Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation

Methylation of CpG islands associated with genes can affect the expression of the proximal gene, and methylation of non-associated CpG islands correlates to genomic instability. This epigenetic modification has been shown to be important in many pathologies, from development and disease to cancer. We report the development of a novel high-resolution microarray that detects the methylation status of over 25 000 CpG islands in the human genome. Experiments were performed to demonstrate low system noise in the methodology and that the array probes have a high signal to noise ratio. Methylation measurements between different cell lines were validated demonstrating the accuracy of measurement. We then identified alterations in CpG islands, both those associated with gene promoters, as well as non-promoter-associated islands in a set of breast and ovarian tumors. We demonstrate that this methodology accurately identifies methylation profiles in cancer and in principle it can differentiate any CpG methylation alterations and can be adapted to analyze other species

Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation

Methylation of CpG islands associated with genes can affect the expression of the proximal gene, and methylation of non-associated CpG islands correlates to genomic instability. This epigenetic modification has been shown to be important in many pathologies, from development and disease to cancer. We report the development of a novel high-resolution microarray that detects the methylation status of over 25 000 CpG islands in the human genome. Experiments were performed to demonstrate low system noise in the methodology and that the array probes have a high signal to noise ratio. Methylation measurements between different cell lines were validated demonstrating the accuracy of measurement. We then identified alterations in CpG islands, both those associated with gene promoters, as well as non-promoter-associated islands in a set of breast and ovarian tumors. We demonstrate that this methodology accurately identifies methylation profiles in cancer and in principle it can differentiate any CpG methylation alterations and can be adapted to analyze other species

Loss of STOP Protein Impairs Peripheral Olfactory Neurogenesis

International audienceIn conclusion, STOP protein seems to be involved in the establishment of synapses in the olfactory glomerulus. Our results indicate that the olfactory system of STOP null mice is a well-suited experimental model (1) for the study of the mechanism of action of STOP protein in synaptic function/plasticity and (2) for pathophysiological studies of the mechanisms of altered neuronal connections in schizophrenia

Systemic chemotherapy with or without cetuximab in patients with resectable colorectal liver metastasis (New EPOC): long-term results of a multicentre, randomised, controlled, phase 3 trial.

BACKGROUND: The interim analysis of the multicentre New EPOC trial in patients with resectable colorectal liver metastasis showed a significant reduction in progression-free survival in patients allocated to cetuximab plus chemotherapy compared with those given chemotherapy alone. The focus of the present analysis was to assess the effect on overall survival. METHODS: New EPOC was a multicentre, open-label, randomised, controlled, phase 3 trial. Adult patients (aged ≥18 years) with KRAS wild-type (codons 12, 13, and 61) resectable or suboptimally resectable colorectal liver metastases and a WHO performance status of 0-2 were randomly assigned (1:1) to receive chemotherapy with or without cetuximab before and after liver resection. Randomisation was done centrally with minimisation factors of surgical centre, poor prognosis cancer, and previous adjuvant treatment with oxaliplatin. Chemotherapy consisted of oxaliplatin 85 mg/m2 administered intravenously over 2 h, l-folinic acid (175 mg flat dose administered intravenously over 2 h) or d,l-folinic acid (350 mg flat dose administered intravenously over 2 h), and fluorouracil bolus 400 mg/m2 administered intravenously over 5 min, followed by a 46 h infusion of fluorouracil 2400 mg/m2 repeated every 2 weeks (regimen one), or oxaliplatin 130 mg/m2 administered intravenously over 2 h and oral capecitabine 1000 mg/m2 twice daily on days 1-14 repeated every 3 weeks (regimen two). Patients who had received adjuvant oxaliplatin could receive irinotecan 180 mg/m2 intravenously over 30 min with fluorouracil instead of oxaliplatin (regimen three). Cetuximab was given intravenously, 500 mg/m2 every 2 weeks with regimen one and three or a loading dose of 400 mg/m2 followed by a weekly infusion of 250 mg/m2 with regimen two. The primary endpoint of progression-free survival was published previously. Secondary endpoints were overall survival, preoperative response, pathological resection status, and safety. Trial recruitment was halted prematurely on the advice of the Trial Steering Committee on Nov 1, 2012. All analyses (except safety) were done on the intention-to-treat population. Safety analyses included all randomly assigned patients. This trial is registered with ISRCTN, number 22944367. FINDINGS: Between Feb 26, 2007, and Oct 12, 2012, 257 eligible patients were randomly assigned to chemotherapy with cetuximab (n=129) or without cetuximab (n=128). This analysis was carried out 5 years after the last patient was recruited, as defined in the protocol, at a median follow-up of 66·7 months (IQR 58·0-77·5). Median progression-free survival was 22·2 months (95% CI 18·3-26·8) in the chemotherapy alone group and 15·5 months (13·8-19·0) in the chemotherapy plus cetuximab group (hazard ratio [HR] 1·17, 95% CI 0·87-1·56; p=0·304). Median overall survival was 81·0 months (59·6 to not reached) in the chemotherapy alone group and 55·4 months (43·5-71·5) in the chemotherapy plus cetuximab group (HR 1·45, 1·02-2·05; p=0·036). There was no significant difference in the secondary outcomes of preoperative response or pathological resection status between groups. Five deaths might have been treatment-related (one in the chemotherapy alone group and four in the chemotherapy plus cetuximab group). The most common grade 3-4 adverse events reported were: neutrophil count decreased (26 [19%] of 134 in the chemotherapy alone group vs 21 [15%] of 137 in the chemotherapy plus cetuximab group), diarrhoea (13 [10%] vs 14 [10%]), skin rash (one [1%] vs 22 [16%]), thromboembolic events (ten [7%] vs 11 [8%]), lethargy (ten [7%] vs nine [7%]), oral mucositis (three [2%] vs 14 [10%]), vomiting (seven [5%] vs seven [5%]), peripheral neuropathy (eight [6%] vs five [4%]), and pain (six [4%] vs six [4%]). INTERPRETATION: Although the addition of cetuximab to chemotherapy improves the overall survival in some studies in patients with advanced, inoperable metastatic disease, its use in the perioperative setting in patients with operable disease confers a significant disadvantage in terms of overall survival. Cetuximab should not be used in this setting. FUNDING: Cancer Research UK

Perioperative platelet and monocyte activation in patients with critical limb ischemia

BackgroundPatients with critical limb ischemia (CLI) have a high rate of adverse cardiovascular events, particularly when undergoing surgery. We sought to determine the effect of surgery and vascular disease on platelet and monocyte activation in vivo in patients with CLI.MethodsAn observational, cross-sectional study was performed at a tertiary referral hospital in the southeast of Scotland. Platelet and monocyte activation were measured in whole blood in patients with CLI scheduled for infrainguinal bypass and compared with matched healthy controls, patients with chronic intermittent claudication, patients with acute myocardial infarction, and those undergoing arthroplasty (n = 30 per group). Platelet and monocyte activation were quantified using flow cytometric assessment of platelet-monocyte aggregation, platelet P-selectin expression, platelet-derived microparticles, and monocyte CD40 and CD11b expression.ResultsCompared with those with intermittent claudication, subjects with CLI had increased platelet-monocyte aggregates (41.7% ± 12.2% vs 32.6% ± 8.5%, respectively), platelet microparticles (178.7 ± 106.9 vs 116.9 ± 53.4), and monocyte CD40 expression (70.0% ± 12.2% vs 52.4% ± 15.2%; P < .001 for all). Indeed, these levels were equivalent (P-selectin, 4.4% ± 2.0% vs 4.9% ± 2.2%; P > .05) or higher (platelet-monocyte aggregation, 41.7% ± 12.2% vs 33.6% ± 7.0%; P < .05; platelet microparticles, 178.7 ± 106.9 vs 114.4 ± 55.0/μL; P < .05) than in patients with acute myocardial infarction. All platelet and monocyte activation markers remained elevated throughout the perioperative period in patients with CLI (P < .01) but not those undergoing arthroplasty.ConclusionsPatients undergoing surgery for CLI have the highest level of in vivo platelet and monocyte activation, and these persist throughout the perioperative period. Additional antiplatelet therapy may be of benefit in protecting vascular patients with more severe disease during this period of increased risk.Clinical RelevancePeripheral arterial disease is increasingly common and is associated with a significant risk of cardiovascular complications, especially at the time of surgery. Despite this, patients are poorly provided with evidence-based therapies such as antiplatelet and lipid-lowering medications. Platelets play a key role in the pathogenesis of atherothrombosis, with elevated levels of in vivo platelet activation prognostic of adverse clinical events. This study demonstrates, for the first time to our knowledge, significantly greater levels of platelet activation in patients with severe peripheral arterial disease compared with patients with acute myocardial infarction or patients undergoing other moderate- to high-risk surgical procedures. This further emphasizes the need for improved risk stratification and cardioprotection of this vulnerable group

An Overview of Three Promising Mechanical, Optical, and Biochemical Engineering Approaches to Improve Selective Photothermolysis of Refractory Port Wine Stains

During the last three decades, several laser systems, ancillary technologies, and treatment modalities have been developed for the treatment of port wine stains (PWSs). However, approximately half of the PWS patient population responds suboptimally to laser treatment. Consequently, novel treatment modalities and therapeutic techniques/strategies are required to improve PWS treatment efficacy. This overview therefore focuses on three distinct experimental approaches for the optimization of PWS laser treatment. The approaches are addressed from the perspective of mechanical engineering (the use of local hypobaric pressure to induce vasodilation in the laser-irradiated dermal microcirculation), optical engineering (laser-speckle imaging of post-treatment flow in laser-treated PWS skin), and biochemical engineering (light- and heat-activatable liposomal drug delivery systems to enhance the extent of post-irradiation vascular occlusion)

The Effects of a simulated acid precipitation on leaf litter decomposition in a lotic ecosystem

Bibliography: p. 122-130

Vulvovaginitis and other common childhood gynaecological conditions.

Paediatric gynaecological problems, especially those involving the vulvar area, are common in childhood. The conditions frequently seen include recurrent bacterial vulvovaginitis, vulvar irritation, labial adhesions and dermatological conditions. The presentation and management of these conditions will be reviewed

Advances in synthetic, structural and reaction chemistry of zinc and zincate complexes containing alkyl and/or amido ligands

One of the most important reactions in chemistry, metallation benefits society in general as it is utilised for the synthesis of many commodities used in daily life. While lithium-based monometallic compounds have long been the reagents of choice for metallation, bimetallic reagents are emerging as second generation metallating agents that can overcome many limitations of lithium reagents. This study extends "synergic" bimetallic chemistry focusing mainly on sodium, zinc and sodium zincate systems, investigating their syntheses, structures and reactions with organic substrates. Probing the metallation of trifluoromethylbenzene, using tBuLi or nBuNa activated by a donor (THF, TMEDA or PMDETA), established the metallation system dictated both regioselectivity and yield. The origin of the increased ortho-selectivity using sodium systems was uncovered through analysis of isolated metallated intermediates [(TMEDA)·Li(C6H4-CF3)]2, 3, [(TMEDA)·Na(C6H4-CF3)]2, 4 and [(PMDETA)·Na(C6H4-CF3)]2, 5. Since pyrrole is important within pharmaceuticals and zinc-pyrrole chemistry is remarkably underdeveloped, we have synthesised the first crystallographicallycharacterized zinc pyrrolyl complexes, with one, two, three or four pyrrole anions attached to zinc in [tBuZn(NC4H4)(TMEDA)·HNC4H4], 7; [Zn(NC4H4)2(TMEDA)], 8; [{(THF)2·NaZn(THF)(NC4H4)3}∞], 9; [{(TMEDA)·Na}2Zn(NC4H4)4], 10; [{(PMDETA)·Na}2Zn(NC4H4)4], 11, as well as for comparison [{(PMDETA)·Na(NC4H4)}2], 12. Improving upon sub-ambient temperature protocols, ambient temperature zincation of N-Boc pyrrolidine was achieved via sodium zincate [(TMEDA)Na(μ-TMP)(μ-tBu)Zn(tBu)]. Metallated intermediate [(TMEDA)Na(TMP)(α-NC4H7NBoc)Zn(tBu)] 13 was crystallographically characterised. Exploring the basicity of 13, in situ sidereactions with toluene, ferrocene or anisole generated [{(TMEDA)Na[OC(NC4H8)CHPh]}2], 14, [(TMEDA)(Boc-NC4H8)Na{(η2-C5H4)Fe(C5H5)}Zn(tBu)2], 16 and [(TMEDA)Na(μ-TMP)(μ-C6H4OMe)Zn(tBu)], 17, respectively. Incorporating multifunctional 2,2'-dipyridylamide (dpa) into a bimetallic system produced neutral zinc, sodium and potassium zincates with unprecedented structures in [{(dpa)Zn(tBu)}2]; 18, [(TMEDA)2Na2(μ-dpa)2Zn(tBu)2]; 19, [{Na(THF)6}+{Zn(tBu)2(dpa)Zn(tBu)2}-]; 20, [{K(THF)6}+{Zn(tBu)2(dpa)Zn(tBu)2}-], 21. Sodium zincates 19 and 20 tert-butylate benzophenone at the challenging para-position, whereas di-tert-butylzinc failed to react. Another difunctional amide, N'-benzyl-N,N-dimethylethylenediamide (BD) generated sodium zincate [(TMEDA)Na(μ-BD)(μ-tBu)Zn(tBu)], 25, which successfully deproto-metallated both N,N-diisopropylbenzamide and anisole.One of the most important reactions in chemistry, metallation benefits society in general as it is utilised for the synthesis of many commodities used in daily life. While lithium-based monometallic compounds have long been the reagents of choice for metallation, bimetallic reagents are emerging as second generation metallating agents that can overcome many limitations of lithium reagents. This study extends "synergic" bimetallic chemistry focusing mainly on sodium, zinc and sodium zincate systems, investigating their syntheses, structures and reactions with organic substrates. Probing the metallation of trifluoromethylbenzene, using tBuLi or nBuNa activated by a donor (THF, TMEDA or PMDETA), established the metallation system dictated both regioselectivity and yield. The origin of the increased ortho-selectivity using sodium systems was uncovered through analysis of isolated metallated intermediates [(TMEDA)·Li(C6H4-CF3)]2, 3, [(TMEDA)·Na(C6H4-CF3)]2, 4 and [(PMDETA)·Na(C6H4-CF3)]2, 5. Since pyrrole is important within pharmaceuticals and zinc-pyrrole chemistry is remarkably underdeveloped, we have synthesised the first crystallographicallycharacterized zinc pyrrolyl complexes, with one, two, three or four pyrrole anions attached to zinc in [tBuZn(NC4H4)(TMEDA)·HNC4H4], 7; [Zn(NC4H4)2(TMEDA)], 8; [{(THF)2·NaZn(THF)(NC4H4)3}∞], 9; [{(TMEDA)·Na}2Zn(NC4H4)4], 10; [{(PMDETA)·Na}2Zn(NC4H4)4], 11, as well as for comparison [{(PMDETA)·Na(NC4H4)}2], 12. Improving upon sub-ambient temperature protocols, ambient temperature zincation of N-Boc pyrrolidine was achieved via sodium zincate [(TMEDA)Na(μ-TMP)(μ-tBu)Zn(tBu)]. Metallated intermediate [(TMEDA)Na(TMP)(α-NC4H7NBoc)Zn(tBu)] 13 was crystallographically characterised. Exploring the basicity of 13, in situ sidereactions with toluene, ferrocene or anisole generated [{(TMEDA)Na[OC(NC4H8)CHPh]}2], 14, [(TMEDA)(Boc-NC4H8)Na{(η2-C5H4)Fe(C5H5)}Zn(tBu)2], 16 and [(TMEDA)Na(μ-TMP)(μ-C6H4OMe)Zn(tBu)], 17, respectively. Incorporating multifunctional 2,2'-dipyridylamide (dpa) into a bimetallic system produced neutral zinc, sodium and potassium zincates with unprecedented structures in [{(dpa)Zn(tBu)}2]; 18, [(TMEDA)2Na2(μ-dpa)2Zn(tBu)2]; 19, [{Na(THF)6}+{Zn(tBu)2(dpa)Zn(tBu)2}-]; 20, [{K(THF)6}+{Zn(tBu)2(dpa)Zn(tBu)2}-], 21. Sodium zincates 19 and 20 tert-butylate benzophenone at the challenging para-position, whereas di-tert-butylzinc failed to react. Another difunctional amide, N'-benzyl-N,N-dimethylethylenediamide (BD) generated sodium zincate [(TMEDA)Na(μ-BD)(μ-tBu)Zn(tBu)], 25, which successfully deproto-metallated both N,N-diisopropylbenzamide and anisole