyopathy, and the cardiac phenotype in mice could be rescued by expression of dystrophin . Further characterization of these mouse models and use of DOT1L inhibitors in preclinical in vivo testing will hopefully clarify the potential utility and safety of this very promising new therapeutic strategy.
Development of other specific inhibitors of histone methyltransferases holds promise in myeloid leukemias. The challenges for this prospect have been twofold: (1) knowledge of the genome-wide and locus-specific effects of histone modifications due to direct genetic abnormalities found in myeloid leukemia patients has been less clear for the majority of non-MLL translocated patients. For instance, loss-of-function mutations in the H3K27 methyltransferase EZH2 has been recently found in patients with MDS and primary myelofibrosis [69–71]. At the same time, loss of the H3K27 demethylase UTX has also been suggested to occur in some of the same disorders making the rationale for targeted changes affecting H3K27 methylation hard to understand . (2) In addition, from a drug development standpoint, potent and selective inhibitors of histone protein methyltransferases have only recently begun. One strategy mentioned earlier is the development of small-molecule inhibitors of the SAM-binding pocket, a universal feature of protein methyltransferase somewhat analogous to the targeting of the ATP-binding pockets of protein kinases. In fact, the first selective and potent protein methyltransferase inhibitor was recently reported using this strategy . This molecule serves to inhibit the arginine methyltransferase CARM1, a protein overexpressed and thought to be important in the pathogenesis of prostate and breast carcinomas [100, 101]. Although empiric use of these medications in early-phase clinical trials has been utilized previously with some success, in-depth characterization of histone posttranslational modifications in patients with MDS/AML may shed light on rational strategy for specific histone methyltransferase inhibition as a therapeutic strategy in these disorders.
The exciting discovery of new genetic abnormalities in patients with myeloid malignancies holds the promise for furthering our understanding of the pathogenesis of these disorders but also in refining our risk stratification and therapeutic management of patients. As highlighted here, a series of studies have rapidly suggested that mutations in TET2, IDH1/2, and DNMT3a will likely refine our current prognostication of patients with AML if borne out repeatedly in large prospective trials of AML patients. Moreover, given the suggested effects of these genetic abnormalities on DNA methylation, the potential importance of these mutations on affecting response to DNMTIs will need to be more thoroughly investigated. The effects of the recurrent DNMT3a R882 mutation will particularly need to be scrutinized given its frequency and the fact that it is always present as a heterozygous mutation. Furthering our understanding of the specific altered epigenetic marks placed by genetic abnormalities in MDS/AML patients is critical as it may result in the development of novel epigenetic targeted therapeutics. The clearest example of this currently is the exciting development of DOT1L inhibitors for MLL-translocated leukemias described here. Further characterization of DOT1L deficiency/inhibition in a variety of in vivo models is greatly anticipated. Moreover, development of additional protein methyltransferase inhibitors is likely forthcoming and prompts for greater understanding of the epigenetic alterations present in patients with MDS and AML.
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In Reply to: Audi announces 7-killer S8!!!! posted by MikeH on October 28, 2000 at 21:39:49:
The A8 is Audi's equivalent to the 740, except that Audi also provides Quattro all wheel drive, and the car has an aluminum space frame.
I had the use of an A8 for a week, and it was not up to 740 standards. But the S8 adds a sport suspension and 360 HP motor. This is what was missing in the A8. Audi's 4050 lb curb weight is considerably under a 7, and it provides the AWD as well. According to the article, the S8 runs the 1/4 mile in 13.9 and has a top speed of 155. And at $74,000, it is reasonably priced.
Attention, please, BMW: put the M5/Z8 engine in the 740 sport and call it an M7. EVERYBODY will want one. It will be the finest luxury sedan in the world. You are being eclipsed by Audi, and there is no reason to let this happen.
Comments? Well, I've got a few!
First, let's not be silly, there will be no M7 in the E38 flavor. The E38 tombstone has already been erected; in the late spring the E65 will be introduced.
While we're at it, the M5 engine will not go into the 7er. Sorry, it's a fact. There is such a strong demand for the M5 and Z8 (despite their pricing) that BMW is having a hard time supplying these engines in the first place. Also, the M5 engine does not have enough power to properly propel the 7er (that's why the M3 and M5 have different engines). If there is an M7, it will be a V12. Displacement? I don't know. Supposedly the new V12 will displace 6 liters, so an M engine should displace more. It would seem rather 70s to have a 6.5 or 7 liter engine in a sedan, so like I said, I don't know how they're going to work that out. I personally think it's very likely that the V12 will be a 5.5, and the M engine will be 6.0.
Now there is a real hope for an M7 in the E65 variant. This is only because Audi is offering up the S8 (actually, it has been for the past 2 or so years, in Europe), and Mercedes has the S55AMG. Honestly, the 740i Sport was a band-aid solution; the cost of developing an M7 so late into the E38 life cycle is not cost effective, and 7 series sport suspensions have been around in Europe since the E32 (late 80s). So just throwing in the sport suspension and a few trinckets was easy. Let's face it, what's so sporty about the 740iS? The suspension. The powertrain is nothing new. (don't get me started on the rear diff.!)
Now my predictions (hopefully will come true):
(1) A new V12. Conventional valve train will be eliminated. Vavles will be opened electro-magnetically. I predict displacement to be 5.5 liters. Also, expect to see the a/c compressor etc electrically operated (ie. not off the crank) so as to sap less power from the engine.
(2) Pricing. If the V12 is 5.5, expect to pay less that the MB V12. About the price of an S55AMG (good plan, get a V12 for the price of a mercedes V8).
(3) Although the M62B44 is getting long in the tooth, it will power the new 740.


text:
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Public Health nurses are available to consult with teachers and school nurses. Information and resources are available on various health topics such as:
Vision and hearing screening clinics are provided when requested by the school. Other services, such as Early Childhood Screening, are provided by contractual agreement.

domain: health


From one top GOP senator openly lamenting the fallout of the ongoing fight over contraception, to the author of the controversial legislation at the heart of that fight effectively conceding defeat in the upper chamber, signs mounted Tuesday that suggest Senate Republicans want to put the birth control controversy to bed.
“You know, I think we’ve got as many votes as I think there were to get on that,” Senate GOP Conference Vice Chairman Roy Blunt told TPM Tuesday afternoon after a weekly Capitol briefing. “I think the House side may take some further action. That debate will go on for a long time, though I don’t know that there’s anything else to happen in the Senate in the near future.”
The concession marks a departure for the GOP leadership, which as recently as last week insisted that Republicans were on the right side of the issue and would fight on.
Last Thursday, after his amendment was narrowly tabled 51-48, Blunt vowed that, “The fight is not over.” He had maintained that he wants to tack it onto legislation the president cannot veto. But on Tuesday, after a meeting with his caucus, he dialed down expectations for any further action in the Senate.Sen. Lisa Murkowski (R-AK) told TPM that she has no indication that leadership wants to continue the fight. “I really don’t know,” she said. “I really don’t have any sense of that.”
Murkowski — a former member of the GOP leadership — originally expressed reservations about the Blunt legislation and the broader fight over health benefits. Though she ultimately voted for the amendment, she has largely disavowed that vote, and conceded Tuesday that Republicans have fallen on the wrong side of the issue.
More evidence that the GOP is ready to relent: Senate Republican leaders have abandoned what they once considered winning turf, and gone silent after weeks of publicly attacking President Obama and Democrats for infringing on religious liberties.
Murkowski added that ripple effects — on the campaign trail and in public comments by conservative commentators — have damaged the GOP as well. She said she’s heard from constituents, and fretted that the ongoing spat is giving voters the sense that her party is on the wrong side of a war on women’s health.
“I heard a lot [from my constituents] because it was in the news this weekend,” she told me. “There’s just an awful lot that’s been going on. There have been some comments made by some of our presidential candidates. There was the incendiary comments made by Rush Limbaugh. I think [these incidents] are just adding to this sense that women have that women’s health rights are being attacked — that in 2012 we’re having a conversation about whether or not contraception should be allowed. I think most thought that we were done with those discussions decades ago. So it’s been kind of an interesting week for women’s health issues.”
Murkowski said she was “just stunned” by Limbaugh’s protracted smear of Georgetown law student Sandra Fluke. “In the end, I’m a little bit disappointed that there hasn’t been greater condemnation of his words by people in leadership positions,” she said. Even Republicans? “Everybody,” she said.
These concessions delight Democrats, who plan to revisit the events of the last several weeks, and the vote on the Blunt amendment, as the election nears.
Sen. Chuck Schumer (D-NY), who devised the Democrats’ legislative and messaging strategy in this fight, told TPM, “I think Republicans know that it hasn’t served them well.”


text:
June Round Lab
The microbiota is now appreciated to have clear and important effects on mammalian health. However, this community is complex including bacterial, fungal and viral members. Moreover, most research in this area is correlative and lacks clear mechanistic studies. My research program is designed to extend beyond the correlative studies and draw causal associations between gut microbes and health. I have designed this program to analyze a diverse array of microbes including bacteriophages (viral), fungi and bacteria and to uncover the mechanistic underpinnings of how these organisms influence disease. Our work extends into many areas of health including gastro-intestinal diseases such as IBD and colorectal cancer, neurological disorders and metabolism. Given this breadth, my research program integrates a wealth of collaborators to ensure well-rounded expertise in all areas. This has allowed us to move into new areas and forge an inter-disciplinary training program for students and post-doctoral fellows that train in my laboratory. My end goal is to exploit interactions with commensal microbes and the host immune system to harness microbial products for therapies.

domain: medical


text:
Despite the election of political centrist Emanuel Macron as French President, European investors would be wrong to conclude that threats to the sustainability of the Euro have now passed. Indeed, a major new threat looms on the horizon – Italian elections. Along with the prospect of higher US interest rates, this threat suggests the Euro could come under renewed downward pressure – which may, in fact, support European equities, especially on a currency hedged basis.
Renzi is Back!
Having resigned as Italian Prime Minister in December last year – following the failure of his proposed constitutional reforms to pass a referendum – Matteo Renzi recently regained leadership of the ruling Democratic Party (PD) after a stunning 70% of party members voted for him in primary elections.
While the victory did not automatically reinstall Renzi as Prime Minister – a position now held by Renzi’s former foreign minister and PD colleague Paolo Gentiloni – it does mean Renzi will lead the party’s efforts in the next national election, which must be held no later than May 2018.
If the Democratic Party wins enough seats at that election, Renzi could indeed be appointed Prime Minister by President Sergio Mattarella, to run what would likely be another coalition government.
Five-Star party a major risk
Yet while Renzi remains a popular figure within this own party, his reformist zeal and combative style has meant his once high national popularity rating has declined, and many Italians harbour suspicions over where he wants to take the country.
Indeed, according to polling, the anti-Euro Five-Star Party is the most popular in Italy, with just over 30 per cent support, or around 6 percentage points more than Renzi’s Democratic Party. At this stage, however, the challenge for the Five Star Party is that it has so far maintained it would not be part of a coalition government – even though current polling suggests it won’t get the 40% popular vote that an ability to govern in its own right would likely require.
Either way, markets are likely to grow apprehensive as the Italian election looms. After all, the Five-Star Party platform pledges to hold a referendum on remaining part of the single currency bloc, and generally espouses anti-establishment trade protectionist policies.
Disenchantment with the political establishment in Italy remains high, as the country continues to suffer from relatively low growth and high unemployment – even by European standards.
Unemployment rates: Italy, Germany and the European Union
An early election remains a possibility
Having regained leadership of the Democratic Party, Matteo Renzi could grow impatient and may push for early elections. As it stands, his party is already at risk of fracturing due to left-wing dissenters. Even the Five Star Party is keen on early elections, though the ultimate decision rests with the President, Sergio Mattarella.
Euro weakness, European equity strength?
Ironically, European equities, in general, could benefit from growing political risk in Italy to the extent it weakens the Euro and boosts the competitiveness of European companies in global trade. Indeed, as a major global exporter, the German economy was a key beneficiary of Euro weakness during Europe’s sovereign debt crisis a few years ago. Italian political risks could also weaken the Euro indirectly to the extent this makes the European Central Bank even more hesitant about winding back monetary stimulus anytime soon. Adding to the downside risks facing the Euro, moreover, is the prospect of further interest rates increases in the US.
All up, the potential downside risks faced by the Euro as the next Italian election looms, presents (perhaps somewhat perversely) upside potential for stocks across the continent. Either way, these risks suggest those seeking European equity exposure might find merit in a currency-hedged vehicle – such as the BetaShares WisdomTree Europe ETF – Currency Hedged (ASX Code: HEUR).

domain: economics


text:
Here’s another St. Patrick’s Day idea: mint fudge. It’s super easy to make (about 10 minutes of prep work) and tastes just like Andes chocolate mints, but in soft fudge form.
I love Andes mints. I get so excited when they are offered at restaurants as an after-dinner-mint or put on the pillows in hotel rooms at night. So I really enjoyed these treats.
I made them as two layers, but if you want them to look more like Andes mints, you can add another layer of dark chocolate on top. I personally didn’t want to spoil the pristine winter green surface, so I chose to keep it as two layers.
I made this fudge in the microwave with just chocolate chips, condensed milk, a little food coloring and some mint extract. Ten minutes of prep work and these were ready to go in the fridge to set.
They keep for quite a while, making them perfect for gifting.
Or maybe for storing in your secret stash so you can slowly eat them all yourself?
More Recipes to Try
Andes Mint Fudge
- 1 1/2 cups semisweet chocolate chips
- 1 1/2 cups white chocolate chips
- 1 (14-oz) can condensed milk divided
- 1 tsp mint extract
- 2-4 drops of green food coloring
- Line a 9 x 9 inch pan with parchment paper (this ensure the fudge will not stick to the pan and can be peeled off easily).
- Combine semisweet chocolate chips and half of the condensed milk in a large microwave-safe bowl. Microwave for 30 seconds and stir with a large spatula. Microwave for another 30 seconds and stir again. If chocolate is not yet completed melted, microwave for an additional 30 seconds. Mine took exactly 1 minute and 30 seconds total. Don't heat the entire 1 minute 30 seconds at once because you may burn your chocolate. Stir until chocolate is completely melted and fudge is smooth.
- Pour chocolate fudge into the baking pan lined with parchment paper. Spread out fudge with spatula until it is evenly spread across the entire pan and the surface is smooth.
- In a separate large microwave-safe bowl, combine white chocolate chips, remaining condensed milk and mint extract. Heat and stir at 30-second intervals, until chocolate is completely melted and smooth (Again, mine took 1 minute and 30 seconds total). Add in a few drops of green food coloring and mix into batter until it reaches the desired green hue you desire.
- Spoon the mint fudge layer on top of semisweet layer, spreading out with spatula until it is evenly spread across the entire pan and the surface is smooth.
- Refrigerate for an hour or more to set the fudge. Slice into rectangles with a sharp knife before serving.
The nutrition information provided are only estimates based on an online nutritional calculator. I am not a certified nutritionist. Please consult a professional nutritionist or doctor for accurate information and any dietary restrictions and concerns you may have.

domain: food


Author Brendan BurkettÑParalympic gold medalist and world-record holderÑpours his love of sport and his expertise as an award-winning engineer and biomechanics professor into this enhanced, revised, and reformatted text. Divided into three parts covering the fundamentals of sport mechanics, mechanical principles, and application of the knowledge to sport contexts, this text highlights real-life sporting achievements and the mechanics behind them. Various pedagogical aids including review questions and practical activities are featured, allowing students and coaches to gauge their understanding of key concepts chapter by chapter.
Publish Date: 11 February 2010
Category: Sports Science


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Second Regular Session 115th General Assembly (2008)
PRINTING CODE. Amendments: Whenever an existing statute (or a section of the Indiana
Constitution) is being amended, the text of the existing provision will appear in this style type,
additions will appear in this style type
, and deletions will appear in
this style type.
Additions: Whenever a new statutory provision is being enacted (or a new constitutional
provision adopted), the text of the new provision will appear in this style type
. Also, the
will appear in that style type in the introductory clause of each SECTION that adds
a new provision to the Indiana Code or the Indiana Constitution.
Conflict reconciliation: Text in a statute in this style type
this style type
between statutes enacted by the 2007 Regular Session of the General Assembly.
HOUSE ENROLLED ACT No. 1220
AN ACT concerning taxation.
Be it enacted by the General Assembly of the State of Indiana:
SOURCE: ; (08)HE1220.1.1. -->
SECTION 1. [EFFECTIVE UPON PASSAGE] (a) The commission
on state tax and financing policy established under IC 2-5-3 shall:
(1) study state and local funding alternatives for the South
Shore West Lake extension; and
(2) report the commission's findings and recommendations to
the legislative council before November 1, 2008.
The report under subdivision (2) must be in an electronic format
under IC 5-14-6.
(b) This SECTION expires January 1, 2009.
SOURCE: ; (08)HE1220.1.2. -->
SECTION 2. An emergency is declared for this act.
HEA 1220 _ Concur
Graphic file number 0 named seal1001.pcx with height 58 p and width 72 p Left aligned

domain: law


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Search in palace articles.
Chehel Sotoun Palace is one of the historical monuments of Isfahan province in Iran. The palace, which covers an area of sixty-seven thousand square meters, was built in the late sixteenth century. Large and very beautiful halls have given a magnificent view to this historical monument. The magnificent painting ceiling of the hall, the eighteen […]
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domain: history


WATERTOWN, Conn. (WTNH) — A man is accused of breaking 50 windows and violating a protective order in Watertown.
Watertown police arrested Anthony Teixeira, 34, on Feb. 2 after officers responded to the report of vandalism on Jan. 3.
Officers found 38 broken windows and a mirror, all broken from the inside, according to police. Police said the detached garage had 12 broken windows.
Police said Teixeira was in the home earlier in the day and had a current protective order from a previous incident protecting a person from abuse and harassment.
Teixeira was arrested and charged with violation of a protective order, first-degree criminal mischief and second-degree breach of peace.
He was arraigned on Feb. 3 and given a $75,000 bond.


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Here’s the tutorial video showing you how to sew the baby doll dress that I showed you in Monday’s post. I suppose it wouldn’t hurt to re-post the links to the patterns you’ll need as well, so here they are:
For anyone who’s wanting to use the pattern for your vintage Velvet doll from Ideal, don’t worry; I’ll post an updated tutorial video later on, featuring Velvet in the same outfit.
We’ll also need patterns and tutorials for making the bloomers. That’s coming soon too!
If you enjoy my free, printable sewing patterns and video tutorials for making doll clothes, please show your appreciation by telling the world about them on social media! (See helpful social media links below…)
Some of my followers have wondered what motivates me to give my patterns away for free.
First of all, I’m a librarian by trade. Librarians love free stuff! We believe that the more a person knows, the more enriched their life will be. So it may sound kind of crazy, but I want ChellyWood.com to become a sort of library of free patterns that help people learn to sew doll clothes.
If you’d like to learn more about my motives, feel free to visit my Chelly’s Books page.
My Gallery Page is the easiest way to search through all of my patterns to find what you want. Each image on the Gallery Page takes you to links for patterns and tutorials.
Need help printing my patterns? This link offers a tutorial showing you how to download and print my FREE patterns using Google Docs. (For the older print-a-pattern tutorial, which uses Microsoft Word, click here.) To review my difficulty scale (demonstrating how hard or easy a pattern is by the number of flowers displayed), take a look back at this blog post.
Please note: you must enlarge my patterns to fit a full-sized piece of American computer paper (8.5 x 11 inches or 216 x 279 mm) without margins, before printing. These designs use a scant 1/4 inch seam (4 mm to be exact).
My patterns are now available through “Creative Commons Attribution.” This means that I created my patterns (and therefore I own rights to them), but I’m willing to share them with everyone who will tell people about my website.
Here are some helpful ways to tell the world about my patterns:
- You can pin them on Pinterest.
- You can like them on Facebook.
- You can tweet about them.
- Use any other form of social media that appeals to you!
Are you new to sewing? I’ve got a playlist of tutorials for the beginning sewists on my YouTube channel. It includes video tutorials showing you how to do a basic straight stitch when sewing by hand, how to use the whipstitch to hem a garment, how to sew on snaps, and even how to design your own doll clothes patterns, for those who are new to design and alterations.
In case you haven’t heard, I have actually designed some commercial patterns for Lammily LLC. You might want to visit the Lammily website to see what they’ve got going on.
If your question wasn’t answered here, feel free to submit a question. I’m always happy to help my followers find what they need, so they, too, can make amazing doll clothes and crafts.
2 thoughts on “Sew a baby #dolls dress w/free #dollClothes #patterns @ ChellyWood.com”
Hi Chelly do have a cape pattern I need to make a Dracula outfit thanks denise
I do have a couple of different cape patterns for 11.5 inch fashion dolls like Barbie/Ken. Please go to my Gallery page and look through all of the images until you find the cape you want. Click on it and click again on the link provided under the image. It will take you to the page with all the patterns you need to download and the video you’ll want to watch to understand how to make the cape you’ve chosen. Best of luck!

domain: artistic


text:
Music Gear News
Eric Clapton 'a brilliant acoustic guitar player'
The manufacturer has revealed the first ten names on its Top 50 Acoustic Guitarists of All Time list, and Old Slowhand came in at number 41.
Correspondent Bryan Wawzeneck explained that many of his younger followers grew up listening to his work on the acoustic guitar due to his Grammy-winning 1992 Unplugged album.
He noted that although he made his name as a blues player on the electric guitar in the 1960s, albums such as From the Cradle earned him a whole new legion of fans.
"The man formerly known as God proved his divine talents translated brilliantly when stripped down – his tasteful solos and clever licks shining through on Robert Johnson covers or a reworked version of Layla," Mr Wawzeneck commented.
Clapton is set to tour the UK and Ireland next year, with dates in London, Glasgow, Belfast, Cardiff and Dublin pencilled in for throughout May 2011.
ÂPosted on 25 Oct 2010 15:18 to category : Band / Celebrity News
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Through a collaboration with Martin, Sheeran's latest signature model features artwork from his latest album '÷' and has been built to his exact specifications.
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Founding guitarist of legendary LA band Y and T Joey Alves passes away due to complications relating to Ulcerative Colitis. Documentary on soon to follow.
Destroyer Cola is to be the latest release in rock veterans Kiss' long line of merchandise currently available to the general public for purchase
In a recent unconfirmed report, frontman of famous rock and roll outfit Guns N' Roses is said to be heading into the studio with Australian rock veterans AC/DC
Never before has music been so accessible. With social media, tab sites and forums at your immediate disposal, learning your favourite song couldn't be easier.
Recent Music News
The latest entry into the acclaimed microphone brands line-up, the sE8 Small Condenser Microphone packs a big punch despite its diminutive and compact frame.
A familiar face in an all new format, the Waza Amplifier Head lets you experience the full power of an established classic in the comfort of your own bedroom.
A visual percussion instrument that promises a completely immersive rhythmic experience, the DR-01S is the first of many in the latest percussive generation.
In celebration of BOSS' 40th anniversary, the company releases a triumvirate of their most famous and sought-after pedals in a limited-edition box-set format.
In celebration of their rich history, VOX launches the AC30HW60 and AC15HW60. Designed to emulate the classics, VOX add impetus to the saying 'old is gold'
Continuing the legacy of Elektron's previous entries, the Analog Four MKII/Analog Rytm MKII marks the beginning of a new era graced with bountiful enhancements


OLDSMOBILE CUTLASS ENGINE PROBLEM
1989 Oldsmobile Cutlass • 100,000 miles
My 1989 cutlass ciera has 100000 miles on it. I have been trying to figure out why the engine wont turn over. For awhile I have had to repeated press on the gas but then noticed the gas pressure being slowly decreased over time. Just yesterday I have zero pressure in the clutch and the engine wont turn over at all. I thought it might have just been brake fluid which would mean no hydraulic pressure, but after checking it, the reservoir is full. My next thing to look at is either the flywheel or see if there is a clutch cable. I haven't really been able to look too much so it is something I am unsure of. The main thing I notices with the service engine light is when I lifted my foot off the gas it would come on so I know it is a pressure issue. I am stumped as to what to look for now.
December 31, 2012.
December 31, 2012.
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DCJ Digital Accessibility Standard
How we design and develop accessible digital content at DCJ
Adhere to WCAG
The Web Content Accessibility Guidelines (WCAG) are used widely around the world as a benchmark for web accessibility.
At DCJ, we adhere – at a minimum – to WCAG 2.0 Level AA.
Our goal is to take our digital accessibility beyond WCAG. In particular, we carefully consider the needs of people with cognitive disability.
WCAG has four main principles:
- Perceivable – Information and user interface components must be presentable to users in ways they can perceive.
- Operable – User interface components and navigation must be operable.
- Understandable – Information and the operation of user interface must be understandable.
- Robust – Content must be robust enough that it can be interpreted reliably by a wide variety of user agents.
For each principle, there are a list of criteria that need to be met to make sure your online content is accessible. There are 38 techniques in total.
The ‘ How to meet WCAG ’ quick reference guide is a helpful starting point.
Then, you need to test and audit the digital product. See the criteria explaining how we always test, validate and verify.
You can also find out more about our criteria for reporting, fixing and reviewing.

domain: design


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March 20, 2019
Containment attempts are futile. It has already started. Brenton unleashed it.
They can’t drown the fire.
The accused Christchurch gunman is being held in New Zealand’s only maximum security prison in solitary confinement.
Brenton Tarrant is under 24 hour surveillance and has been refused access to newspapers, television and radio, The Department of Corrections in New Zealand confirmed.
Without access to outside world information, Tarrant will be unable to obtain reports on the Christchurch mosque massacre he’s accused of spearheading last Friday.
Authorities have tried to shroud the 28-year-old’s exact whereabouts in secrecy.
In a statement issued to the publication on behalf of The Department of Corrections, it confirmed Tarrant will not receive visitors.
‘He is being managed in accordance with the provisions set out in the Corrections Act 2004 and our international obligations for the treatment of prisoners.
‘At this time he has no access to television, radio or newspapers and no approved visitors,’ the statement read.
It’s not yet confirmed as to whether Tarrant will appear in person his next court appearance. Or if he’ll appear via video link from prison.
No visitors, solitary confinement, possibility of him not appearing in person in court… it’s obvious that they’re afraid that people may realize Brenton is a normal man and one of their own. They’re afraid Brenton may speak convincing words because so far that’s what Brenton has done; he spoke and wrote convincing words.
Brenton Tarrant knew something like this would happen. That’s why he recorded what he did for you to watch and wrote what he thought for you to read.
The video of his mosque tour is more entertaining than anything you could find on Netflix or the cinema and it also has better production values. People love it and want to share it with everyone, and that prompted huge censorship attempts by the people that want to keep you from witnessing reality. They want to restrict people’s access to information.
It’s always about keeping the goyim from knowing.
It’s too late for that.
The deed is done. There are videos of the deed. There are memes about the deed. The Internet will never be the same. The West will never be the same.
Nothing they could do to Brenton can undo what Brenton did. The Knight Templar knows this.
He was prepared for this.
Others… not so much.


text: Activation of the nuclear hormone receptor perioxisome proliferator-activated receptor gamma (PPARgamma) inhibits cell growth and induces apoptosis in several human cancers. We have hypothesized that PPARgamma mRNA levels could be predictors of the differentiation and survival of lung cancer. The study included 77 lung cancer cases. The mRNA levels were quantified by real time reverse transcription-polymerase chain reaction (RT-PCR) using LightCycler. The PPARgamma mRNA levels were decreased in tumor tissues from lung cancer (0.579 +/- 1.255) compared to the normal adjacent lung tissues (4.191 +/- 2.868) (P = 0.0001). No significant difference in PPARgamma mRNA levels was found among gender, age, and pathological subtype. The PPARgamma mRNA levels were higher in tumor tissues from higher differentiated lung cancer. The NSCLC patients with low PPARgamma mRNA expression (< 0.5) had significantly worse survival than the patients without low PPARgamma mRNA levels (P = 0.0438, Breslow-Gehan-Wilcoxon test; P = 0.0168, Cox's proportional-Hazards regression model). Thus, PPARgamma mRNA levels may serve as a prognostic marker in lung cancer. Using the LightCycler RT-PCR assay, the determination of PPARgamma mRNA levels might provide a potential marker for treatment of lung cancer by PPARgamma agonist. However, further studies and a longer follow up are needed to confirm the impact of PPARgamma in the biological behavior of the tumor.


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We strive to provide a generous box of food that allows the recipient to prepare well-rounded meals for 5-7 days. In addition to standard dry staples such as beans, rice, and canned goods, we include bakery items, fresh produce, frozen meat, butter and treats as available.
The pantry has many opportunities to purchase items at a greatly reduced cost. In addition, we currently prepare meals for about 150 men and women on a weekly basis.
Why a Food Pantry?
The purpose of the organization was to provide a central distribution center for companies, groups and individuals who wished to help provide food for hungry people in Pinellas County Fl. Second Harvest's mission is to feed hungry people and work to solve hunger issues in our community.
Second Harvest’s vision of ending hunger is more important than ever. Hunger is a big problem, and we need everyone’s help to solve this issue. According to the USDA, an estimated 49 million Americans are at risk of hunger, and almost 14 million U.S. children under 18 are food insecure.

domain: sociology


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Pros: Rachel in Santa Rosa location was very friendly and helpful.
Cons: I originally ordered the car to be picked up at Petaluma location. No communication was received regarding the change of pick up location.
Closed before reservation time. Novato, CA
Cons: The office closed at my reservation time of noon. I couldn't get my car and had to borrow a car for the 3 day weekend
Cons: I felt the charge for a one way rental was excessive (double the going rate), since I intend to do another one from the airport at the end of my trip. Hertz has been much more reasonable in the past, typically around $35 or $40
Good company outlet
Pros: Very nice reception, nice car, easy in and out.
Cons: By booking through Kayak, we didn't get the ability to return the car early and get a refund. The interface between the companies makes this a bit of a problem....Hertz couldn't change it.
Great Service. Poor Product.
Pros: Hertz staff was helpful, timely, and efficient.
Cons: Vehicle was of poor quality and lacked certain basic features, such as cruise control and coat hooks in the rear seating area. I expected a Ford Focus and the Mitsubishi Mirage was a disappointment.
Pros: Quick check in n check out plus drop off service was great. Free upgrade as the mid size auto wasn't available.
Cons: We picked up the car the day of our reservation. The cost was final, taxes and charges included. When we arrived at the agency, we were charged $90 extra because the type of car requested was not available at that agency. We were told that we were supposed to have called the agency after we reserved the car to make sure that they had that type of car. Nothing was mentioned on the website to warn us about that. So we had to pay the upgrade. For sure, we will not go back to this rent at Herz again.

domain: travel


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Pune, often referred to as the “Oxford of the East” and the cultural capital of Maharashtra, is a city steeped in history and spirituality. Beyond its bustling urban life, Pune offers a serene escape to the devout and curious souls seeking spiritual solace. Within a 100-kilometer radius of Pune, one can find a treasure trove of temples that resonate with ancient legends, architectural marvels, and deep-rooted traditions. In this blog, we will embark on a spiritual journey to explore some of the most enchanting Temples Near Pune Within 100 KM, each offering a unique blend of devotion, history, and natural beauty.
Dagdusheth Halwai Ganpati Temple
Distance from Pune: 1.5 kilometers
Our spiritual voyage to Temples Near Pune Within 100 KM begins with the iconic Dagdusheth Halwai Ganpati Temple, situated in the heart of Pune. This revered temple is dedicated to Lord Ganesha and is known for its intricate architecture, majestic idol, and the 10-day Ganesh Chaturthi festival, which attracts thousands of devotees from all over the world.
Jejuri Khandoba Temple
Distance from Pune: 48 kilometers
A scenic drive of about two hours from Pune takes you to Jejuri, a small town famous for its Khandoba Temple. This temple is dedicated to Lord Khandoba, an incarnation of Lord Shiva, and is renowned for its vibrant turmeric festival (Jejuri Yatra) where devotees smear turmeric on each other and on the temple’s premises.
Distance from Pune: 24 kilometers
Alandi, a tranquil town on the banks of the Indrayani River, is known for the Alandi Temple dedicated to the 13th-century Marathi saint, Dnyaneshwar. Devotees visit this temple to pay homage to the saint and seek his blessings. The peaceful ambiance of Alandi makes it an ideal place for introspection and spiritual rejuvenation.
Theur Ganpati Temple
Distance from Pune: 26 kilometers
Theur, another spiritual gem near Pune, is home to the Chintamani Ganpati Temple. This temple, dedicated to Lord Ganesha, is one of the Ashtavinayak temples and is believed to fulfill the wishes of its devotees. The serene surroundings of Theur and the captivating architecture of the temple make it a must-visit destination.
Distance from Pune: 51 kilometers
Nestled amidst the picturesque hills of Yavat, the Bhuleshwar Temple is a Shiva temple known for its unique architecture and historical significance. The temple’s structure is a fine example of Hemadpanti style architecture, and the ancient carvings on its walls tell stories from Hindu mythology.
Pataleshwar Cave Temple
Distance from Pune: Within the city
Located in the heart of Pune city, the Pataleshwar Cave Temple is a hidden gem that transports you to a bygone era. Carved out of a single rock, this 8th-century temple is dedicated to Lord Shiva and is a peaceful retreat amid the bustling city.
Ranjangaon Mahaganpati Temple
Distance from Pune: 50 kilometers
Ranjangaon is home to the Ranjangaon Mahaganpati Temple, another significant Ashtavinayak temple dedicated to Lord Ganesha. The temple’s serene ambiance and intricate architecture are a testament to the rich cultural heritage of Maharashtra.
Siddhivinayak Temple, Siddhatek
Distance from Pune: 100 kilometers
Just a little over 100 kilometers from Pune lies the Siddhivinayak Temple in Siddhatek, another Ashtavinayak temple dedicated to Lord Ganesha. The temple is situated on a hill, offering panoramic views of the surrounding landscape.
Alandi Devachi Temple
Distance from Pune: 28 kilometers
Alandi Devachi, a small village near Alandi, is home to the Samadhi Temple of Sant Dnyaneshwar. Devotees visit this temple to pay their respects to the saint, whose teachings continue to inspire generations.
Distance from Pune: 66 kilometers
Narayanpur, a quaint village in Pune district, houses the ancient Narayaneshwar Temple. This temple is dedicated to Lord Shiva and is a peaceful spot for meditation and spiritual reflection.
The Temples Near Pune Within 100 KM radius offer not only a glimpse into the rich cultural heritage of Maharashtra but also a serene escape from the hustle and bustle of city life. Each of these temples has a unique charm, be it their architecture, legends, or natural surroundings.
Whether you are a devout pilgrim or someone seeking inner peace, these temples are a testament to the enduring spirituality and cultural diversity of the region. So, take a spiritual journey and explore the divine retreats that await you near Pune.

domain: geography


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Release Date: 30/04/2021
Real Groove is taken from the UK #1 album, ‘DISCO’. Collectively, Kylie’s singles have spent over 300 weeks in the Official UK Singles Chart Top 40 and she has sold more than 80 million albums worldwide. Kylie has multiple awards and accolades to her name, including 3 BRIT Awards, 2 MTV Music Awards and a Grammy. Her 2019 Glastonbury performance was the most-watched TV moment in the festival’s history. Kylie broke the record as the first female artist to earn a #1 UK album in five consecutive decades with the release of ‘DISCO’ in 2020.
A. Real Groove
B. Real Groove ft Dua Lipa (Studio 2054 Remix)


text: And it intends collective efforts to promote peace and security, protect human rights, provide humanitarian help, and advance sustainable growth. Terrorism is an affront to mankind. Neo-Nazi, white supremacist motions are becoming more dangerous by the day. They now represent the main internal security threat in several countries — and the quickest growing. Terrorist and violent extremist groups are finding fertile surface on the Internet to spew their own vicious venom. Through placing human rights at the core of all counter-terrorism policies. Today and every day time, let us work together to build more peaceful, inclusive, and stable societies in which terror plus violent extremism have no home. Through inclusion, by ensuring counter-terrorism methods reflect a wide array of voices — especially minorities, women, and young people. We must become more vigilant than ever. It undermines the beliefs that bind us together. It goals people of all ages, cultures, beliefs and nationalities. Following is UN Secretary-General António Guterres’ message for that International Day for the Avoidance of Violent Extremism when Conducive to Terrorism, observed on 12 February: We have to act to confront the task. Through prevention, by addressing the underlying circumstances that can lead to terrorism to begin with.