A randomised sham controlled trial of vertebroplasty for painful acute osteoporotic vertebral fractures (VERTOS IV)

Background

Combination of erlotinib and bevacizumab is a promising regimen in advanced non-squamous non-small-cell lung cancer (NSCLC). We are conducting a single arm phase II trial which aims to evaluate the efficacy and safety of this regime as a second- or third-line chemotherapy.

Methods

Key eligibility criteria were histologically or cytologically confirmed non-squamous NSCLC, stage III/IV or recurrent NSCLC not indicated radical chemoradiation, prior one or two regimen of chemotherapy, age 20 years or more, and performance status of two or less. The primary endpoint is objective response rate. The secondary endpoints include overall survival, progression-free survival, disease control rate and incidence of adverse events. This trial plans to accrue 80 patients based on a two-stage design employing a binomial distribution with an alternative hypothesis response rate of 35% and a null hypothesis threshold response rate of 20%. A subset analysis according to EGFR mutation status is planned.

Discussion

We have presented the design of a single arm phase II trial to evaluate the efficacy and safety of combination of bevacizumab and erlotinib in advanced non-squamous NSCLC patients. In particular we are interested in determining the merit of further development of this regimen and whether prospective patient selection using EGFR gene is necessary in future trials.

Trial registration

This trial was registered at the UMIN Clinical Trials Registry as UMIN000004255 (http://www.umin.ac.jp/ctr/index.htm).     

The interaction of lipophilic drugs with intestinal fatty acid-binding protein

Intestinal fatty acid-binding protein (I-FABP) is a small protein that binds long-chain dietary fatty acids in the cytosol of the columnar absorptive epithelial cells (enterocytes) of the intestine. The binding cavity of I-FABP is much larger than is necessary to bind a fatty acid molecule, which suggests that the protein may be able to bind other hydrophobic and amphipathic ligands such as lipophilic drugs. Herein we describe the binding of three structurally diverse lipophilic drugs, bezafibrate, ibuprofen (both R- and S-isomers) and nitrazepam to I-FABP. The rank order of affinity for I-FABP determined for these compounds was found to be R-ibuprofen approximately bezafibrate > S-ibuprofen > nitrazepam. The binding affinities were not directly related to aqueous solubility or partition coefficient of the compounds; however, the freely water-soluble drug diltiazem showed no affinity for I-FABP. Drug-I-FABP interaction interfaces were defined by analysis of chemical shift perturbations in NMR spectra, which revealed that the drugs bound within the central fatty acid binding cavity. Each drug participated in a different set of interactions within the cavity; however, a number of common contacts were observed with residues also involved in fatty acid binding. These data suggest that the binding of non-fatty acid lipophilic drugs to I-FABP may increase the cytosolic solubility of these compounds and thereby facilitate drug transport from the intestinal lumen across the enterocyte to sites of distribution and metabolism.     

Nesprin-2-dependent ERK1/2 compartmentalisation regulates the DNA damage response in vascular smooth muscle cell ageing

Prelamin A accumulation and persistent DNA damage response (DDR) are hallmarks of vascular smooth muscle cell (VSMC) ageing and dysfunction. Although prelamin A is proposed to interfere with DNA repair, our understanding of the crosstalk between prelamin A and the repair process remains limited. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have emerged as key players in the DDR and are known to enhance ataxia telangiectasia-mutated protein (ATM) activity at DNA lesions, and in this study, we identified a novel relationship between prelamin A accumulation and ERK1/2 nuclear compartmentalisation during VSMC ageing. We show both prelamin A accumulation and increased DNA damage occur concomitantly, before VSMC replicative senescence, and induce the localisation of ERK1/2 to promyelocytic leukaemia protein nuclear bodies (PML NBs) at the sites of DNA damage via nesprin-2 and lamin A interactions. Importantly, VSMCs treated with DNA damaging agents also displayed prelamin A accumulation and ERK compartmentalisation at PML NBs, suggesting that prelamin A and nesprin-2 are novel components of the DDR. In support of this, disruption of ERK compartmentalisation at PML NBs, by either depletion of nesprin-2 or lamins A/C, resulted in the loss of ATM from DNA lesions. However, ATM signalling and DNA repair remained intact after lamins A/C depletion, whereas nesprin-2 disruption ablated downstream Chk2 activation and induced genomic instability. We conclude that lamins A/C and PML act as scaffolds to organise DNA-repair foci and compartmentalise nesprin-2/ERK signalling. However, nesprin-2/ERK signalling fidelity, but not their compartmentalisation at PML NBs, is essential for efficient DDR in VSMCs.DNA damage is a major driving force during cellular ageing, and it has been implicated in hastening the development of cardiovascular diseases, including atherosclerosis where the accumulation of senescent cells has been shown to accelerate disease.¹ Normally, DNA damage is efficiently repaired by the DNA damage response (DDR), a complex signalling cascade of proteins that include sensors (NBS1/MRE11), transducers (ataxia telangiectasia-mutated protein (ATM)/ataxia telangiectasia- and Rad3-related protein (ATR)) and effectors (p53/p21). However, if damage is overwhelming or repair is inefficient, the accumulation of unrepaired DNA damage leads to persistent DNA damage signalling and premature senescence.² Recently, the nuclear lamina has been implicated in the DDR, and its disruption is associated with accelerated cardiovascular ageing.³The nuclear lamina is composed of A-type (lamins A/C) and B-type (lamins B1/B2) lamins that underlie the nuclear envelope (NE) and extend throughout the nucleoplasm⁴ to form a scaffold that is essential for the compartmentalisation and the integrity of nuclear signalling.⁴ The pathological accumulation of lamin A precursors such as prelamin A or progerin causes Hutchinson Gilford Progeria Syndrome (HGPS), a disease of accelerated ageing where patients develop severe early-onset arteriosclerosis characterised by vascular smooth muscle cell (VSMC) attrition.⁴ During the DDR, lamin A interacts with Ku70 and γH2AX, and forms a framework that is essential for the positional stability of repair foci.^{5, 6} Prelamin A accumulation interferes with DNA-repair processes and has been shown to delay the recruitment of DNA-repair proteins such as 53BP1 to double strand breaks (DSBs)⁷ and cause mislocalisation of DNA PK_cs.⁸ Fibroblasts and induced pluripotent stem (iPS) cells derived from HGPS patients show persistent DNA damage signalling and premature senescence.⁸ Importantly, aged VSMCs, both in vitro and in vivo, also accumulate prelamin A and activate persistent DDR, suggesting a key role for the nuclear lamina in vascular ageing.²The nesprin family of spectrin-repeat (SR) proteins were first identified as NE lamin A-binding proteins. However, both nesprin-1 and nesprin-2 show extensive alternate splicing, and variants have been shown to localise to multiple nuclear and cytoplasmic compartments.⁹ One such variant, nesprin-2βΔKASH1, retains a lamin A-binding region and localises to promyelocytic leukaemia protein nuclear bodies (PML NBs) in VSMCs.¹⁰ Previously, we demonstrated that nesprin-2βΔKASH1 scaffolds extracellular signal-regulated kinases 1 and 2 (ERK1/2) at PML NBs and acts to regulate nuclear ERK1/2 activity and downstream VSMC proliferation.¹⁰ Importantly, both ERK and PML NBs have also been implicated in the DDR; PML and ERK1/2 localise at DNA lesions where ERK1/2 enhance ATM- and ATR-mediated repair.^{11, 12, 13} In addition, PML and ERK1/2 are essential for regulating the cell cycle in response to DNA damage; PML forms nucleolar cap structures that sequester MDM2 and activate DNA damage-mediated p53 signalling, while ERK1/2 are essential for efficient G2/M checkpoint activation.^{14, 15, 16} Similar to nesprin-2, PML and ERK1/2 have also been shown to associate with the nuclear lamina suggesting that the nuclear lamina, potentially via nesprin-2βΔKASH1, may regulate ERK compartmentalisation during the DDR.^{17, 18}In this study, we identify a novel signalling complex that regulates compartmentalisation of ERK1/2 during the DDR in VSMCs. We show that the nuclear lamina tethers PML NBs and spatially organises nuclear signalling events. Disruption of this organisation results in ATM mislocalisation from DNA-repair foci and impairs downstream DNA-repair signalling, ultimately leading to genomic instability.     

MIDAS: software for analysis and visualisation of interallelic disequilibrium between multiallelic markers

Background  

Various software tools are available for the display of pairwise linkage disequilibrium across multiple single nucleotide polymorphisms. The HapMap project also presents these graphics within their website. However, these approaches are limited in their use of data from multiallelic markers and provide limited information in a graphical form.     

Characterization of human spermidine/spermine N1-acetyltransferase purified from cultured melanoma cells

Extreme inducibility of spermidine/spermine acetyltransferase (SSAT) by bis-ethyl derivatives of spermine in human large cell lung carcinoma and melanoma cells has prompted biochemical characterization of the purified enzyme. Treatment of human MALME-3 melanoma cells with 10 microM N1,N11-bis(ethyl)norspermine (BENSPM) for 48-72 h increased SSAT activity by some 1000- to 4000-fold and enabled purification of the enzyme by established procedures--binding on immobilized spermine and elution with spermine followed by binding on Matrex Blue A and elution with coenzyme A. The enzyme showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a single subunit species and molecular weight of approximately 20,300 Da. By gel permeation chromatography, the holoenzyme was found to have a molecular weight of 80,000 Da, suggesting a total of four identical subunits. Purified SSAT had a specific activity of 285 mumol/min/mg for spermidine and Km values of 5.9 microM for acetylcoenzyme A, 55 microM for spermidine, 5 microM for spermine, 36 microM for N1-acetylspermine, 1.6 microM for norspermidine, and 4 microM for norspermine. Homologs of BENSPM were found to be competitive inhibitors of spermidine acetylation, with Ki values of 0.8 microM for BENSPM, 1.9 microM for N1,N12-bis-(ethyl)spermine and 17 microM for N1,N14-bis-(ethyl)-homospermine. Correlation of these values with the relative abilities of the homologs to increase SSAT in intact cells suggests that formation of an enzyme inhibitor complex may play a contributing role in enzyme induction.     

Growth and survival of Bordetella bronchiseptica in natural waters and in buffered saline without added nutrients.

Bordetella bronchiseptica showed increases in viable count when incubated in phosphate-buffered saline (PBS), in reagent-grade water, and in local lake and pond waters, all without added nutrients. Within 48 to 72 h at 37 degrees C in PBS and in lake and pond waters, stationary-phase populations of around 2.7 x 10(6) CFU/ml developed from washed B. bronchiseptica inocula of around 2 x 10(3) CFU/ml. Increases in CFU on the order of five- and eightfold, respectively, were observed in reagent-grade water and in seawater from the same sizes of inocula. The organisms remained viable for at least 3 weeks in PBS and in lake waters at 37 degrees C. The possibility that carry-over of nutrients was responsible for growth was discounted by showing serial transfer of B. bronchiseptica in PBS under conditions in which Escherichia coli tested in parallel rapidly died out.     

中国五加科植物的分类学研究: 罗伞属和鹅掌柴属的新种和新异名

在编写《Flora of China》五加科Araliaceae时,发现两个新种,即拟榕叶罗伞Brassaiopsis pseudoficifolia Lowry＆C．B．Shang和光华鹅掌柴Scheffiera zhuana Lowry＆C．B．Shang。前者与榕叶罗伞B.ficifolia Dunn很相似,其区别在于,叶裂片数目较多,花序上具较多的伞形花序,花序轴具刺,主要分布于云南,广西偶见。后者与白花鹅掌柴S．leucantha R．Vig．相似,其区别在于,子房心皮数目较多,果时具明显的萼缘,花盘扁平,叶质地较薄,局限分布于西藏墨脱县。同时发现越南产的Brassaiopsis gaussenii Bui和中国产的细序鹅掌柴Scheffiera tenuis H．L．Li、球序鹅掌S.glomerulata H．L．Li均不能成立,处理为异名。