Regional chemotherapy allows further exploitation of the steep dose response curve of most chemotherapeutic agents, while systemic toxicity remains tolerable. We investigated the difference in maximally tolerated dose, pharmacokinetics and antitumour effect comparing administration of melphalan as a bolus in isolated liver perfusion (ILP) or via hepatic artery infusion (HAI). For these in vivo studies an experimental model for liver metastases in male WAG/Ola rats is obtained by subcapsular inoculation of CC531 rat colon carcinoma cells. In this system, ILP allowed administration of a two times higher dose than HAI (12 mg kg-1 vs 6 mg kg-1). In both treatment modalities systemic toxicity (leukopenia) was dose limiting. No hepatic toxicity was observed. Bolus administration of the maximally tolerated doses of melphalan in HAI (6 mg kg-1) and ILP (12 mg kg-1) resulted in four times higher concentrations in both liver and tumour tissue of the ILP treated rats. However, the ratio of mean drug concentration in liver vs tumour tissue appeared to be 1.5 times that found for HAI. In the range of the in tumour tissue measured melphalan concentrations the CC531 cells showed a steep dose response relationship in vitro. Whereas HAI resulted in significant tumour growth delay, complete remissions were observed in 90% of the rats treated with ILP. This study shows that with 12 mg kg-1 melphalan in ILP highly effective drug concentrations are achieved in CC531 tumour tissue; although the melphalan concentration in liver tissue shows an even higher increase than in tumour tissue, hepatic toxicity is negligible in this dose range.(ABSTRACT TRUNCATED AT 250 WORDS)


We describe melphalan pharmacokinetics in 26 patients treated by isolated limb perfusion (ILP). Group A (n = 11) were treated with a bolus of melphalan (1.5 mg kg-1), and in a phase I study the dose was increased to 1.75 mg kg-1. The higher dose was given as a bolus to Group B (n = 9), and by divided dose to Group C (n = 6). Using high performance liquid chromatography (HPLC) the concentrations of melphalan in the arterial and venous perfusate (during ILP) and in the systemic circulation (during and after ILP) were measured. Areas under the concentration time curves for perfusate  and systemic (AUCs) data were calculated. In all three groups the peak concentrations of melphalan were much higher in the perfusate than in the systemic circulation. The pharmacokinetic advantages of ILP can be quantified by the ratio of AUCa/AUCs, median value 37.8 (2.1-131). AUCa and AUCv were both significantly greater in Group B than in Group A . In Groups B and C acceptable 'toxic' reactions occurred but were not simply related to melphalan levels. Our phase I study has allowed us to increase the dose of melphalan to 1.75 mg kg-1, but we found no pharmacokinetic advantage from divided dose administration.


Systemic toxicity is usually the dose-limiting factor in cancer chemotherapy. Regional chemotherapy is therefore an attractive strategy in the treatment of liver metastasis. Two ways of regional chemotherapy, hepatic artery infusion (HAI) and isolated liver perfusion (ILP), were compared investigating the difference in toxicity with tissue and biofluid concentrations of mitomycin C (MMC). In wistar derived WAG rats the maximally tolerated dose of mitomycin C via HAI was 1.2 mg kg-1. Body weight measurements after HAI with doses higher than 1.2 mg kg-1 suggest both an acute and delayed toxic effect of mitomycin C since the time weight curves were triphasic: a rapid weight loss, a steady state and a second fall in weight phase. These rats died due to systemic toxicity. ILP with 4.8 mg kg-1 was associated with no signs of systemic toxicity and only transient mild hepatotoxicity. ILP with 6.0 mg kg-1 was fatal mainly due to hepatic toxicity. The four times higher maximally tolerated dose in ILP resulted in a 4-5 times higher peak concentration of mitomycin C in liver tissue, while the plasma concentration remained significantly lower than in the HAI treated rats. In the tumour tissue a 500% higher concentration of mitomycin C was measured in the ILP with 4.8 mg kg-1 than in HAI with 1.2 mg kg-1 treated rats. We demonstrated that when mitomycin C was administered by ILP a 400% higher dose could be safely administered and resulted in a five times higher tumour tissue concentration. In view of the steep dose-response curve of this alkylating agent this opens new perspectives for the treatment of liver metastasis.


P<0.0001) and were significant predictors of tissue origin (P<0.0001). In solid tissues (n = 119) we found striking, highly significant CpG island–dependent correlations between age and methylation; loci in CpG islands gained methylation with age, loci not in CpG islands lost methylation with age (P<0.001), and this pattern was consistent across tissues and in an analysis of blood-derived DNA. Our data clearly demonstrate age- and exposure-related differences in tissue-specific methylation and significant age-associated methylation patterns which are CpG island context-dependent. This work provides novel insight into the role of aging and the environment in susceptibility to diseases such as cancer and critically informs the field of epigenomics by providing evidence of epigenetic dysregulation by age-related methylation alterations. Collectively we reveal key issues to consider both in the construction of reference and disease-related epigenomes and in the interpretation of potentially pathologically important alterations.Epigenetic control of gene transcription is critical for normal human development and cellular differentiation. While alterations of epigenetic marks such as DNA methylation have been linked to cancers and many other human diseases, interindividual epigenetic variations in normal tissues due to aging, environmental factors, or innate susceptibility are poorly characterized. The plasticity, tissue-specific nature, and variability of gene expression are related to epigenomic states that vary across individuals. Thus, population-based investigations are needed to further our understanding of the fundamental dynamics of normal individual epigenomes. We analyzed 217 non-pathologic human tissues from 10 anatomic sites at 1,413 autosomal CpG loci associated with 773 genes to investigate tissue-specific differences in DNA methylation and to discern how aging and exposures contribute to normal variation in methylation. Methylation profile classes derived from unsupervised modeling were significantly associated with age ( The causes and extent of tissue-specific interindividual variation in human epigenomes are underappreciated and, hence, poorly characterized. We surveyed over 200 carefully annotated human tissue samples from ten anatosites at 1,413 CpGs for methylation alterations to appraise the nature of phenotypically, and hence potentially clinically important epigenomic alterations. Within tissue types, across individuals, we found variation in methylation that was significantly related to aging and environmental exposures such as tobacco smoking. Individual variation in age- and exposure-related methylation may significantly contribute to increased susceptibility to several diseases. As the NIH–funded HapMap project is critically contributing to annotating the human reference genome defining normal genetic variability, our work raises key issues to consider in the construction of reference epigenomes. It is well recognized that understanding genetic variation is essential to understanding disease. Our work, and the known interplay of epigenetics and genetics, makes it equally clear that a more complete characterization of epigenetic variation and its sources must be accomplished to reach the goal of a complete understanding of disease. Additional research is absolutely necessary to define the mechanisms controlling epigenomic variation. We have begun to lay the foundations for essential normal tissue controls for comparison to diseased tissue, which will allow the identification of the most crucial disease-related alterations and provide more robust targets for novel treatments. While all somatic cells in a given individual are genetically identical (excepting T and B cells), different cell types form highly distinct anatomic structures and carry out a wide range of disparate physiologic functions. The vast repertoire of cellular phenotypes is made possible largely via epigenetic control of gene expression, which is known to play a critical role in cellular differentiation. Epigenetics is the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence We have previously distinguished normal and tumor tissues using methylation profiling et al. observed significant variation among individuals when bisulfite sequencing a particular CpG island, and suggested that larger-scale studies are required to determine the extent of interindividual variability in methylation patterns et al. suggests a more complex picture. These authors found both increased and decreased intra-individual global methylation levels (enriched for promoter regions) in peripheral blood cell DNA over time Efforts to describe the methylation profiles of normal tissues are now underway. Recent genome-wide studies of methylation in normal human tissues have shown that DNA methylation profiles are tissue-specific and correlated with sequence elements In this study we used Illumina's GoldenGate methylation platform to investigate cytosine methylation in 217 normal human tissue specimens from 10 different anatomic sites in order to begin to understand variation both between and within tissues across individuals. Profiling CpG methylation of normal human tissues allowed us to begin characterizing the role of aging and environmental exposures in interindividual methylation variation, as well as specific gene-loci determinant of normal tissue-specificity. This work highlights the dynamic nature of epigenomes, and begins to disentangle the roles of aging, environmental factors, and innate variability among individual epigenomic profiles, both within, and across tissues.P<0.0001). Further, age was significantly associated with methylation classes (P<0.0001). Separating samples into groups as placenta, blood, or other solid tissue, we found a significant association between group and methylation profile classes (P<0.0001).Array methylation data were first assembled for exploration and visualization with unsupervised hierarchical clustering using Manhattan distance and average linkage for the 500 most variable autosomal CpG loci . EpigeneP<0.0001). Not unexpectedly, tissue types with larger sample sizes showed a significantly reduced misclassification error rates (P<0.05). The mean and standard deviation of average beta values for all autosomal CpG loci in each tissue type, and values for the decrease in random forest classification accuracy with locus removal are given in P<0.0001), and the mean and standard deviation of average beta values for all autosomal CpG loci in each of placenta, blood, or solid tissue, and values for the decrease in random forest classification accuracy with locus removal are given in Random Forests (RF) classification of all samples based on methylation average beta values at all autosomal loci returned a confusion matrix showing: which samples are correctly classified, which are misclassified, and the misclassification error rate for each sample type . OverallP<0.005), though we did not detect significant associations between methylation class and smoking status, packyears, or alcohol consumption. An RPMM of lung tissues (n = 53) resulted in five methylation classes (P<0.07).Variation in tissue-specific methylation relative to differences between tissue types was first explored visually. Scatter plots of methylation values for representative samples from two different tissues were less well correlated than similar plots of two representative samples from the same tissue type, though variation in tissue-specific methylation was also evident . Tissue- classes  where cl classes , and claQ<0.05, MLH1 (Q<0.0001), and RIPK3 (Q<0.002) methylation; and over 30 CpG loci had significantly altered methylation in never versus ever drinkers  were also observed. Additionally, and in contrast to the predominantly increased age associated methylation at other gene-loci, there was a significant age-related decrease in CpG methylation of the de novo methyltransferase DNMT3B; and unlike the vast majority of other CpGs tested, DNMT3B_P352 was not located in a CpG island , after correcting for multiple comparisons, over 300 CpG loci had age-related methylation alterations Q<0.05, . RestricP = 7.0E-04; P = 5.2E-05, There is now a considerable literature that suggests that genome structure affects both the initial placement of DNA methylation marks in development class-specific mean associations between age and methylation and plotted the estimates with their 95% confidence intervals. In a class-specific model for solid tissue samples, there was a positive correlation between age and methylation in classes whose loci were predominantly located in CpG islands  with RPMM (aiming to examine classes of CpGs with similar methylation profiles in more detail), grouping CpGs with similar methylation into eight separate classes. The CpG island status of all loci was plotted, and illustrates the well known tendency for CpGs located in islands to be unmethylated, while non-island CpGs tend to be methylated . We agaiRARA_P176 (P = 0.003), DNMT3B_P352 (P = 0.008), and LIF_P383  was validated by pyrosequencing  based on aging and the environment has the potential to dramatically improve the success of studies of epigenetic alterations in disease. Hence, our work characterized methylation of phenotypically important CpG loci across several human tissue types, elucidating interindividual tissue-specific variation in methylation profiles and the contribution of CpG island context to age associated methylation alterations. This work increases our appreciation for the dynamic nature of the epigenome, and begins to define basic tenets to follow in pursuit of both constructing reference epigenomes and elucidating epigenetic alterations truly indicative of disease states.Using recursively-partitioned mixture modeling and random forests approaches, we differentiated tissues based on CpG methylation profile, consistent with other recent studies conducting genome-wide DNA methylation profiling Factors known to contribute to methylation alterations include carcinogen exposures, inflammation, and diet. Several carcinogen exposures such as tobacco, alcohol, arsenic, and asbestos have been associated with methylation-induced gene-inactivation in various human cancers including bladder cancer, head and neck squamous cell carcinoma, and mesothelioma et al. described an association between aging colonic mucosa and estrogen receptor methylation et al. reported that 29 loci had age-related methylation alterations, with 23 loci displaying increased methylation with age and 6 decreasing with age et al. found both increased and decreased methylation levels dependent on the individual, with over 50% of participants exhibiting >5% change in methylation Cancer is a disease of aging, and initial studies of age-related methylation in normal tissues were motivated in large part by studies of methylation in cancer et al. and Bjornsson et al. who showed bi-modal age-related methylation in normal tissues. A direct comparison, by examination of the data of Bjornsson et al., indicated that a high percentage of their top 50 most age-altered loci  are not located in CpG islands; among 24 of 30 autosomal CpGs in their Stratifying our data on CpG-island status of loci, we showed that both the direction and strength of correlation between age and methylation were largely dependent upon CpG island status. More specifically, we found a propensity for CpG-island loci to gain methylation with age, and non-island CpGs to lose methylation with age. Our data are consistent with the literature that has demonstrated age-related increases in methylation at gene-loci found within CpG islands nd functional allele). Future population-based studies addressing the potential of quantifying age and/or exposure associated methylation alterations indicative of disease risk are necessary.The observed pattern of age associated methylation was irrespective of tissue-type, suggesting a common mechanism or dysregulation to explain these alterations. Reduced fidelity of maintenance methyltransferases with aging is one potential explanation for age related decreases in methylation; while age related increases in methylation could potentially reflect the accumulation of stochastic methylation events over time. As the examined tissues do not have a pathologic phenotype, methylated CpGs in these cells may not indicate dramatic functional consequences upon gene expression. However, the (in part selective) accumulation of alterations without readily detectable functional consequences should not be interpreted as biologically insignificant. Age-related drift of normal epigenomes without prominent changes in gene expression may nonetheless confer significantly increased risk of conversion to a pathologic phenotype by enhancing both the likelihood and frequency of methylation events that ultimately result in aberrant expression or altered genomic stability. For example, in the context of acquired “non-functional” CpG methylation in the promoter region of an aged individual, continued stochastic methylation events (e.g. “methylation spreading”) increase the chance of methylation induced silencing at that promoter (or silencing of another locus through action at a distance via silencing of other important regions such as enhancers), and hence, progression to a pathologic phenotype. Certainly, this hypothesis is especially plausible for the many diseases of aging. Alternatively, aberrant CpG methylation that silences a gene on a single allele may not appear to have a functional consequence if the complementary allele can provide compensatory expression. As a result, for example, clusters of cellular clones with mono-allelic gene expression could contribute to an increased risk of progression to a pathologic phenotype . Briefly, normal brain tissues (n = 12) were contributed by the Wiencke lab at UCSF through the San Francisco Adult Glioma Study et alFresh frozen tissue and whole blood DNA was extracted using the QIAamp DNA mini kit according to the manufacturer's protocol . DNA was modified by sodium bisulfite to convert unmethylated cytosines to uracil using the EZ DNA Methylation Kit  according to the manufacturer's protocol. Illumina GoldenGate methylation bead arrays were used to simultaneously interrogate 1505 CpG loci associated with 803 cancer-related genes. Bead arrays have a similar sensitivity as quantitative methylation-specific PCR and were run at the UCSF Institute for Human Genetics, Genomics Core Facility according to the manufacturer's protocol and as described by Bibikova Quantification of cytosine percent methylation was performed by pyrosequencing bisulfite-converted DNA using the PyroMark MD pyrosequencing system (Biotage). Specific pyrosequencing primers were designed to amplify array CpG sites and as many downstream CpGs as conditions permitted  using Biotage Assay Design Software v1.0.6.FZD9), and 10× PCR buffer with 15 mM MgCl2 under the following conditions: 95°C 15′,  30″, 72°C 1′) × 45 cycles, and 72°C 5′. Final reaction primer concentration for PCR and sequencing was 0.3 µM, primer details are in All PCR reactions were carried out in 25 µl, utilized Qiagen Hot Star Taq polymerase, 5× Q solution )/(|Cy3|+|Cy5|+100), the average methylation (β) value is derived from the ∼30 replicate methylation measurements. Raw average beta values were analyzed without normalization as recommended by Illumina. Each array CpG is annotated with the gene name followed by the CpG location  seq to reference a specific sequence) and its physical distance from the transcription start site. At each locus for each sample the detection P-value was used to determine sample performance; 5 samples (2%), had detection P-values >1.0E-05 at more than 25% of CpG loci and were removed from subsequent analysis. Similarly, CpG loci with a median detection P–value >0.05 , were eliminated from analysis. Finally, all CpG loci on the X chromosome were excluded from analysis. The final dataset contained 217 samples and 1413 CpG loci associated with 773 genes. The manufacturer recommended CpG island designation of array CpGs was used and follows the definition of CpG island in Data were assembled with BeadStudio methylation software from Illumina . All array data points are represented by fluorescent signals from both methylated (Cy5) and unmethylated (Cy3) alleles, and methylation level is given by β = (max(hclust with Manhattan metric and average linkage. To discern and describe the relationships between CpG methylation and tissue type (sample clustering), and the relationships between CpGs with coordinate methylation (CpG clustering), a modified model-based form of unsupervised clustering known as recursively partitioned mixture modeling (RPMM) was used as described in Subsequent analyses were carried out using the R software m out of the total M variables is chosen and the best split is found among the m variables. We utilized the default value for m in the Random Forest R package, √M (√1413 = 38). The misclassification error rate is the percentage of time the RF prediction is incorrect.The R Package was also used to build classifiers with the Random Forest (RF) approach. RF is a tree-based classification algorithm similar to Classification and Regression Tree (CART) et al.a priori hypothesis existed were tested separately, such as those that have been previously associated with aging in normal tissues WRN. Array-wide scanning for CpG loci associations with sample type or covariate used false discovery rate estimation and Q–values computed by the qvalue package in R Associations between covariates and methylation at individual CpG loci were tested with a generalized linear model. The beta-distribution of average beta values was accounted for with a quasi-binomial logit link with an estimated scale parameter constraining the mean between 0 and 1, in a manner similar to that described by Hsuing To test the hypothesis that there are associations of age and exposure with methylation, we constructed measures of partial methylation sets, in analogy with global DNA methylation, which is measured on repeats Sequencing data were processed using Pyro Q-CpG software v1.0.9 (Biotage) under default analysis parameters and exported for subsequent analysis in R software Figure S1Pairwise plots comparing average beta values (A) between all blood and all head & neck samples, (B) individual blood sample versus an individual head & neck sample, comparisons within tissue type between individual samples for (C) blood and (D) head & neck. Average beta value scatterplots between tissue types indicate significant differences between tissues, and scatterplots within tissue type indicate relative similarity in the presence of interindividual variation. A) Mean of average betas for all blood samples (n = 30) versus mean of average betas for all head and neck samples (n = 11), indicates relatively high variability between tissue types, R2 = 0.84. B) Representative blood sample 1 average betas versus representative head and neck sample 1 average betas indicate similarly high variability between tissue types at the individual sample level, R2 = 0.87. C) Representative blood sample 2 versus representative blood sample 3 indicates relative similarity between individuals within a tissue type in the presence of interindividual variation, R2 = 0.97. D) Representative head and neck sample 1 versus representative head and neck sample 2 indicates relative similarity between individuals within a tissue type in the presence of interindividual variation, R2 = 0.96.(0.07 MB TIF)Click here for additional data file.Figure S2Bisulfite pyrosequencing mean percent methylation across all CpGs measured for RARA, DNMT3B, and LIF versus their respective CpG of interest on the array. Bisulfite pyrosequencing mean percent methylation across all CpGs measured for RARA, DNMT3B, and LIF versus their respective CpG of interest on the array. A) Mean bisulfite pyrosequencing percent methylation across array target CpG RARA_P176 and 5 downstream CpGs plotted versus Illumina GoldenGate methylation array average beta demonstrates a significant correlation between sequencing and array methylation . B) Mean bisulfite pyrosequencing percent methylation across array target CpG DNMT3B_P352 and 2 downstream CpGs plotted versus Illumina GoldenGate methylation array average beta demonstrates a significant correlation between sequencing and array methylation . Mean bisulfite pyrosequencing percent methylation across array target CpG LIF_P383 and 2 downstream CpGs plotted versus Illumina GoldenGate methylation array average beta demonstrates a significant correlation between sequencing and array methylation .(0.04 MB TIF)Click here for additional data file.Table S1Autosomal CpG locus mean (sd) of average beta and values for the decrease in random forest classification accuracy with locus removal, all tissues.(0.68 MB XLS)Click here for additional data file.Table S2Autosomal CpG locus mean (sd) of average beta and values for the decrease in random forest classification accuracy with locus removal, for solid tissues, blood, and placenta.(0.29 MB XLS)Click here for additional data file.Table S3CpG loci with significantly altered methylation by reported asbestos exposure in pleural samples (n = 18).(0.04 MB DOC)Click here for additional data file.Table S4CpG loci with significantly altered methylation in never versus ever alcohol drinkers in blood (n = 29).(0.05 MB DOC)Click here for additional data file.Table S5CpG loci with significantly altered methylation by smoking in lung tissue (n = 53).(0.13 MB DOC)Click here for additional data file.Table S6Top 100 CpG loci associated with age by tissue type.(0.84 MB DOC)Click here for additional data file.Table S7Pyrosequencing assay primers.(0.05 MB DOC)Click here for additional data file.


The corpus callosum, which is the largest white matter structure in the human brain, connects the 2 cerebral hemispheres. It plays a crucial role in maintaining the independent processing of the hemispheres and in integrating information between both hemispheres. The functional integrity of interhemispheric interactions can be tested electrophysiologically in humans by using transcranial magnetic stimulation, electroencephalography, and functional magnetic resonance imaging. As a brain structural imaging, diffusion tensor imaging has revealed the microstructural connectivity underlying interhemispheric interactions. Sex, age, and motor training in addition to the size of the corpus callosum influence interhemispheric interactions. Several neurological disorders change hemispheric asymmetry directly by impairing the corpus callosum. Moreover, stroke lesions and unilateral peripheral impairments such as amputation alter interhemispheric interactions indirectly. Noninvasive brain stimulation changes the interhemispheric interactions between both motor cortices. Recently, these brain stimulation techniques were applied in the clinical rehabilitation of patients with stroke by ameliorating the deteriorated modulation of interhemispheric interactions. Here, we review the interhemispheric interactions and mechanisms underlying the pathogenesis of these interactions and propose rehabilitative approaches for appropriate cortical reorganization. The corpus callosum, which is the largest white matter structure in the human brain, connects the homologous and nonhomologous areas of the 2 cerebral hemispheres , 2. It pResearch on the functions of interhemispheric interactions is based on studies of brain lateralization, which is thought to allow each hemisphere to process information without the interference of the contralateral hemisphere , 16. SevHowever, processing tasks that share and integrate the information between hemispheres  require facilitative communication between hemispheres . Even inThe ability to perform precisely coordinated movements using both hands is an important aspect of particular human abilities, such as tying a string, peeling a fruit with a knife, typing, and playing a musical instrument. It is now known that modulations of interhemispheric interactions are involved in the control of the unimanual and bimanual coordinations that generate the spatially and temporally precise coordinated limb movements that enable humans to perform different movements . MoreoveRecent studies have revealed that the modulation of interhemispheric interactions relates to neural plasticity, which refers to the ability of the brain to develop new neuronal interconnections, acquire new functions, and compensate for impairments –25. HoweIt has been estimated that the corpus callosum is the pathway through which one hemisphere can inhibit the other, thus facilitating brain lateralization. Alternatively, the corpus callosum integrates information across the cerebral hemispheres and serves an excitatory function in interhemispheric communication , 3, 15. The inhibitory theory posits that the corpus callosum maintains independent processing between the hemispheres, hinders activity in the opposing hemisphere, and allows the development of hemispheric asymmetries . A TMS sHandedness may be related to inhibitory interhemispheric interactions. Although it remains controversial whether interhemispheric inhibition from the dominant motor cortex differs from the nondominant motor cortex under resting condition –30 physiThe excitatory theory posits that the corpus callosum shares and integrates information between the hemispheres, resulting in greater connectivity, which decreases brain lateralization by masking the underlying hemispheric asymmetries in tasks that require interhemispheric exchange , 31. ThiAs a motor system, the excitatory interhemispheric interaction plays an important role in the adjustment of movement onset. A TMS study revealed that interhemispheric interaction from the nonactive to the active motor cortex translates from inhibitory to excitatory effects around movement onset . This exHowever, the findings of interhemispheric interactions during in-phase movements support the inhibitory theory. The maximum speed of bimanual in-phase movements was the highest in subjects that exhibited weak inhibition of both homologous motor cortices . InterheThese findings suggest that, depending on the motor task, the interhemispheric interactions may be inhibitory or excitatory, so that homologous muscles are adjusted . This isThe degree of connectivity between the hemispheres is reflected in the size of the corpus callosum , 31, 42.Several studies have revealed a correlation between interhemispheric interactions and age –47. The Aging also influences interhemispheric interactions. Several MRI studies have reported that aging increases the atrophy of the corpus callosum , 46. MorHowever, the role of the overactivation of cortices in older adults may vary according to the brain region involved in tasks. The results of previous studies supported the idea that overrecruitment of bilateral prefrontal activation compensates cognitive tasks in older adults , 57. In Several studies have reported morphological and microstructural differences in the corpus callosum between men and women. The relative size of the corpus callosum proportional to cerebral volume was larger in women compared to men , 62, butAs described previously, modulation of interhemispheric interactions influences human movement patterns, such as handedness. In contrast, motor training itself can change interhemispheric interactions. Changes in interhemispheric interactions mediated by motor training have been reported, especially in musical training –76. MusiIn addition to bimanual training, interhemispheric interactions may contribute to motor acquisitions, such as intermanual transfer, as it is well known that motor learning using one hand improves the performance of the other hand , 78. A pIn contrast to motor training, the nonused limb may also influence interhemispheric interactions. A recent study revealed that transient arm immobilization reduced the interhemispheric inhibition from the immobilized to the nonimmobilized motor cortex . MoreoveStudies of callosotomy or callosal lesions have provided much insight into the functions of interhemispheric interactions via the impairment of the corpus callosum , 15, 33.Lesions of the corpus callosum are commonly detected in patients with traumatic brain injury –83. DiffMultiple sclerosis is an inflammatory disease that affects myelinated axons and leads to neurological and cognitive impairments. Therefore, the corpus callosum, which is the largest white matter structure in the brain, is considered a target for inflammation. Corpus callosum degeneration, which has been described frequently –88, can Impairments of interhemispheric inhibition detected using TMS have been reported in patients with Parkinsonian syndromes, including patients with corticobasal degeneration and progressive supranuclear palsy , 90. MRISeveral studies using MRI reported the atrophy and reduction in microstructural connectivity of the corpus callosum in patients with schizophrenia , 94. PreSeveral studies have reported that stroke lesions indirectly disrupt interhemispheric interactions , 98, 99.In addition to stroke, recent studies revealed that indirect changes in interhemispheric interactions through the corpus callosum occur after changes in peripheral organs, such as limb amputation , 106. ThIt has been reported that several techniques alter interhemispheric interactions. In particular, noninvasive brain stimulation (NIBS), which can modulate cortical excitability, may enhance neural plasticity by altering interhemispheric interactions. Moreover, paired associative stimulation of the homologous motor cortices using TMS induces a neural plasticity that is dependent on Hebbian mechanisms through interhemispheric interactions. In this section, we discuss the neural plasticity that is induced by changes in interhemispheric interactions.Repetitive TMS and transcranial direct current stimulation are NIBS techniques that can alter the excitability of the human cortex for several minutes . In partA recent study reported that paired associative stimulation of the homologous motor cortices using TMS is a new interventional protocol that induces an increase in excitability in the conditioned motor cortex . The paiAs mentioned previously, excessive interhemispheric inhibition from the unaffected hemisphere deteriorates the motor function of the paretic hand in patients with stroke. Therefore, improvement of the motor deficits of these patients may be achieved by decreasing the excitability of the unaffected hemisphere using NIBS , 102. InAlthough it has been reported that inhibitory NIBS over the unaffected hemisphere facilitates motor recovery during the acute stage of stroke , 128, a This paper focused on the mechanisms underlying motor control and neural plasticity that relate to interhemispheric interactions to suggest approaches for appropriate cortical reorganization. Inhibitory or excitatory interactions that occur via interhemispheric communication may vary depending on the different time points during the movement and different cortical areas that are involved in the processing demands of the motor task. The age-related degeneration of the corpus callosum may induce the engagement of both hemispheres partly because of the failed inhibition of the contralateral hemisphere. Female hormones may exert positive effects on the interhemispheric communication that is related to maintaining independent processing between the hemispheres in the motor system. Plastic developmental changes that are caused by extensive bimanual training during childhood result in more symmetrical brains and equally efficient connections between the hemispheres. Several neurological disorders, such as traumatic brain injury, multiple sclerosis, and Parkinsonian syndromes, directly alter interhemispheric interactions by impairing the corpus callosum. Stroke lesions indirectly disrupt interhemispheric inhibition, which is highly relevant to the research on motor recovery after stroke. In addition, amputations may indirectly alter interhemispheric interactions between sensorimotor cortices. Inhibitory NIBS reduces the interhemispheric inhibition from the stimulated motor cortex to the non-stimulated motor cortex. The paired associative stimulation of the homologous motor cortices using TMS induces a neural plasticity that is dependent on Hebbian mechanisms that occur via interhemispheric interactions. Inhibitory NIBS over the unaffected hemisphere in patients with stroke can improve the motor function of the paretic hand by reducing the interhemispheric inhibition from the unaffected hemisphere to the affected hemisphere. However, it should be noted that inhibitory NIBS might worsen bimanual movements by reducing the interhemispheric inhibition that controls them. Assessments of interhemispheric interactions have provided information on the mechanisms underlying the physiological processes involved in motor control and have allowed the formulation of interventional strategies that can improve motor function in neurological disorders, which is a critical issue in clinical neurorehabilitation.


Here we study the possible control exerted by the stationary phase sigma factor RpoS on the bistability decision. The gene for RpoS in P. knackmussii B13 was characterized, and a loss-of-function mutant was produced and complemented. We found that, in absence of RpoS, ICEclc transfer rates and activation of two key ICEclc promoters (Pint and PinR) decrease significantly in cells during stationary phase. Microarray and gene reporter analysis indicated that the most direct effect of RpoS is on PinR, whereas one of the gene products from the PinR-controlled operon (InrR) transmits activation to Pint and other ICEclc core genes. Addition of a second rpoS copy under control of its native promoter resulted in an increase of the proportion of cells expressing the Pint and PinR promoters to 18%. Strains in which rpoS was replaced by an rpoS-mcherry fusion showed high mCherry fluorescence of individual cells that had activated Pint and PinR, whereas a double-copy rpoS-mcherry–containing strain displayed twice as much mCherry fluorescence. This suggested that high RpoS levels are a prerequisite for an individual cell to activate PinR and thus ICEclc transfer. Double promoter–reporter fusions confirmed that expression of PinR is dominated by extrinsic noise, such as being the result of cellular variability in RpoS. In contrast, expression from Pint is dominated by intrinsic noise, indicating it is specific to the ICEclc transmission cascade. Our results demonstrate how stochastic noise levels of global transcription factors can be transduced to a precise signaling cascade in a subpopulation of cells leading to ICE activation.Conjugative transfer of the integrative and conjugative element ICE clc normally resides in the chromosome of its bacterial host, but can excise from the chromosome and prepare for conjugation. Interestingly, the decision to excise ICEclc is made in only 3%–5% of cells in a clonal population in stationary phase. We focus specifically on the question of which mechanism may be responsible for setting this threshold level of ICEclc activation. We find that ICEclc activation is dependent on the individual cell level of the stationary phase sigma factor RpoS. The noise in RpoS expression across a population of cells thus sets the “threshold” for ICEclc to excise and prepare transfer.Horizontal gene transfer is one of the amazing phenomena in the prokaryotic world, by which DNA can be moved between species with means of a variety of specialized “elements” and/or specific host cell mechanisms. In particular the molecular decisions that have to be made in order to transfer DNA from one cell to another are fascinating, but very little is known about this at a cellular basis. Here we study a member of a widely distributed type of mobile DNA called “integrative and conjugative elements” or ICE. ICE For example, several ICE carry genes for antibiotic resistance clc in Pseudomonas knackmussii B13 must be the consequence of a bistable switch that culminates in the activation of the intB13 integrase promoter (hereafter named Pint) in 3% of cells during stationary phase clc is a 103-kb sized element with strong homologies to a large number of genomic islands in Beta- and Gammaproteobacteria, and is named after its propensity to provide the host cell with the capacity to metabolize chlorinated catechols, encoded by the clc genes clc copies reside in the chromosome of strain B13, which are interspaced by 340 kb . Activation of the intB13 integrase leads to excision and formation of a closed circular ICEclc intermediate oriT) on ICEclcint activation was preceded by and dependent on expression of a protein named InrR (for INtegRase Regulator) in the same individual cell (clc under control of another bistably expressed promoter (PinR) Bacillus subtilis, which lead to phenotypically differentiated cells Although several ICE have been genetically and functionally characterized, and their general importance for bacterial evolution and adaptation is now widely appreciated, still very little is known about their cell biology ual cell . InrR isclc becomes active. We focused our attention on both Pint and PinR promoters, which are expressed during stationary phase and only in a subpopulation of cells clc transfer in stationary phase cells further suggested involvement of a specific sigma factor such as RpoS (σs). RpoS is the stress-starvation sigma factor that in P. aeruginosa controls the expression of some 772 genes at the onset of stationary phase rpoS in P. aeruginosa does not result in a dramatically changed phenotype, although such mutants survive 50-fold less well to heat and salt shocks than wild-type, and produce less extracellular proteins such as elastase, exotoxin A, and alginate clc, we identified an rpoS-gene in P. knackmussii B13 and studied the effects of interruption and subsequent complementation using single-cell reporter gene fusions to Pint and PinR. Interestingly, a B13 wild-type equipped with a second rpoS gene copy displayed a much higher subpopulation of cells expressing both Pint and PinR promoters. To study whether actually individual cell levels of RpoS could be somehow deterministic for the activation of ICEclc we replaced native rpoS by a gene for an active RpoS-mCherry fusion protein. Finally, we measured contributions of intrinsic and extrinsic noise on Pint and PinR promoters from covariance in the expression of double gene reporters placed in single copy on different parts of the B13 chromosome int and PinR, which suggests that the stochastic variation in RpoS levels across a population of cells is transduced into ICEclc activation and transfer in a small subpopulation.The goal of the underlying work was to explore whether noisiness may lay at the basis of determining the proportion of cells in which ICErpoS gene of P. knackmussii strain B13 we used PCR amplification with primers designed against conserved regions in a multiple alignment of rpoS sequences of P. aeruginosa, P. putida KT2440 and P. fluorescens , which showed that the rpoS region of strain B13 is syntenic to that in P. aeruginosa PAO1 with a gene for a lipoprotein (nlpD) upstream of rpoS, and an rsmZ-like gene and a gene for a ferredoxin (fdxA) downstream . Maximum specific growth rates of strain B13-2671 (rpoS) on MM with 5 mM 3CBA were similar as B13 wild-type , but the onset of exponential growth was slightly delayed in B13-2671 (rpoS) (A single crossover 1 (rpoS) . Reversi1 (rpoS) .clc are solely expressed in stationary phase P. knackmussii B13 cells inR promoter clc stationary phase expression. Inactivation of rpoS in B13 indeed resulted in reduced expression of both PinR and Pint promoters. This was evident, first of all, from a reduced proportion of cells in a B13-2673 (rpoS) compared to B13 wild-type population expressing eCherry and eGFP above detection threshold from single copy transcriptional fusions to PinR and Pint, respectively (rpoS) produced a lower average reporter fluorescence signal than wild-type cells (inR and Pint were expressed in the same cell (rpoS) cells examined after 24 h in stationary phase, but after 72 h a small fraction of cells still developed eGFP and eCherry fluorescence (rpoS) compared to B13-78 wild-type to reach stationary phase (int and PinR in B13-2673 (rpoS) was not due to reversion of the rpoS mutation  was significantly lower than in B13 wild-type and the rpoS-complemented strain (B13-2993), RpoS is necessary for achieving native transcription levels from the PinR promoter . On the other hand, RpoS is not absolutely essential, since cells with interrupted rpoS gene eventually (96 h) express PinR and Pint, which was not due to reversion of the rpoS mutation  in the rpoS mutant could be the result of either less InrR being formed from PinR, or of a direct control by RpoS of Pint, we compared eGFP expression from a single copy Pint-egfp transcriptional fusion in B13, the B13 rpoS mutant (B13-2976) and a B13 lacking both inrR copies , and correlated eGFP to eCherry expression. Since this strain would be devoid of InrR-mediated expression of Pint, we expected that expression of egfp from Pint in absence of rpoS would be lower than expression of echerry from PinR. Indeed, there was a slight tendency for the mean proportion of cells expressing eGFP (from Pint) in strain B13-3091  to be lower than that expressing eCherry (from PinR), although this was only poorly significant after 96 h (P = 0.04), again because of the very low subpopulation sizes . This suggests that transcription from Pint is both indirectly (via InrR) and directly dependent on RpoS.Since the proportion of cells expressing eGFP from Putations . For this <0.5%, . Purifieint and PinR promoters on ICEclc, we also determined ICEclc core gene expression and transfer frequencies from B13 wild-type or derivatives as donor and P. putida UWC1 as recipient. Expression of the ICEclc core genes in stationary phase cells measured by microarray analysis was lower (up to 27-fold) for both B13-2671 (rpoS) and B13-2201 (inrR−/−) compared to B13 wild-type (inrR operon was not only downregulated in B13-2671 (rpoS) but also in B13-2201 (inrR−/−) (Whereas expression of the reporter gene fusions was interpreted as being representative for the behaviour of the native Pild-type . InteresnrR−/−) , suggestclc core gene expression but also transfer frequencies were significantly lower at all time points from B13-2673 (rpoS) or B13-3091  than from B13-2581 wild-type or the rpoS-complemented B13 rpoS mutant (B13-2993) as donor (clc transfer frequencies from the complemented B13 rpoS mutant were not significantly different than those from B13 wild-type. Transfer frequencies from B13-2673 (rpoS) as donor were significantly higher than from B13-3091  as donor, but only after 96 h mating time . mCherry expression from PrpoS in stationary phase is normally distributed among all cells with a mean around 50 RFU . RpoS-mCherry but not an N-terminal mCherry-RpoS fusion protein complemented B13-rpoS for bistable Pint or PinR-dependent eGFP expression (data not shown). This indicated that the RpoS-mCherry fusion protein functionally replaces B13 wild-type RpoS. Significantly, only B13-3564 and B13-3555 cells expressing the highest RpoS-mCherry levels had also activated eGFP from Pint or PinR, respectively, although not all cells with high RpoS-mCherry levels expressed high levels of eGFP  from the original ribution , and eGF of eGFP . This suinR and Pint, an additional rpoSB13 copy under control of its own promoter was introduced by mini-Tn5 transposition  expressed eGFP from Pint and eCherry from PinR compared to 5% in B13-2581 wild-type (clc transfer from B13-3260 (+rpoS) as donor to P. putida UWC1 as recipient was twice as high as with B13 wild-type after the same mating contact time, although this was not a statistically significant difference  did not significantly differentially express both reporter genes from PinR and Pint  and in the same strain into which another single copy of rpoSB13-mcherry was transposed (B13-3712). Indeed, the mean mCherry fluorescence in B13-3712 was almost twice as high as in B13-3564  from two individual single copy transcription fusions to Pint or PinR, placed at different positions of the B13 chromosome as suggested in Elowitz et al.int promoter than on PinR . Also adding an additional copy of inrR resulted in a lowering of the total noise, although the proportion of cells expressing eGFP and eCherry in the inrR+ strain was not increased compared to wild-type  as sole carbon and energy source measurably express PinR and Pintclc excision and transfer at population level clc element.One of the mysteries in ICE gene transfer among bacteria is the mechanism that controls the  frequency by which they become excised in clonally identical populations of donor cells. ICE conjugation must start with its excision and therefore the cellular decision that determines conjugation is binary: ICE excision or not. Low transfer frequencies  suggest that the binary ‘ON’-decision is only made in a very small proportion of donor cells. Indeed, our previous results on ICEP. knackmussii is a stationary phase sigma factor controlling transcription of the PinR- and Pint-promoters and thus, indirectly, transfer of ICEclc to P. putida. Addition of an extra rpoSB13 gene copy led to an increased proportion of stationary phase cells in which the PinR- and Pint-promoters are activated, which suggested that the expression level of RpoS is important for controlling the bistable switch leading to ICEclc activation. Indeed, by expressing an RpoS-mCherry fusion instead of RpoS wild-type protein in strain B13 we showed that PinR- or Pint-egfp expression in stationary phase preferably occurred in individual cells with the highest levels of RpoS-mCherry fluorescence  must be responsible for the activation or derepression of PinR  InrR and the relatively minor role of (a widely abundant) RpoS directly on Pint-expression. This effect may actually have been overestimated by a bias introduced by the measurement technique . In contrast, and in the same ‘biased’ setting , the total noise is significantly lower on the PinR-promoter and the relative contribution of the extrinsic noise is higher . This would make sense since individual cells would overall contain higher levels of RpoS permitting more direct interaction with Pint. Adding a third copy of inrR also reduced the level of intrinsic noise on Pint, but in this case because such cells would produce more InrR, diminishing the noise effect by ‘small numbers’ of regulatory factors . Noise in individual cell RpoS levels is thus not propagated to noise in expression of downstream regulons, as was shown recently for global transcription factors in yeast clc factors to a precise activation cascade leading to ICEclc excision and transfer.This conclusion is further supported by noise measurements on the Promoters . Intrinss higher , which irpoS copy number strongly increased the proportion of cells in the population expressing Pint and PinR from 3% to almost 20%, although the transfer frequency of ICEclc only doubled  to Pint and other ICEclc core genes. Microarray analysis confirmed the important role of InrR for the overall activation of ICEclc core functions, and indicated a possible feedback loop on its own expression , although it is not highly conserved Intriguingly, doubling  doubled . In contneration . In thispression . Importa70 family, which is widely distributed among prokaryotes, although RpoS regulons can be quite different in individual species 4652 activity in P. putidatnpA transposition frequency since Tn4652 becomes at least 10 times more activated in an rpoS-defective strain rpoS mRNA levels in P. aeruginosa biofilms, but this occurred rather as a consequence of physico-chemical gradients within the biofilm rbsV-rbsW-sigB operon for the stress response sigma factor SigB in Bacillus subtilis. Interestingly, sigBp expression proceeds in a ‘burst-like’ fashion with a higher pulse frequency under stress than under normal growth condition sigB operon feedback on itself and on its anti- and anti-anti-sigma factors RbsW and RbsV, respectively.As far as we are aware, this is the first time that RpoS has been implicated in controlling horizontal gene transfer of a conjugative element. RpoS homologs are part of a large protein cluster called the σclc are wired within noise in a global transcription factor but can transduce this noise to a precise activation cascade, and thus may have been selected for their capacity to successfully exploit the noise.Gene expression noise is ubiquitous and plays an essential role in a variety of biological processes, triggering stochastic differentiation in clonal populations of cells Escherichia coli DH5α  was routinely used for plasmid propagation and cloning experiments. E. coli HB101 (pRK2013) was used as helper strain for conjugative delivery of mini-transposon constructs P. knackmussii strain B13 clc element (ICEclc), of which it carries two copies E. coli, whereas LB and type 21C mineral medium (MM) P. knackmussii. 3-Chlorobenzoate (3CBA) was added to MM to a final concentration of 5 or 10 mM. When necessary, the following antibiotics were used at the indicated concentrations (µg per ml): ampicillin, 500 (for P. knackmussii) or 100 (for E. coli); kanamycin, 50 and tetracycline, 100 (for P. knackmussii strain B13 derivatives) or 12.5 (for E. coli). P. knackmussii strain B13 was grown at 30°C; E. coli was grown at 37°C.Luria-Bertani (LB) medium 9 donor cells (P. knackmussii B13 or one of its derivatives) and 500 µl suspension of around 109 recipient cells (P. putida UWC1) on membrane filters for 24, 48, 72 or 96 h, as described earlier P. putida UWC1 with ICEclc) were selected on MM plates with 5 mM 3CBA as sole carbon and energy source (to select for ICEclc) and 50 µg per ml rifampicin (resistance marker of the recipient). Transfer frequencies were expressed as number of transconjugant colony forming units (CFU) per number of donor CFU.Self-transfer was tested by mixing 500 µl suspension of around 10E. coli and restriction enzyme digestions were all carried out according to standard procedures Polymerase chain reaction (PCR), reverse transcription RT-PCR, plasmid and chromosomal DNA isolations, RNA isolation, DNA fragment recovery, DNA ligations, transformations into rpoS genes of P. aeruginosa, P. putida and P. fluorescens . The B13 rpoS gene region was submitted to GenBank under accession number AB696604.Primers were designed for conserved regions obtained in a nucleotide sequence alignment among orescens . A singlrpoSB13 gene was amplified with a forward primer (080304) carrying a BamHI, and reverse primer (080303) carrying an EcoRI restriction site . Separate experiments to delete rpoS by using recombination with a DNA fragment in which rpoS was fully deleted were not successful either (not shown). The same strategy was then used to produce a single recombinant disruption of rpoS in P. knackmussii strain B13 that lacked both inrR copies rpoS-pME3087 allele to wild-type rpoS in stationary phase cultures was tested by specific PCR  was amplified from strain B13 purified genomic DNA using primers 091206 and 090902 . As this strain carried a mini-Tn5 insertion already it was necessary to remove the Km gene cassette associated with it. Hereto the strain was transformed with plasmid pTS-parA rpoS-rpoS fragment, which was designated B13-2993 . Three independent clones with possible different mini-transposon insertion sites were examined for ICEclc transfer and reporter gene expression.A 2.2-kbp fragment containing the d 090902 . The amporf95213 and inrR genes plus PinR was amplified by PCR using primers  and cloned in pBAM1 using SphI and EcoRI. The resultant suicide plasmids were introduced into B13 or its derivatives by electroporation, from where the transposition was selected by plating on MM plus 3CBA and kanamycin. Bona fide single copy transposition was verified by PCR. At least three independent clones with possibly different insertion positions were used for further experiments.A 1700-bp fragment containing int promoter in front of intB13 and the egfp gene, or Pint and a promoterless echerry gene have been described previously orf95213, inrR, ssb gene cluster (PinR) and either egfp or echerry have been detailed elsewhere inR and Pint promoters simultaneously, we used a previous construct with PinR-echerry in one and Pint-egfp in the opposite direction rpoS promoter (PrpoS), a 1200-bp fragment upstream of rpoS including the nlpD gene was amplified from strain B13 by PCR  as a template and primers (101003 and 101004), in which the start codon of mcherry was replaced by a short nucleotide sequence encoding 15 amino acids (KLPENSNVTRHRSAT) as a linker peptide. The fragment was then cloned in HindIII and SpeI sites on the mini-Tn5 delivery plasmid pBAM1, resulting in pBAM-link-mCherry. A 2.1 kb region containing PrpoS and rpoS lacking its stop codon was amplified using B13 genomic DNA and primers 101001 plus 010102. This fragment was digested wtih EcoRI and HindIII, and cloned into the same sites on pBAM-link-mCherry (designated pBAM-rpoS-mcherry), After transformation in E. coli and purification, this plasmid was introduced into strain B13 or its derivatives by electroporation. Single copy transposon insertions of the rpoS-mcherry fusion construct were selected by plating cells on MM plus 3CBA and kanamycin. If required for introduction of subsequent mini-transpositions the kanamycin gene cassette was removed by ParA resolvase action (see above). At least three independent clones with possibly different insertion positions were used for further experiments.To produce a C-terminal fusion of RpoS to mCherry, a ∼750 bp fragment containing the rpoS of B13 by the gene for the RpoS-mCherry fusion protein we used double recombination by crossing-over. Hereto, a ∼1 kb downstream region of rpoS was first amplified using B13 genomic DNA and primers 110524 plus 110525, which was digested using XbaI and SalI and ligated wtih pJP5603-ISceIv2 EcoRI and SpeI, an inserted upstream of the amplified fragment in pJP5603-ISceIv2 which was hereto digested with EcoRI and XbaI. After transformation in E. coli and purification, the resulting plasmid was electroporated into strain B13-78 (rpoS.To replace n B13-78 . Single P. knackmussii strain B13 or B13 rpoS carrying the PrpoS-mcherry fusion were grown in 96-well black microtiter plates (Greiner Bio-one) with a flat transparent bottom. Each well contained 200 µl of MM medium with 5 mM 3CBA and was inoculated with 2 µl of a bacterial preculture grown overnight in LB medium. Microtiter plates were incubated at 30°C with orbital shaking at 500 rpm. At each given time point both culture turbidity (A600) and fluorescence emission (excitation at 590 nm and emission at 620 nm) were measured from triplicate cultures using a Fluostar fluorescence microplate reader (BMG Lab Technologies). Cultures of P. knackmussii strain B13-78 wild-type served for background fluorescence correction.To image eGFP, eCherry or mCherry expression in single cells, culture samples of 4 µl were placed on regular microscope slides, closed with a 50 mm long and 0.15 mm thick cover slip, and imaged within 1–2 minutes. Fluorescence intensities of individual cells were recorded on image fields not previously exposed to UV-light to avoid bleaching. For most imaging series, except data shown in int and PinR) was then calculated as 100% - the percentile of the breakpoint. The average expression intensity over the highest expressing subpopulation was calculated as the mean AGV over the percentile range between that of the breakpoint and 100%. Fluorescence images for display were adjusted for brightness to a level +143, cropped to their final size and saved at 300 dpi with Adobe Photoshop (Version CS4). Corresponding phase-contrast images were ‘auto contrasted’ using Photoshop.Subpopulation expression was determined from cumulative ranking of all objects according to their AGV. The ‘breakpoint’ between subpopulations on cumulative distribution curves  was deteint and PinR promoters, two identical copies were fused to distinguishable reporter genes (i.e. egfp and echerry) and integrated into separate locations on the chromosome of B13 or its derivatives using mini-Tn5 delivery. Three independent clones with different insertional positions were maintained. Stationary phase cells of such double-reporter strains grown in MM with 3CBA were examined in epifluorescence microscopy, and their eGFP and eCherry fluorescence intensities were measured as outlined above (AGVs). AGVs of both markers in each cell were scaled to subtract background AGV of digital EFM images and normalized to the highest AGV in a population (100%). Only cells belonging to the subpopulations of having higher eGFP or eCherry fluorescence than the breakpoint in the respective cumulative curves , extrinsic noise (ηext), and total noise (ηtot) were then calculated according to previous definitions given in Elowitz et al.g and c denote the normalized eGFP and eCherry AGV, respectively, observed in the nth single cell. Angled brackets denote a mean over the sample population.To identify and quantify noise in expression of the Pes e.g.,  were useSignificance of different treatments was examined by pair-wise t-test or ANOVA followed by a Tukey post hoc test. To test the effect of subpopulation size on noise calculations, data sets were randomly resampled using bootstrap procedures (1000 times), upon which the intrinsic, extrinsic and total noise were calculated and finally, averaged over all resampled populations of the same data set.P. knackmussii B13-78 (wild type), B13-2671 (rpoS) and B13-2201 (inrR−/−) cultures after 48 h in stationary phase after growth on 3CBA as sole carbon and energy source, by using the procedure described previously Total RNA was isolated from Figure S1rpoS genes from Pseudomonas putida (P. p.), P. fluorescens (P. f.) and P. aeruginosa (P. a.). Rectangular boxes represent the region chosen to design primers for the amplification of rpoS from strain B13. Inosine was used in the oligonucleotides at non-conserved positions. Genbank numbers: P. putida KT2440, NC_002947.3; P. fluorescens Pf-5, NC_004129.6; P.aeruginosa PAO1, NC_002516.2.Alignment of (TIF)Click here for additional data file.Figure S2Pseudomonas strains. (A) MegAlign alignment (DNAStar Lasergene package v.8) and indication of consensus per position. (B) Dendrogram  showing the closest neighbourhood clustering of the strain B13 rpoS gene.Comparison of the predicted RpoS amino acid sequence from strain B13 and orthologues from four other (TIF)Click here for additional data file.Figure S3rpoS in strain B13 by a single recombination event. (A) rpoS gene region. (B) Amplification of a 600-bp internal rpoSB13 fragment by PCR whilst creating BamHI and EcoRI restriction sites. Insertion of the rpoSB13 fragment into the suicide vector pME3087. (C) Genetic structure produced by single homologous recombination and inactivation of rpoS on the B13 chromosome.Strategy for inactivating (TIF)Click here for additional data file.Figure S4P. knackmussii B13-78 wild-type and B13-2671 (rpoS) in MM with 5 mM 3CBA. Data points are the average from three independent biological replicates ± one calculated standard deviation. Maximal specific growth rates in exponential phase for B13-78 were 0.22±0.01 versus 0.26±0.01 h−1 for B13-2671 (rpoS). Note that growth medium for B13-2671 included Tc to select for the rpoS-pME3087 allele. (B) Semi-quantification of the presence of rpoS revertants in B13-2671 (rpoS) cultures by PCR. 25 ng of genomic DNAs isolated from B13-2671 culture with Tc at 24 h (lane 5), 48 h (lane 6), 72 h (lane 7), or 96 h (lane 8) were used as templates. A serially diluted B13-78 (wild-type) DNA was used as control: lane 1, 0.25 ng; lane 2, 0.5 ng; lane 3, 2.5 ng; lane 4, 25 ng. Intact rpoS (upper panel) and fdxA  alleles were amplified using primer pairs 090206+090902 and 110524+110525, respectively. Lane M, molecular mass marker . The positions and sizes of the expected PCR fragments are indictaed. Note that some reversion of rpoS-pME3087 to wild-type rpoS must occur (lane 7–9) but at less than 1% in the population (lane 1).Growth of (TIF)Click here for additional data file.Figure S5rpoS or double inrR disruption on expression of a Pint-egfp fusion in P. knackmussii. (A) Relevant construction details of the mini-Tn construct delivering the single copy Pint-egfp fusion. (B) Micrographs showing the subpopulation of cells expressing eGFP from Pint amidst a large number of silent cells for B13-1346 (wild-type), B13-2976 (rpoS) or B13-2979 (−/−inrR) cultured on 3CBA after 24 h into stationary phase. (C) As B, but after 72 h in stationary phase. Shown are phase-contrast micrographs at 1,000× magnification and corresponding epifluorescence images. For quantification, see Comparison of effects caused by (TIF)Click here for additional data file.Figure S6clc gene expression compared among P. knackmussii B13-78 (wild-type), B13-2201 (−/−inrR) and B13-2671 (rpoS). A) Log2 fold-change in negative-strand probe signals on an ICEclc micro-array. Inset shows detail around inrR-operon. B) Positive-strand probe signals. Open reading frames of ICEclc plotted along its length; white boxes: genes oriented on the positive strand, grey boxes: negative strand. Known ICEclc functional genes or regions indicated by name for reference.ICE(TIF)Click here for additional data file.Figure S7rpoS promoter in P. knackmussii. (A) Relevant construction details of the mini-Tn construct used to place a single copy PrpoS-mCherry transcriptional fusion in the B13 genome. (B) Culture-density normalized mCherry fluorescence as a function of culture density (open circles) and incubation time in B13-3165 (wild-type) B13-3228 (rpoS), or B13-3654 (rpoS-mCherry). (C) Corresponding phase contrast (PhC) and epifluorescence micrographs of B13-3165 cells 24 h into stationary phase. Note how expression from PrpoS is RpoS independent and how expression of RpoS-mCherry from PrpoS is detectable slightly later than that of mCherry alone, suggesting post-transcriptional effects.Growth phase dependent expression from the (TIF)Click here for additional data file.Figure S8int or PinR above threshold and representative for activating the ICEclc element. (A) Finding the breakpoint between the larger non-active subpopulation of cells and the smaller ICEclc-active subpopulation of cells on a cumulative distribution curve of reporter fluorescence values from Pint or PinR. (B) Scaling and normalizing of eCherry and eGFP expression for noise calculations. Only cells falling in the grey zones  are considered for noise calculation.Calculation of the subpopulation (size and mean reporter fluorescence expression) of B13-cells expressing P(TIF)Click here for additional data file.Table S1clc from P. knackmussii strain B13, the inrR deletion and the rpoS deletion mutants to P. putida UWC1 as recipient.Transfer frequencies of ICE(DOC)Click here for additional data file.Table S2clc from P. knackmussii strain B13 and the rpoS+ strain to P. putida UWC1 as recipient.Transfer frequencies of ICE(DOC)Click here for additional data file.Table S3int-copies in different places on the chromosome of P. knackmussii derivatives.Effect of subpopulation size on noise calculation from two identical P(DOC)Click here for additional data file.Table S4Primers used in this study.(DOC)Click here for additional data file.


Hypoxia-ischemia brain insult induced significant brain weight reduction, profound cell loss, and reactive gliosis in the damaged hemisphere. Hypoxic preconditioning significantly attenuated glial activation and resulted in robust neuroprotection. As early as 2 h after the hypoxia-ischemia insult, proinflammatory gene upregulation was suppressed in the hypoxic preconditioning group. In vitro experiments showed that exposure to 0.5% oxygen for 4 h induced a glial inflammatory response. Exposure to brief hypoxia (0.5 h) 24 h before the hypoxic insult significantly ameliorated this response. In conclusion, hypoxic preconditioning confers strong neuroprotection, possibly through suppression of glial activation and subsequent inflammatory responses after hypoxia-ischemia insults in neonatal rats. This might therefore be a promising therapeutic approach for rescuing neonatal brain injury.Perinatal insults and subsequent neuroinflammation are the major mechanisms of neonatal brain injury, but there have been only scarce reports on the associations between hypoxic preconditioning and glial activation. Here we use neonatal hypoxia-ischemia brain injury model in 7-day-old rats and  Hypoxia-ischemia injury is the final common mechanism in different kinds of brain damage resulting from trauma. Hypoxia-ischemia injury is also the major cause of brain damage in neonates . The incPathophysiological features of cerebral hypoxia-ischemia are unique to the immature brain and provide the potential for clinical intervention . Multipl in vivo and in vitro.Hypoxic preconditioning, or hypoxia-induced tolerance, refers to a brief period of hypoxia that protects against an otherwise lethal insult occurring minutes, hours, or days later . Gidday  ad libitum access to water and food. The pups were housed with their dams until weaning on postnatal day 21 (P21). Only male rat pups were used in this study.All experiments were performed in accordance with the National Institutes of Health Guidelines on Laboratory Animal Welfare and were approved by the Animal Care and Use Committee of the College of Medicine, National Taiwan University. Ten to twelve Sprague-Dawley pups per dam were used in this study and housed in institutional standard cages on a 12-hour light/12-hour dark cycle, withA modified Rice-Vannucci model was used for the induction of hypoxia-ischemia (HI) brain damage in 7-day-old (P7) neonatal rats . P7 ratsn = 6 in each group). After removal of the brainstem and cerebellum, the forebrain was sectioned at the midline, and both hemispheric weights were determined. A previous study showed that the extent of unilateral reduction in hemispheric weight is highly correlated with biochemical, electrophysiological, and morphometric markers of tissue injury, in the same animal model [On day 14 after birth (P14), the pups were anaesthetized with isoflurane and decapitated and the brains were removed . The pups were anaesthetized with isoflurane and perfused transcardially with ice-cold saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). Brains were then removed, postfixed overnight in 4% paraformaldehyde, and then transferred into a 30% sucrose solution for 72 h. At this time, brains were embedded and frozen in Neg-50  for storage at −80°C. Coronal sections were cut at 20 μm on a Leica cryostat and collected serially onto gelatin-coated slides for storage at −80°C.Brains were collected 24 h after HI injury (P8) for immunohistochemistry assessment of glial activation and Nissl staining was performed for evaluation of structural and cellular damage  was used as a marker of activated astrocytes. Briefly, sections were thawed and treated with 3% hydrogen peroxide for 30 min. After blocking in 4% nonfat milk containing 0.4% Triton X-100 for 60 min at room temperature, the sections were incubated overnight at 4°C with primary antibodies against CD11b  and GFAP . Sections were then washed several times in PBS and incubated with a biotinylated secondary antibody and then treated using an avidin-biotin complex kit . Finally, the labeling was detected by treatment with 0.01% hydrogen peroxide and 0.05% 3,3′-diaminobenzidine . The DAB reaction was stopped by rinsing tissues in PBS. Labeled tissue sections were then mounted and analyzed under a bright-field microscope.7 cells into 75 cm2 flasks in Dulbecco's modified Eagle's medium  containing 10% heat-inactivated fetal bovine serum , 2 mM L-glutamine, 1 mM sodium pyruvate, 100 mM nonessential amino acids, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air, and medium was replenished twice a week. These cells were used for experiments after reaching confluence (7-8 days).Primary glial cultures were prepared according to a previous report . In brie2 and 95% air. Confluent cultures were passaged by trypsinization. All the cells were plated on 12-well plates at a density of 2.5 × 105 cells/well for nitrite assays or on 6-well plates at a density of 5 × 105 cells/well for reverse transcriptase-polymerase chain reaction (RT-PCR). The cells were then cultured for 2 days before experimental treatment.The murine BV-2 cell line was cultured in DMEM with 10% FBS at 37°C in a humidified incubator in 5% CO2, 94.5% N2, and 5% CO2 , and control cultures were incubated under normoxic conditions for the same duration. After the indicated hypoxic period, reoxygenation was performed by transferring the cells into a regular normoxic incubator , and cells were incubated for another 24 h for nitrite assays. For RT-PCR, total RNA was extracted from the cells after termination of hypoxia, using the same method outlined above.Hypoxia/reoxygenation was performed as previously described with some modifications . To geneμM). After incubation for 24 h, 20 μL CellTiter 96 AQueous One Solution Reagent  was added to culture wells for 30 min. One hundred microliters of the culture medium from each well was transferred to an ELISA 96-well plate, and absorbance at 490 nm was measured with a 96-well plate reader. The absorbance at 490 nm is directly proportional to the number of living cells in culture.Cell viability was assessed by the 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay. Cells cultured in 12-well plates were exposed to hypoxia  or lipopolysaccharide  in 96-well cell culture plates for 10 min at room temperature in the dark. Nitrite concentrations were determined by using standard solutions of sodium nitrite prepared in cell culture medium. The absorbance at 550 nm was determined using a microplate reader .Accumulation of nitrite in the medium was determined using a colorimetric assay with Griess reagent. Briefly, 100 n = 4 in each group) or cell culture (n = 5 in each group) using an RNeasy mini Kit . Two micrograms of RNA was used for RT-PCR. The reaction mixture contained 1 μg Oligo (dT)15 Primer, 0.02 mM deoxynucleotide triphosphate (dNTP), 40 U RNase Inhibitor, 100 U M-MLV Reverse Transcriptase, and 5x Reaction Buffer . PCR was performed using an initial step of denaturation (5 min at 94°C), 25 cycles of amplification , and an extension (72°C for 7 min). PCR products were analyzed on 1.5% agarose gels. The mRNA of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal control for sample loading and mRNA integrity. All of the mRNA levels were normalized to the level of GAPDH expression. The oligonucleotide primers are shown in Total RNA was extracted from brain tissue homogenates (α) and interleukin-1 beta (IL-1β), were detected using ELISA kits , according to the manufacturer's instructions. Briefly, pups were sacrificed by decapitation at 2, 6, 12, 24, and 72 h after HI (n = 4 for each time point per group) and protein samples of the ipsilateral hemispheric cortex from each pup were collected. All protein concentrations were determined by the Bradford method . Data were acquired using a 96-well plate reader (BioTek). The cytokine content is expressed as pg cytokines/mg protein.Two major proinflammatory cytokines, tumor necrosis factor-alpha . The mRNA levels and protein concentrations at different time points were analyzed by two-way ANOVA to compare the HI and HP + HI groups. Individual groups were then compared using Bonferroni's post hoc tests, as appropriate. Statistical analyses were performed using Prism GraphPad v.5.0 , with a significance level of The mean ratio of brain weight reduction of the right hemisphere, measured 7 days after right carotid artery ligation and exposure to 2 h of hypoxia in the HI group, was 34.6 ± 2.1%; this reduction was significantly greater than in the HP + HI group. The ratio of brain weight reduction was not statistically different between HP + HI and sham groups. The percentages for brain weight reduction are individually illustrated in To identify the effect of hypoxia preconditioning on glial response after hypoxia-ischemia injury, we used CD11b immunostaining to determine microglial activation and GFAP immunostaining to determine astrocytic activation . Twenty-β upregulation were significantly reduced as early as 2 h after the hypoxia-ischemia insult . We thus extracted total protein from the right cortex and further evaluated TNF-α and IL-1β protein expression by ELISA at different time points (α were very low in the sham group (0.08 ± 0.02 pg/mg protein) and were increased markedly at 2 h after the hypoxia-ischemia insult . These levels peaked at 6 h  and were decreased after 12 h. Hypoxia preconditioning resulted in a significant reduction of the TNF-α level at 6 h . The level of IL-1β was also very low at baseline , slightly increased 2 h after the hypoxia-ischemia insult , and markedly elevated 6 h after the hypoxia-ischemia insult . Hypoxia preconditioning before the hypoxia-ischemia insult markedly decreased the IL-1β levels at 6 h . Therefore, hypoxia preconditioning was able to suppress the hypoxia-ischemia injury-induced inflammatory response in neonatal rat brain.We next examined the effect of hypoxia preconditioning on the inflammatory response induced by hypoxia-ischemia injury. Comparing the mRNA levels of the right cortex in the HP + HI group with the HI group, inducible NOS (iNOS) and IL-1a insult . Six houe points . The levTo further clarify the role of glial cells in the anti-inflammatory mechanisms of hypoxia preconditioning, we next examined the glial response to hypoxia preconditioning at the cellular level. Firstly, nitrite production and cell viability after hypoxia exposure for different time durations were determined in a primary mixed glial culture and in the microglial cell line, BV-2 . Exposur in vivo model of hypoxia preconditioning. The production of nitrite after reoxygenation was decreased significantly by hypoxia preconditioning for 0.5 h, but not for 1 or 2 h  and the expression of the inflammatory related gene, COX-2. Our results also showed that a longer period of hypoxia preconditioning had less of an anti-inflammatory effect, which implies the importance of selecting the appropriate duration for hypoxia preconditioning. The induction of proinflammatory cytokine genes, including TNF-α and IL-1β, was not as prominent as for the in vivo model. The difference in cytokine response may be due to the recruitment of circulating white blood cells. In the animal model, hypoxia-ischemia will induce migration and infiltration of circulating macrophages and monocytes, further increasing proinflammatory cytokine release [α and IL-1β were all significantly increased after hypoxia exposure for 4 hours, and hypoxia preconditioning for the optimal duration (0.5 h) was able to attenuate the activation effects. Again, longer durations of hypoxia preconditioning decreased the anti-inflammation effects, as in primary mixed glia cultures.It may be argued that a diminished inflammatory reaction arises from a reduction in neuronal damage and is not a direct cellular response. We thus developed an release . The majGlial cells, in particular astrocytes, are usually viewed as supporters of neuronal function. However, numerous studies are increasingly demonstrating the important role of glial cells in preserving brain function under physiological and pathological conditions . The genThe results of the present study can also be extrapolated to other mechanisms of neonatal brain injury. An increasing body of evidence has demonstrated a link between inflammation and long-term brain dysfunction. A recent meta-analysis of 26 articles has shown a positive association between infection and cerebral palsy, in both preterm and full-term infants . In addiIn conclusion, hypoxic preconditioning induced significant neuroprotection against neonatal hypoxia-ischemia brain insults and suppressed astroglial and microglial activation in the ischemic cortex and hippocampus. Pretreatment with sublethal exposure to hypoxia before prolonged hypoxia injury prevented the cellular inflammatory response in the primary glial culture and microglial cell line BV-2. These results further address the importance of anti-inflammatory strategies in preventing neonatal brain injury.


Declarations on end-of-life issues are advocacy interventions that seek to influence policy, raise awareness and call others to action. Despite increasing prominence, they have attracted little attention from researchers. This study tracks the emergence, content, and purpose of declarations concerned with assisted dying and euthanasia, in the global context. The authors identified 62 assisted dying/euthanasia declarations covering 1974–2016 and analyzed them for originating organization, geographic scope, format, and stated viewpoint on assisted dying/euthanasia. The declarations emerged from diverse organizational settings and became more frequent over time. Most opposed assisted dying/euthanasia. Euthanasia and certain forms of assisted dying are currently legal or decriminalized in just a few countries. The Netherlands (2001), Belgium (2002), and Luxembourg (2009) have legalized euthanasia   such declarations group around a common purpose. They capture the goals of interest groups, make statements of intent, point to a more desirable state of affairs, and encourage greater awareness to achieve a stated goal. These declarations have no legal mandate but do have potential for influencing laws, policies, systems and processes on end-of-life issues. They have become a part of the landscape of end-of-life care, and the debates that swirl around it.We refer to advocacy interventions of this type as At the same time, they are poorly documented and largely ignored by researchers. Yet they are important markers in the evolution of end-of-life discourse. They give perspective on the changing discussion around specific issues and have some importance within the culture of many end-of-life care organizations. They merit research scrutiny, in particular, when declarations on the same topic take up opposing or differing perspectives.Building on an earlier study of declarations in support of palliative care development  included national and international medical and nursing associations, specific fields of medicine such as palliative care or geriatric care, and societies representing particular patient groups, such as the Association for Persons with Severe Handicaps, Parkinson’s UK and The Arc of the United States. Religious organizations (16) were all Christian in orientation, including Methodist, Baptist, Catholic, the Salvation Army, the Reformed Churches, and the Christian Medical and Dental Association. Others included political parties (three) and those organizations instituted to advocate for (eight) or against (four) euthanasia/assisted dying.Seven out of the eight declarations in the group established to advocate for euthanasia/assisted dying were issued by the World Federation of Right to Die Societies. The first of these was in 1976 and the remaining six were issued between 1996 and 2006 at 2-year intervals, corresponding with the biannual conferences of the Federation.Nearly three quarters of the declarations (45/62) were against euthanasia/assisted dying and were issued by associations of palliative care and other health care disciplines, associations of patient groups, and churches. Nine declarations advocated for the introduction of euthanasia/assisted dying, of which seven were issued by the World Federation of Right to Die Societies. Among the eight declarations that expressed a neutral position, two were from political parties calling for further discussion on the subject. Others included health care associations representing divided views of members, organizations that expressed their commitment to equal treatment of all patients irrespective of their position on euthanasia, and those that refrained from taking a position because euthanasia/assisted dying was illegal in their respective countries.All declarations issued by religious organizations were against euthanasia/assisted dying. Among health care organizations, 24 were against and 5 were neutral. Two declarations from political parties took a neutral position and one was for euthanasia/assisted dying.Analyzing the 51 declarations where the year of publication could be identified, we found different viewpoints showing prominence over specific periods of time. The first two declarations from 1974 (against) and 1976 (for) represented either side of the argument. With two exceptions, all declarations issued in the 1990s were against legalizing euthanasia/assisted dying. Five out of the nine declarations published between 2000 and 2010, were in support. The period from 2011 to 2016, which showed highest activity (23), was dominated by declarations against euthanasia/assisted dying (18). The first declaration with a neutral stance appeared in 2001 and, after a break of 10 years, five declarations were issued between 2012 and 2016.The majority of declarations (39) were oriented to national audiences: United States (14), United Kingdom (12), Canada (eight), New Zealand (three), and the Netherlands (one). Many were published because of a proposed change in legislation or a judicial decision. The international declarations (19) were all issued by organizations or churches with a global presence, such as the World Medical Association, The World Federation of Right to Die Societies, The Christian Medical and Dental Associations, The Salvation Army International, and the Sacred Congregation for the Doctrine of Faith. Two declarations involved two countries only , and two involved a specific region within a country .The 62 declarations came in several formats. Most common was a statement of convictions (38) expressing beliefs and opinions. Others made recommendations (23) to governments, policy makers, health care professionals and the wider public, expressed specific concerns (10), made a call to action (seven) for governments, health institutions or the public, made an explicit position statement (six) of the organizations’ stand, described their action plan (three), and recorded their commitment to a cause or an aspect of care (two). Many declarations contained more than one of these formats.Most declarations indicated the ethical or practical reasons for their position on euthanasia/assisted dying  and inclThe recommendations in the declarations varied in relation to the “viewpoint” adopted: for, against or neutral. Recommendations from declarations for euthanasia or assisted dying included decriminalization of voluntary medically assisted death; legalizing medically hastened death; respecting the voluntarily expressed will of individuals as an intrinsic human right; openness to and acceptance of terminal sedation as a form of assisted dying; inclusion of assisted dying within the mandate and practice of palliative care.The most prominent recommendation from declarations against euthanasia/assisted dying was for improvement in the provision of palliative care. This was followed by recommendations about access to and the administration of medications for adequate pain relief. They asserted that good palliative care and physical symptom control minimize the number of requests for hastened death and that governments should pay attention to lack of relevant health and social support, equality, and justice. Asserting that misconceptions about suffering at the end of life fuel the public demand for legalizing euthanasia, some declarations recommended public education about palliative care.Our study has demonstrated that the practice of issuing declarations on euthanasia/assisted dying has emerged as a significant phenomenon within the field of end-of-life care. We have shown an increasing incidence of such declarations over time and their growing prominence as an advocacy tool. The declarations take specific (though varied) positions on the issue of legalization of euthanasia/assisted dying and aim to promote these to gain public support and/or favorable actions from governments. Despite their emerging significance, no commentary exists to our knowledge on such advocacy documents and their role in end-of-life debates and discourse. As the discussion on these issues spreads to more countries we are likely to see the appearance of further declarations of this type.Our analysis shows a specific geographic range in the declarations identified. They all emanate from the United States, Canada, Western Europe, Australia, and New Zealand. These are countries where active measures have taken place to consider the value of legalizing euthanasia/assisted dying or where such legalization has already taken place. The absence of declarations from other parts of the word, including Asia and Africa is notable. Although discussions and studies exploring perceptions on the issue of euthanasia and assisted dying are emerging from these parts of the world Saadery, , relevanThe diversity of viewpoints on euthanasia/assisted dying is strikingly depicted in these declarations. Declarations for euthanasia/assisted dying range from those which endorse the decriminalization of assisted dying, to those which demand it as a fundamental human right. Declarations against range from those suggesting that assisted dying may not be the right solution to the problem of suffering, to others which strongly condemn initiatives to legalize euthanasia.Although declarations for and against use some terminologies in common, the extent of their meaning and use differs significantly. Respecting the contents of a living will is a commonly recognized issue in end-of-life care. Yet although declarations favoring euthanasia extend the value of the living will to those expressing the wish to die, those against do not support its use to facilitate medical assistance to end life.Although all declarations express their intention to promote dignified death, those for euthanasia consider respecting autonomous decisions of the individual on the timing, place, and manner of death as aspects of dignity. Declarations against euthanasia, however, present dignity as an equal and inviolable quality inherently possessed by human beings. They present the view that intentional killing of a human being, even at their voluntary request due to intractable suffering, undermines human dignity.Despite their wide ranging characteristics and divided perspectives, euthanasia/assisted dying declarations share some of the wider principles of advocacy. They identify with disadvantaged populations, promote their cause, and invoke responses from positions of authority and professional groups, as well as from wider communities (Gray & Jackson, We acknowledge certain limitations to our study. Although the search for declarations was conducted in a systematic way, it is possible there may be other declarations we did not find, for example declarations could have used different terminology in their titles to our keywords or declarations may have been issued in other languages than English. Therefore, while capturing the landscape of declarations to a significant degree, there may be other declarations on euthanasia/assisted dying that are not covered in this study. We consider this a small possibility however. The findings of our study are also limited by the contents of these advocacy documents. We acknowledge that these may not necessarily represent the views of all individuals that make up these organizations, though they are the declared organizational position on the issue. It is also possible that there may be other organizations concerned about the legalization of euthanasia/assisted dying that have not considered it a high enough priority to issue a declaration.Declarations relating to euthanasia and assisted dying represent the views and demands of diverse communities of interest concerned about suffering at the end of life, often with a determination to make their voices heard and to advocate for change. Our study has catalogued the emergence of this particular form of intervention as an advocacy tool in the wider debates about end-of-life issues. We have identified the various organizations involved, the positions represented and the recommendations made. In so doing, we have opened up a space for further analytic work and more comparative analysis of declarations across a range of end-of-life issues. Further exploration of these declarations in the light of their respective contexts will help understand their significance and impact.