BRCA2-8765delAG mutation was firstly described in breast cancer families from French-Canadian and Jewish-Yemenite populations; it was then reported as a founder mutation in Sardinian families. We evaluated both the prevalence of the BRCA2-8765delAG variant in Sardinia and the putative existence of a common ancestral origin through a haplotype analysis of breast cancer family members carrying such a mutation.The BRCA2 gene locus were used for the haplotype analysis. Screening for the 8765delAG mutation was performed by PCR-based amplification of BRCA2-exon 20, followed by automated sequencing.Eight polymorphic microsatellite markers  spanning the BRCA2-8765delAG variant among unselected and consecutively-collected breast cancer patients originating from the entire Sardinia revealed that such a mutation is present in the northern part of the island only [9/648 (1.4%) among cases from North Sardinia versus 0/493 among cases from South Sardinia].Among families with high recurrence of breast cancer (≥ 3 cases in first-degree relatives), those from North Sardinia shared the same haplotype whereas the families from French Canadian and Jewish-Yemenite populations presented distinct genetic assets at the BRCA2 locus. Screening for the BRCA2-8765delAG has an independent origin in geographically and ethnically distinct populations, acting as a founder mutation in North but not in South Sardinia. Since BRCA2-8765delAG occurs within a triplet repeat sequence of AGAGAG, our study further confirmed the existence of a mutational hot-spot at this genomic position .The  Breast Cancer (BC) is the most common malignancy among women in industrialized countries. Germline mutations in BRCA1 (MIM 113705) and BRCA2 (MIM 600185) genes account for cancer predisposition in majority of families with BC recurrence. Positive family history is indeed recognized as a high risk factor for developing the disease; about 10% of all breast cancers arise in individuals carrying a germline mutation in one of such two genes , further confirming that occurrence of the BRCA2 mutations is strictly associated with the presence of a higher familial recurrence of the disease.To evaluate the prevalence of BRCA2-8765delAG in breast cancer cases across the entire Sardinia, we screened genomic DNA obtained from both peripheral blood and paraffin-embedded normal tissues (see Methods) of 1,141 unselected and consecutively-collected BC patients originating from either the northern (N = 648) or southern N = 493) part of the island. The 2-bp deletion in exon 20 of the  part of BRCA2-8765delAG as a founder mutation in this geographical area. Haplotype analysis revealed that Sardinian mutation carriers shared majority of allele markers with only one of the two Jewish-Yemenite families; the remaining Jewish-Yemenite family and the French-Canadian families did not present the same genotypic asset . As a confirmation of this hypothesis, other authors have described different nucleotide deletions at this genomic level among families with breast and/or ovarian cancer [In the BIC database , the BRCite-Jews , allow tn cancer ,14,17,18BRCA2-8765delAG variant among the unselected patients from the Sardinian population, our extensive screening clearly indicated that such a mutation is recurrent in North Sardinia  but e island . For bremelanoma , these fBRCA2-8765delAG mutation in breast cancer families was lower than that of other BRCA1-2 founder mutations observed in different genetically-homogeneous populations  declare that they have no competing interests.GRP participated in the design of the study and performed the molecular analyses. ACOS performed the data management. EF participated in the families' collection. MB participated in the analysis and interpretation of data. AF participated in the patients' collection. ACON participated in the patients' collection. MP performed some haplotype analyses. PB performed some screening analyses. MCS performed some screening analyses. FT participated in the design of the study. GIP conceived of the study and drafted the manuscript.The pre-publication history for this paper can be accessed here:


A tumour model consisting of the highly metastatic mammary 13762 parental line, the non-metastatic and 6-thioguanine-resistant (TgR) variant line, and two TgR revertant lines  that were occasionally metastatic, were used to determine whether the release of N-acetylneuraminic acid (NANA) was related to tumour metastasis. For comparative purposes, the occasionally metastatic R3230AC and the nonmetastatic DMBA8 tumour lines were studied. The NANA was considered to be in bound form, because acid hydrolysis was required to release it for high-pressure liquid chromatographic analyses. Sera of animals bearing the 13762 and R3230AC tumours had high levels of bound NANA. No differences were found in serum NANA levels in animals bearing metastatic or non-metastatic R3230AC tumours. Low levels of bound NANA were found in animals bearing the other tumour lines regardless of whether metastasis occurred or not. The experiments in vitro substantiated the in vivo findings. The phenotypic expression of bound NANA shedding did not correlate well with metastatic potential of the mammary tumour line. Our analyses suggest that this phenotypic marker cannot be used as a reliable indicator of metastasis.


Determining the relative contribution of intrinsic and extrinsic factors to fluctuations in population size, trends and demographic composition is analytically complex. It is often only possible to examine the combined effects of these factors through measurements made over long periods, spanning an array of population densities or levels of food availability. Using age-structured mark-recapture models and datasets spanning five decades (1950–1999), and two periods of differing relative population density, we estimated age-specific probabilities of survival and examined the combined effects of population density and environmental conditions on juvenile survival of southern elephant seals at Macquarie Island.First-year survival decreased with density during the period of highest population size, and survival increased during years when the Southern Oscillation Index (SOI) anomaly (deviation from a 50-year mean) during the mother's previous foraging trip to sea was positive . However, when environmental stochasticity and density were considered together, the effect of density on first-year survival effectively disappeared. Ignoring density effects also leads to models placing too much emphasis on the environmental conditions prevailing during the naïve pup's first year at sea.Our analyses revealed that both the state of the environment and population density combine to modify juvenile survival, but that the degree to which these processes contributed to the variation observed was interactive and complex. This underlines the importance of evaluating the relative contribution of both the intrinsic and extrinsic factors that regulate animal populations because false conclusions regarding the importance of population regulation may be reached if they are examined in isolation. A central aim in population biology is to discern the relative contribution of intrinsic (density-regulated) and extrinsic  factors to fluctuations in population size and demographic composition, with increasing emphasis placed on quantifying the complex interplay between the two -4. The mGiven that populations of large, long-lived mammals tend to have a relatively low capacity for growth due to their long generation times and low reproductive output , it is hChanges in the population size of large marine predators is potentially indicative of larger ecosystem changes given that their predominate regulator appears to be environmental stochasticity influencing food availability over vast oceanic foraging regions e.g., -22). Upp. Upp22])Mirounga leonina) at Macquarie Island, has been the focus of intensive demographic studies for over fifty years  = 5881; Table ER = 4732, Table ĉ, > 74 % of the model weight was allocated to models with first-year survival varying with SOI measured during the pregnant mother's pre-partum foraging trip  during the year of the mother's pre-partum foraging trip describe an important component of the variation in first-year survival (ER > 3.5).For the high-density era, there was strong evidence that the probability of both first-year and adult survival varied with the standardized SOI  [see Additional file ĉ value from the high-density era to contrast models; this revealed that the models with the most support (combined QAIC weights > 99 %) incorporated the mother's foraging trip SOI, and there was little evidence for a density or pup-year SOI anomaly effect  was examined without the effects of density, the most parsimonious model predicted that the conditions during an individual's first year of life best explained variation in survival when population size was high Table . HoweverWe must also consider that the weak effects of population density on first-year survival are unlikely to capture the full mechanistic component of density regulation in this population, especially given the strong phenomenological evidence for density dependence in both relative-density periods. In addition to vital rates such as survival, density-dependent regulation may apply to other aspects of a species' biology, such as growth, behaviour, incidence of disease and distribution ,62,63. EOur results have important implications for the assessment of environmental change in the Southern Ocean and Antarctic region. Given their status as upper trophic-level predators foraging over vast areas of the subantarctic and Antarctic oceanic zone, variation in abundance and life history parameters in this species may be indicative of larger changes occurring throughout the Antarctic ecosystem . The extOur study quantified the degree to which the likely drivers of variation in abundance interact in a single population of a wide-ranging oceanic predator. The population and demographic models that were constructed provided a quantitative assessment of the complex interactions of extrinsic and intrinsic factors regulating the population, and the processes described provide a comprehensive overview of large mammal dynamics when they are exposed to highly variable environments. Understanding the complex nexus that emerges between these two major forces is a vital precursor for predictions of the influence of rapid climate change on animal populations worldwide. As such, species amenable to long-term monitoring within highly stochastic environments such as the Southern Ocean can act as climatic 'canaries' that chronicle catastrophic ecosystem degradation resulting from human-mediated climate change.nf) breeding there was approximately 5000 and (2) the low-density era between 1993 and 1999 when nf was ca. 2500 to 3000 [Capture-mark-recapture (CMR) datasets were collected from the elephant seal populations breeding on the northern isthmus of Macquarie Island  over two periods of different population density: (1) the high-density era between 1951 and 1965 when the number of breeding females   data, we applied the technique of Brook and Bradshaw  to each l models  used to Nt = population size at time t, r = realized population growth rate, rm = maximal intrinsic population growth rate, K = carrying capacity, θ permits a nonlinear relationship between rate of increase and abundance. The term εt has a mean of zero and a variance (σ2) that reflects environmental variability in r. For each high-density and low-density time series we used maximum-likelihood estimation to fit model parameters  weights, was used to assign relative strengths of evidence to each model [where  model – ). An indch model . The relch model .ϕ, the apparent survival probability  of individuals between the nth and (n + 1)th year , and p, the re-sight probability for all individuals in the nth year  [Capture history matrices were constructed from the re-sighting histories of individual seals, with multiple re-sights within a year treated as a single sighting. Capture matrices were analyzed using the capture-mark-recapture (CMR) program MARK  which pr,..., k) .ĉ, was calculated from this procedure and used to correct AICc values [c [c, denoted QAICc [ER) [c weight of any one model divided by a simpler comparison model QAICc weight. The ER therefore estimates how many more times likely the model in question is over the model(s) to which it is being compared [We tested whether the CJS-model assumptions were met with parametric goodness-of-fit (GOF) tests implemented by the simulation procedures available in MARK . Here, ec values . Differealues [c ,78. We aed QAICc . Models ICc [ER) . The evicompared .We examined if there were any differences in survival probability between the sexes; there was little evidence for a difference (see Results), so the sexes were pooled. First-year survival is a good indicator of potential recruitment given that naïve elephant seals have the highest risks of dying compared to other age classes . Therefoa priori model sets to examine their relative support (weaning mass data for the entire dataset were unavailable). Two separate CJS model sets were constructed using these two expressions of the SOI conditions: (1) the first set employed the SOI values relating to the first-year seals' first foraging trip ; (2) the second set used the SOI corresponding to the environmental conditions prevailing during the mother's pre-partum foraging .We used annual averages of the Southern Oscillation Index (SOI)  to examiPublished counts of female seals on the isthmus during the breeding season were used to measure the effect of population density on survival . Both thϕ) is the logit-transformed survival estimate of a seal with the covariate x, β is the logit function calculated in MARK for x and SD is the standard deviation of x. This function is fixed in the log-likelihood for survival as in a logistic regression. This model assumes that there is an optimal value of the covariate and that there are some selective penalties associated with extreme values [where logit ranking of models examining the effects of density, age, sex, time and the Southern Oscillation Index (SOI) on apparent survival (ϕ) and recapture probability (p) of southern elephant seals (Mirounga leonina) at Macquarie Island with increasing values of over-dispersion (ĉ).Additional tables showing the capture-mark-recapture model rankings using increasing values of over-dispersion. The additional tables demonstrate the change in information-theoretic (Click here for file


The field of molecular evolution provides many examples of the principle that molecular differences between species contain information about evolutionary history. One surprising case can be found in the frequency of short words in DNA: more closely related species have more similar word compositions. Interest in this has often focused on its utility in deducing phylogenetic relationships. However, it is also of interest because of the opportunity it provides for studying the evolution of genome function. Word-frequency differences between species change too slowly to be purely the result of random mutational drift. Rather, their slow pattern of change reflects the direct or indirect action of purifying selection and the presence of functional constraints. Many such constraints are likely to exist, and an important challenge is to distinguish them. Here we develop a method to do so by isolating the effects acting at different word sizes. We apply our method to 2-, 4-, and 8-base-pair (bp) words across several classes of noncoding sequence. Our major result is that similarities in 8-bp word frequencies scale with evolutionary time for regions immediately upstream of genes. This association is present although weaker in intronic sequence, but cannot be detected in intergenic sequence using our method. In contrast, 2-bp and 4-bp word frequencies scale with time in all classes of noncoding sequence. These results suggest that different genomic processes are involved at different word sizes. The pattern in 2-bp and 4-bp words may be due to evolutionary changes in processes such as DNA replication and repair, as has been suggested before. The pattern in 8-bp words may reflect evolutionary changes in gene-regulatory machinery, such as changes in the frequencies of transcription-factor binding sites, or in the affinity of transcription factors for particular sequences. One of the foundations of molecular evolution is the idea that more closely related species are more similar on the molecular level. One example that has been known for several years is the genomic composition of short words  of DNA. Given a sample of genome sequence, one can count the occurrences of all words of a certain length. It turns out that closely related species have more similar word frequencies. The pattern of how these frequencies change over evolutionary time is likely to be influenced by the many functions of the genome . Bush and Lahn investigated the influence of genomic function on word-frequency variation in 13 animal genomes. Using a method designed to isolate the effects acting at particular word sizes, the authors examined how word frequencies vary in different categories of noncoding sequence. They found that interspecies patterns of word-frequency variation change depending on word size and sequence category. These results suggest that noncoding sequence is subject to different functional constraints depending on its location in the genome. An especially interesting possibility is that the patterns in longer words may reflect evolutionary changes in gene regulatory machinery. Biological macromolecules accumulate changes over evolutionary time. Because of this, molecular differences between species contain information about evolutionary history . FrequenAnother level of organization where this has been studied is the composition of short words in genomic DNA. Di- and trinucleotide frequencies are more similar in more closely related species ,5. PhyloHowever, evolutionary patterns in the frequencies of short words are also of interest because of what they reveal about function in the genome. Within genomes, word-frequency variation has been linked to functional variation –16. The An important challenge is to try to sort out the different processes that may be influencing word-frequency variation . Here weWe sought to develop a method for determining interspecies distances in word frequencies which would remove the effect of smaller constituent words. For example, when we compare two species in terms of their composition of 4-bp words, we want to do so while removing any effects due to variation in the frequencies of 1-bp words  and 2-bp words. Our method relies on the fact that 4-bp words can be divided into groups where each group consists of words that have the same GC content and dinucleotide frequencies. We call these iso GC/dinucleotide groups of 4-bp words. One example of such a group is the set TGAC, TGTC, and TCAC. In addition to having the same GC content, these words  share the same six dinucleotides. This is illustrated in Next we calculate a distance based on a comparison of word frequencies within these iso-groups. To illustrate, let us continue with the 4-bp iso GC/dinucleotide group given in Homo sapiens (human), Mus musculus (mouse), Canis familiaris (dog), Monodelphis domestica (opossum), Gallus gallus (chicken), Xenopus tropicalis (frog), Danio rerio (zebrafish), Tetraodon nigroviridis (pufferfish), Ciona intestinalis (sea squirt), Ciona savignyi (sea squirt), Drosophila melanogaster (fly), Anopheles gambiae (mosquito), Caenorhabditis elegans (worm). For each species we obtained equal amounts of genomic DNA from each of four categories: 1) promoter sequence 2 kb upstream to each gene, 2) intronic sequence, 3) randomly chosen intergenic sequence, and 4) coding sequence. For each category within a species we obtained just over 5.5 megabases of nonrepetitive sequence. We then exhaustively counted all 2-bp, 4-bp, and 8-bp words in this sequence. We used these counts to calculate IFW distances between all possible pairs of species for each of the four categories of sequence. The resulting distances are given in We applied our method to repeat-masked genomic sequence from 13 metazoan species. These species were chosen to provide a range of phylogenetic distances. They were: Our interest is in seeing how IFW distances vary over evolutionary time. To examine this, it is useful to compare with a variable such as the rate of protein evolution, which is strongly associated with the degree of relatedness between species. We therefore calculated the number of amino acid replacements per site for all possible pairs of species in our set. We used 89 sets of unique best-reciprocal-hit orthologs, each set consisting of one protein from each of our 13 species. For these we downloaded high-scoring segment pair (HSP) protein alignments from Ensembl and used them to calculate the number of amino acid replacements per site for all possible pairs of our 13 species. Consistent with previous observations, The most interesting result in p-value = 0.002). For 8-bp words in intronic sequence, the association is also present (p = 0.023). But in intergenic sequence, it is not significant using our methods (p = 0.262). This is in contrast to 2-bp and 4-bp words that have significant trends in all noncoding categories . Coding sequence also shows a significant association between IFW distance and amino acid replacements for 8-bp pair words (p = 0.005).We can test associations between distances statistically using a permutation test such as the Mantel test. For 8-bp words, the association between IFW distances and amino acid replacements is significant in promoter sequence . The species used were listed above. We used the Ensembl PERL API to download four different categories of genomic sequence. In all sequences, repeats were masked by Repeat Masker [For each of 13 metazoan species, we obtained genomic sequence from version 39 of Ensembl  that were annotated by Ensembl as one-to-one orthologs with each other. Then for each pairwise comparison in this set, we obtained the HSP protein alignment from Ensembl via the PERL API. We then calculated the amino acid replacement distances (corrected for multiple hits) between every two species using the 89 HSPs together. To do this we used PAML  with theWe tested the statistical significance of associations between IFW distances and amino acid replacements using the Mantel test with 100,000 permutations.http://genome.ucsc.edu/). We first made a custom track consisting of 2 kb upstream to RefSeq genes in each species (in Xenopus we used gene annotations from JGI). We then did a bp-wise intersection of this with the complement of the following tracks: species-specific and xeno mRNA, the species-specific ESTs, ensembl genes, simple repeats, and repeat masker. With this sequence we then repeated the analysis as described above.To make sure our result was not an artifact of surreptitious coding sequence, we repeated it with more stringent efforts to remove potential coding sequence. We did this in human, mouse, chicken, and frog, using the table browser Web site of the University of California at Santa Cruz Click here for additional data file.Table S1(123 KB PDF)Click here for additional data file.


Intrinsically fluorescent proteins have revolutionized studies in molecular cell biology. The parallel application of these proteins in dual- or multilabeling experiments such as subcellular localization studies requires non-overlapping emission spectra for unambiguous detection of each label. In the red spectral range, almost exclusively DsRed and derivatives thereof are used today. To test the suitability of the red fluorescent protein eqFP611 as an alternative in higher plants, the behavior of this protein was analyzed in terms of expression, subcellular targeting and compatibility with GFP in tobacco.When expressed transiently in tobacco protoplasts, eqFP611 accumulated over night to levels easily detectable by fluorescence microscopy. The native protein was found in the nucleus and in the cytosol and no detrimental effects on cell viability were observed. When fused to N-terminal mitochondrial and peroxisomal targeting sequences, the red fluorescence was located exclusively in the corresponding organelles in transfected protoplasts. Upon co-expression with GFP in the same cells, fluorescence of both eqFP611 and GFP could be easily distinguished, demonstrating the potential of eqFP611 in dual-labeling experiments with GFP. A series of plasmids was constructed for expression of eqFP611 in plants and for simultaneous expression of this fluorescent protein together with GFP. Transgenic tobacco plants constitutively expressing mitochondrially targeted eqFP611 were generated. The red fluorescence was stably transmitted to the following generations, making these plants a convenient source for protoplasts containing an internal marker for mitochondria.In plants, eqFP611 is a suitable fluorescent reporter protein. The unmodified protein can be expressed to levels easily detectable by epifluorescence microscopy without adverse affect on the viability of plant cells. Its subcellular localization can be manipulated by N-terminal signal sequences. eqFP611 and GFP are fully compatible in dual-labeling experiments. PCRs with one primer binding in the vector backbone and the other one in the CaMV 35S promoter or smGFP4 coding sequence in our psmGFP4 clearly show that the multiple cloning site is not orientated like in pUC118 and pUC18 but like in pUC119 and pUC19 (data not shown).The vector backbone of psmGFP4  has been reported to be based on pUC118 and to contain the sequence ggatccaaggagatataaca U70495] . Our plaThe absence of a 473 bp fragment in a digestion of the plasmid pIVD144-eqFP611 with RsaI (data not shown) rather indicates a pUC19-like instead of a pUC119-like configuration of the psmGFP4-derived vector-backbone.All PCRs were performed with BD Advantage™ 2 Polymerase Mix , Phusion™ High-Fidelity DNA Polymerase  or self-produced Taq polymerase, respectively. Amplifications were done in 22 to 35 cycles under conditions recommended by the manufacturer . Reactions with self-produced Taq polymerase were done following standard protocols .All PCR-derived DNA fragments were sequenced after cloning, except the RFP-expression cassette in pIVD144-eqFP611-IVD145-smGFP4. In this case, only the IVD144 mitochondrial presequence was analyzed by sequencing.PEG-mediated transient transfection of protoplasts was essentially carried out as described previously . For traNicotiana tabacum L., cv Petit Havana plants were generated essentially as described elsewhere [0, T1, T2 and T3 plants was followed by fluorescence microscopic analysis of parts of the lower epidermis of leaves.Transgenic lsewhere . ExpressN. benthamiana by leaf infiltration was performed as described before [Agrobacteria-mediated transformation of d before .A. tumefaciens was used for experiments requiring T-DNA transfer.Strain GV2260 of A Carl Zeiss Axioplan I microscope and the axiovision software  were used for visualization and documentation of eqFP611 and GFP fluorescence. The microscope was equipped with FITC (fluorescein isothiocyanate) (HQ 470/40/HQ 500 LP) and MitoTracker (HQ545/30/HQ 610/75) filter sets obtained from AHF  for GFP and eqFP611 analysis, respectively.JF designed and constructed the plasmids, carried out the microscopic analyses and drafted the manuscript.SB conceived and supervised the project and worked over the draft version of the manuscript.


Broadband recordings were filtered to yield LERG and multi unit as well as single unit spike signals. RFs were calculated from responses to multifocal pseudo-random spatiotemporal visual stimuli registered at the retinal surface by a 7-electrode array.Receptive fields of retinal neural signals of different origin can be determined from extracellular microelectrode recordings at the inner retinal surface. However, locations and types of neural processes generating the different signal components are difficult to separate and identify. We here report epiretinal receptive fields (RFs) from simultaneously recorded spikes and local electroretinograms (LERGs) using a semi-chronic multi-electrode en route to the optic disk while LERG-RFs are related to superimposed postsynaptic potentials of cells near the electrode tip.LERGs exhibit spatially unimodal RFs always centered at the location of the electrode tip. Spike-RFs are either congruent with LERG-RFs (N = 26/61) or shifted distally (N = 35/61) but never proximally with respect to the optic disk. LERG-RFs appear at shorter latencies  than those of spikes . Furthermore, OFF-center spike-RFs precede and have shorter response rise times than ON-center spike-RFs. Our results indicate that displaced spike-RFs result from action potentials of ganglion cell axons passing the recording electrode Besides contributing to the understanding of retinal function we demonstrate the caveats that come with recordings from the retinal surface, i.e., the likelihood of recordings from mixed sets of retinal neurons. Implications for the design of an epiretinal visual implant are discussed. This may have obscured the contributions of shifted axonal spike-RFs. Recently, Segev and coworkers [in vitro salamander retina. They mostly recorded somatic spikes and reported only rare observations of axonal spikes presumably due to the arrangement of axons in tight bundles. The few axonal RFs they observed were displaced with respect to the somatic RFs. We here present data of broadband epiretinal recordings in in vivo cat retina that allowed us to compare the spatio-temporal RFs of distinct spike sources and LERG signals measured simultaneously with the same microelectrode.The intact retina of animals and humans provides several sources of electrical responses to visual stimulation. Among them, the receptors, horizontal, bipolar, and to some extent the amacrine cells generate graded local potentials . Their doworkers  used mulThe superficial arrangement of ganglion cell axons is also expected to play a role in the design of an epiretinal visual implant ,16. Epir140, 302, and 033: right eye; 142: left eye). Each set of data was recorded using seven epiretinal microelectrodes simultaneously in one or more electrode array positions per experiment. RF characteristics for each electrode position were typically confirmed more than once but they only once contribute to the present study resulting in a total of 44 different retinal recording positions.We present data from experiments in four adult cats  as well as the automated RF mapping (colored blob) points at an upper left position within the RF map. We were able to estimate RFs for six of the seven retinal LERG signals in this example, all being congruent with the projected electrode positions. The location of electrode 5 was only mapped using our back-projection protocol together with manual RF mapping based on spike activity (cf. Methods).The receptive fields estimated for epiretinally recorded signals often exhibited multiple, spatially segregated peaks  comprises spikes from several neurons and exhibits multiple RFs that were found to be aligned along the estimated course of fiber bundles at the respective retinal eccentricity. This can be seen in Fig. Methods). We found that spikes separated by an amplitude threshold criterion belong to functionally distinct ganglion cells that differ in RF center locations, polarities, and/or time courses. We detected 81 spike- (or MUA-) RFs at 34/44 (77 %) of the recording positions. Thus, on average, we found about 1.8 spike-RFs per retinal recording position.Multi unit activity ) or shifted distally (N = 35/61 (57 %)) but never proximally with respect to the optic disk. This strongly indicates that displaced spike-RFs result from the recording of spikes from passing axons en route to the optic disk. This is illustrated in Fig. When LERG- and spike-RFs were detected simultaneously (in 28/44 (64 %) recording positions, 61 spike-RFs) we compared the relative position of RFs: Spike-RFs were either congruent with LERG-RFs (= 26/61 4 %) or shSpike-RFs were found at all distal retinal separations from the retinal electrode tip that were mapped by the visual stimulus. They were either of ON-type  or OFF-type , whereas LERG-RFs were always of OFF-type (N = 34).Methods). Only the broadband recordings were appropriate (N = 3 experiments) as they provided sufficient temporal resolution for the detection of small latency differences. In one experiment we lost the exact temporal information about LERG due to wrong filter settings reducing the number of experiments with broadband recorded LERG to two (N = 18 LERG-RFs). Broadband recorded spikes were available in three experiments (N = 53 spike-RFs).Apart from the spatial aspects of retinal RFs we also analyzed their temporal characteristics to further elucidate the origin of LERG- and shifted spike-RFs. For this we plotted the averaged time course of the retinal response to stimulation within the respective RF center (this is the stimulus-response cross-correlation) and calculated the response latency . Both spike signals comprise two components as is evident from the two RFs in each map. The spatial shift of the spike-RFs with respect to the projected electrode tip position (crosshair) is to the left as the recording was made paracentrally in the right eye. The averaged time courses of the retinal signals in response to stimulation in their respective RF center (stimulus grid coordinates given in the lower right corners) are shown in Figure Typical data from two experiments are presented in Fig. 142, left eye). Here the left spike-RF map comprises three components (two OFF and one ON) and the right map only one (ON) as is evident from the peaks in the correlation maps. The spatial shift of spike-RFs with respect to the projected actual electrode position (crosshair) is now to the right as the recording was made paracentrally in the left eye. The latencies are 15.9 and 16.5 ms for the OFF-responses and 17.4 and 16.9 ms for the ON-responses. OFF-responses again evolve in a stepwise fashion with small rise times, whereas ON-responses evolve more smoothly with longer rise times. Moreover, the rightmost ON-response has two ON-correlation peaks. We found such double-ON-peaks in 5–7 of 53 spike-RFs .Similar results can be derived from Figure local RFs  or pooled local and distal RFs . This is shown in Figure local RFs (brown). Spike responses with distal RFs tended to peak later than those with local RFs most probably due to the additional axonal conduction time. Therefore, LERG latencies can best be compared with latencies of local spikes with latency differences of 5.4 ms ± 0.9 ms  = 6.23, p < 10-6).LERG responses generally had shorter latencies  when compared to spike-responses with local RFs) than ON-spike latencies . Latencies differed insignificantly by 1.9 ms ± 1.4 ms (t (15) = 1.35, p < 0.20). We estimated congruent ON- and OFF-spike RFs simultaneously with the same electrode at four recording positions. The mean latency difference in these cases was 2.4 ms ± 0.8 ms . The OFF-spike RFs temporally preceded the ON-spike RFs in all cases.We further compared the response latencies for ON- and OFF-spikes. OFF-spike latencies were shorter , the averaged time courses of spike OFF-responses to stimulation in the respective RF center appeared stepwise  OFF-responses appeared barely stepwise with rise times not significantly different (ON: 3.3 ms ± 0.1 ms (N = 25), OFF: 3.7 ms ± 0.5 ms (N = 5)).In two of the three experiments with broadband recordings 12, 033, tise Fig. . In the A general prerequisite for the generation of LERGs is the constructive superposition of extracellular field currents of individual neurons and glia cells ,18. Parta-wave, [The intact retina as used in our study provides several sources of graded local potentials, namely the receptor (outer nuclear layer), horizontal, bipolar, and to some extent the amacrine cells (inner nuclear layer) . It is wa-wave, ). OFF-bia-wave,  and perha-wave,  add to ta-wave, . Thus, aa-wave,  argue tha-wave, ,19.All LERG-RF time courses showed negative initial peaks that were sometimes followed by a positive deflection (N = 34). Thus, field potentials were negative-going after ON-stimuli ("1" in the stimulus time course) and/or positive-going after OFF-stimuli ("-1" in the stimulus time course). This is typical of recordings with the recording electrode positioned intraretinally rather than in the vitreous humour ,24.The LERG yielded RFs that were always located at the position of the electrode tips projected to visual space. This strongly suggests that LERG are generated near the electrode tip. Interestingly, the localized nature of the LERG suggests that their RFs can be used as spatial reference points for the analysis of spike-RF positions. This can be very useful in experiments involving many retinal electrodes where the back-projection technique for the analysis of electrode positions would be cumbersome. However, a prerequisite is that the electrodes do not occlude the incoming light from the retina. The seven fiber electrodes in our setup were optically diffused as they were very thin and lying in a different focal plane than the photoreceptors. Their presence therefore had only minimal influence on the retinal light intensity distribution of the stimulus.Another slow negative potential that can be invoked in the dark-adapted state with very dim light stimuli is the scotopic threshold response (STR) . Howeverfollow the spikes, instead LERG signals have shorter latencies than spikes. Secondly, LERG-RFs would have to be located at the same positions as the RFs of (large) spikes, which often was not the case.We can further exclude that LERGs are merely artifacts due to impulse responses of the hardware filter to large action potentials because firstly, due to the causality of the used filters, LERG signals then needed to local RFs, 43 % of 61 spike-RFs) or tended to line up in a way that correlated well with the estimated fiber trajectories . This strongly indicates that displaced spike-RFs result from the recording of axonal spikes originating at more distal locations along the fiber bundle and passing the recording electrode en route to the optic disk. It is not surprising that we registered axonal spikes since axons were in much closer proximity to the recording electrodes than the ganglion cell somata in situations where the electrode tip just touched the inner limiting membrane [The RFs of single unit spikes simultaneously registered by the same electrode were either located at the site of the corresponding LERG-RF of this electrode , it was a challenge to map s . Higherlocal RFs). In these cases, however, LERG-RFs appeared at shorter latencies than spike-RFs. Since for local RFs optimal stimulation was done with the same stimulus pixels , we can exclude stimulus timing issues as an explanation for the latency difference.Spikes and LERGs simultaneously registered with the same electrode often had congruent RFs  cells, comparable to cat Y-cells) including response kinetics. In particular they found that ON cells had a 10–20% shorter time-to-peak, trough and zero-crossing in the biphasic temporal impulse response than OFF cells. However, they also note that the response latency which they estimated as the time to 5% of the peak response was on average 1–2 ms shorter for OFF cells than for ON cells. The latter is in good accord with our estimation of a 1.9 ms latency difference between ON and OFF responses (time to 70% of peak response). The reason why we did not analyze the response peak times in more detail is that we found the OFF peaks to exhibit stepwise time courses that deemed it inappropriate to define a peak time because of their flat peak plateau . Here, rise times for ON- and OFF-responses were similar and both longer than for stepwise OFF responses. From the limited number of observations we cannot deduce why OFF-responses were stepwise in some recordings and not in others.This asymmetry in the retinal ON/OFF pathways may also be accountable for the stepwise time course of cross-correlation functions between stimulus and OFF-responses in two of the three experiments with broadband recordings Fig. . Here, Olocal and distal RFs one can in principle deduce the spike conduction time as the difference of their response latencies. This is only true for RF-pairs with the same polarity as we argued that the signal transduction kinetics for ON- and OFF-responses differ and thus add different temporal delays to the overall signal latency. Response latencies varied considerably  and further indicates that the source of the LERG response to visual stimulation must be precedent to spike initiation.Based on the knowledge of spike responses recorded by the same electrode with bly Fig. , though,d Levick  found thut 1 m/s , the obsOur results may have important implications for the design of epiretinal implants that aim at substituting retinal function by eliciting localized visual percepts ("phosphenes") via focal electrical stimulation ,16,33. ROur data demonstrate multi-site epiretinal RFs suggesting that epiretinal electrical stimuli might unspecifically activate retinal axons that originate in an elongated area of the retina distal to the stimulation site, potentially resulting in multi-site or elongated visual percepts. One way of dealing with multiple axonal stimulation would be to reduce the distance between electrode and ganglion cell somata by advancing small-tipped cone electrodes to the cell body layer. This would increase the probability of activating ganglion cell somata rather than axons. One could in principle test RFs of neurons from "quasi-intraretinal" recordings for contributions of multiple sources and adjust the recording depth until only a local component remains. We therefore tested the retinal RFs' dependence on the recording depth in one pilot measurement: We measured four LERG-RFs and one corresponding spike-RF. The LERG-RFs exhibited a slight latency decrease with penetration depth (appr. 5–10 ms/100 μm). Additionally, the polarity of LERG-RFs abruptly reversed at a certain recording depth (appr. 250 μm from the inner limiting membrane) which was accompanied by an increase in LERG-RF size. This is a typical situation if the choroidal side of Bruch's membrane is penetrated . In contMcIntyre & Grill  approachWhether or not epiretinal electrical stimulation yields multi-site visual sensations must ultimately be tested in alert subjects that can report evoked visual percepts. Very promising data have been collected by Humayun and coworkers  who implOur data demonstrate the feasibility of obtaining highly specific receptive field data from different neural sources by epiretinal broadband recordings with microelectrodes. We could separate distinct retinal signals from the epiretinal broadband recordings which enabled us to study the RF properties of simultaneously recorded signal components from different retinal sources. The caveats that come with recordings from the retinal surface, i.e., the likelihood of mixed contributions from different retinal neurons, may also have implications for the design of an epiretinal visual implant.2O / O2 (70 % / 30 %) and isofluorane (0.5 – 1.5 %). The level of anaesthesia was continuously controlled by monitoring the rectal temperature (38°C), endexpiratory CO2 (3.8 – 4.2 %), ECG, EEG, and muscle reflexes. For head fixation we implanted two bolts in cavities of the forehead by dental acrylic cement that enabled positioning in a standard Horsley-Clark support without pressure points typically induced by the use of ear bars. The operated eye was stabilized with the help of the electrode array and two sutures that were pierced through the conjunctiva and fixed to the Horsley-Clark support. Refraction of the eyes was corrected for the eye-to-monitor distance of 1.3 m using contact lenses. Besides during the ophthalmic surgery it was not necessary to give further analgesia between recording sessions as was assured by monitoring the cat's vital functions. One of the four cats was not sacrificed at the end of the recording sessions. In this case, sclera and conjunctiva were closed with polyglactin sutures and the animal prophylactically received penicillin. This semi-chronic preparation enabled repeated experiments in the same eye after allowing 2–3 weeks for convalescence. At the end of the other three experiments the animals were deeply anesthetized with isofluorane (3 %) followed by an intravenous injection of a lethal dose of T61 . All animal experiments were conducted in accordance with the German animal welfare law, the guidelines of the European Community Council Directives (86/609/EEC) and the NIH Principles of Laboratory Animal Care .For semichronic preparation adult cats  received atropine sulphate (0.03 – 0.04 mg/kg) to reduce salivation. Anaesthesia was induced by intramuscular injection of a mixture of ketamine hydrochloride  and xylazine hydrochloride . After orotracheal intubation, anaesthesia was maintained by ventilation with N302 and 033). The electrodes were hexagonally arranged to provide for a two-dimensional recording and to minimize the scleral opening by a dense packing of electrodes . Retinal electrodes could be individually moved in axial direction under computer control. This allowed for precise epiretinal positioning of the electrode tips under visual inspection with an ophthalmoscope. Additionally, the whole electrode manipulator could be spherically moved around the electrodes' point of insertion into the eye so that a wide range of retinal positions could be approached without mechanical stress to the eye [Epiretinal recording was performed with fiber microelectrodes  with con the eye . The tip the eye .2 were continuously monitored during the experiments. Data were recorded and considered for analysis only when vital functions were normal and stable. Between the measurement sessions, all electrodes were individually retreated to a safe distance in the vitreous body, the electrode array was moved to another retinal site, and the electrodes individually positioned on the retinal surface again. This took at least 30 minutes. The typical interval between sessions was 60 minutes.Vital functions of the cat, including ECG, EEG, body temperature, and expiratory CO140) we hardware-filtered the retinal signals to obtain local electroretinograms  and multi unit activity . These filtered signals were then sampled at 500 Hz  and stored for offline data analysis. Since we wanted to separate single unit spike trains contained in the raw signal we recorded broadband signals (1–4000 Hz) at 20 kHz sampling rate  in all subsequent experiments . For the sake of comparison with our first data set (140), LERGs were then extracted off-line by convolving the broadband signals with the impulse response of our laboratory hardware filter. Using an amplitude thresholding approach enabled us to extract spikes from high-pass filtered (0.5–1 kHz) broadband data: First, local signal peaks of a certain range of widths were detected by the analysis of the first and second derivatives of the signal time series. In a second step, the peaks were sorted into ranges of amplitudes and stored into separate event data files. However, it cannot be ruled out that one sorted spike population comprised more than one spiking neuron.In our first experiment (2 and the luminance of the black pixels 0.1 cd/m2). The multi-focal pseudo-random stimuli were based on binary m-sequences with 4095 steps [140) or 15 times  to improve averaged cross-correlations between stimulus and response. The temporal resolution of the stimulus was limited by the monitor frame rate (9.9 ms frame duration). We therefore resampled the stimulus and signal time courses at 50 times the stimulus' temporal resolution  prior to calculating the cross-correlation functions in order to provide for sufficient temporal interpolation.Monocular RFs were qualitatively evaluated with a hand-held projector by stimulating with an adjustable light bar on a tangent screen 1.3 m in front of the cat. This distance also left enough room to quickly access the eyes for medical treatments between the recording sessions. For the quantitative characterization of the position, size, subfields, and orientation of RFs we used an automated mapping technique in which the luminance of a computer monitor was randomly spatio-temporally modulated covering the RF positions of all retinal recording locations simultaneously . The RF center positions were estimated from the peak maximum of 2-dimensional RF maps . Once the RF center was located, we analyzed the time course of the RF as given by the cross-correlation function among the time course of the optimal stimulus pixel and the retinal signal. In order to improve the signal-to-noise ratio of the estimated RF time course, we averaged the time courses for the optimal stimulus pixel and its adjacent eight stimulus pixels. This was possible since most RFs extended over more than one stimulus pixel width. Visual RF center positions were compared by superposition in a common frame of reference in visual and retinal space Fig. .The polarity of a RF (ON- vs OFF-center neuron) was defined as the polarity of the first major signal deflection in the cross-correlation function. Moreover, we calculated the time elapsed from the stimulus onset to 70% of the RF peak amplitude as a measure for the retinal response latency. We also analyzed the steepness of the rising and falling phase of the time course for averaged ON and OFF responses, respectively. As a reliable measure for this we used the signal rise time from 30% to 70% of the RF peak amplitude.The calculated RF center positions were compared to the back-projected electrode tip positions. This way we were able to check for any deviations of functional RF positions from the geometrically expected ones. Based on the work of Stone & Holländer , we furtMW performed the experiments, analyzed the data, and drafted the manuscript. RE contributed to experiment conception, design, and the critical revision of the article.


Begomovirus) are phytopathogens that cause heavy losses to crops worldwide. Efforts to engineer resistance against these viruses are focused mainly on silencing of complementary-sense virus genes involved in virus replication. Here we have targeted a virion-sense gene (AV2) to develop resistance against Tomato leaf curl New Delhi virus, a bipartite begomovirus prevalent throughout the Indian subcontinent. We show that tobacco plants transformed with an antisense construct targeting this gene are resistant to the virus. Following challenged with the virus, transgenic plants remained symptomless, although viral DNA could be detected in some plants by PCR. This is the first report of transgenic resistance against a bipartite begomovirus obtained by targeting a virion-sense gene. The relatively conserved nature of the gene suggests that the technology may be useful to develop broad-spectrum resistance which is required because of the fact that plants are often infected with multiple begomoviruses in the field.Whitefly-transmitted geminiviruses (genus  Geminiviridae) are a group of phytopathogenic viruses having circular, single-stranded (ss) DNA genomes packed within twinned quasi-icosahedral (geminate) particles. These viruses replicate in the nucleus and are transmitted by insect vectors. Vector specificity for transmission is determined by the coat protein [Begomovirus. These viruses have either two genomic components (DNA A and DNA B) or a single genomic component equivalent to DNA A. Some geminivirus diseases in the Old World are caused by monopartite begomoviruses which require a ssDNA satellite, known as DNA β, to infect and cause symptoms in the hosts from which they were isolated [The geminiviruses , a bipartite begomovirus that occurs widely across the Indian subcontinent in a number of hosts [Begomoviruses are amongst the most devastating phytopathogenic viruses and cause great yield losses throughout the world, particularly in warmer climates that are more suitable for their whitefly vector, reported . Most ofof hosts .U15015) [GGATCCTGGCTATACATTCTGTACAT-3') and ASR (5'-AGTCTAGAATGTGGGATCCATTATTG-3') having BamHI and XbaI restriction endonuclease recognition sequences (underlined) were used in PCR reactions containing the ToLCNDV DNA A clone as the template. The resulting 339 bp PCR product was cloned into a plasmid vector and sequenced. Subsequently, the fragment was directionally cloned at BamHI and XbaI sites into the expression vector pFGC5941 [Agrobacterium tumefaciens strain LBA4404 by electroporation. The transgene and agroinfiltrated gene constructs were sequenced to confirm the integrity of the inserts. Nicotiana tabacum (cv. Samsun) was transformed by the Agrobacterium-mediated leaf disk method [Cauliflower mosaic virus (CaMV) 35S promoter. The number of integration sites of the transgene in plants was determined by genomic Southern hybridization. For this purpose total DNA isolated from six transgenic plants was digested with EcoRI (for which there is only a single recognition sequence within the construct) and Southern blotted to nylon membranes. A α32PdCTP-labelled probe of ToLCNDV AV2 gene was prepared by the oligo-labelling method and hybridized to the membranes. All six plants tested showed the presence of the transgene with one to two copies  . The prik method . The puts Figure .32PdCTP-labelled probe of ToLCNDV DNA A. A semi-quantitative PCR was also done to assess the relative level of viral DNA for plants in which viral DNA could not be detected by Southern hybridization. The primers used, NSPf (5'-GTCCCGGGTATGTCACGTATCCATCA-3') and NSPr (5'-GTATCGATTTATCCAATGTAATTAAGAAT-3'), were designed to amplify the DNA B-encoded nuclear shuttle protein gene and amplify an approximately 550 bp fragment [Transformed and non-transformed plants were inoculated with the cloned components of ToLCNDV by agroinfiltration, as described previously . Replicafragment .N. tabacum plants, primary transformants resulting from independent transformation events, were selected randomly for further evaluation. All tested plants were positive for the presence of the transgene as well as the CaMV 35S promoter. The presence of the transgene had no apparent adverse effect on the growth of plants, which were morphologically normal and indistinguishable from non-transformed plants. 10 T0 transgenic and 5 non-transgenic N. tabacum plants were agroinfiltrated with ToLCNDV components and maintained in an insect-proof glasshouse. All 5 non-transgenic plants developed symptoms typical of ToLCNDV, consisting of leaf curling and stunting, within three weeks of inoculation  and serial dilutions of the template DNA (Ten fold dilutions). These analyses detected the presence of virus in four of the ten transgenic plants, but only at low dilutions (high DNA concentrations); whereas in non-transgenic infected plants viral DNA was detected at all dilutions Figure . These rIndian cassava mosaic virus (ICMV), another bipartite begomovirus from the Indian subcontinent, is involved in virus spread to neighbouring cells [The aim of this study was to explore the possibility of obtaining resistance to ToLCNDV by silencing the virion-sense AV2 gene. ToLCNDV encodes two genes in the virion-sense, of which only AV2 is essential for the movement of the virus in plants . A recenng cells , whereasng cells ,14.The AV2 sequence is well conserved among begomoviruses from the Old World . This suThe authors declare that they have no competing interests.MM and MH conducted all the experiments. SM wrote the manuscript and coordinated the research efforts. YZ conceived the study and edited the paper.


The nucleolus is a subnuclear organelle in which rRNAs are transcribed, processed, and assembled with ribosomal proteins into ribosome subunits. Mass spectrometry combined with pulsed incorporation of stable isotopes of arginine and lysine was used to perform a quantitative and unbiased global analysis of the rates at which newly synthesized, endogenous proteins appear within mammalian nucleoli.Newly synthesized ribosomal proteins accumulated in nucleoli more quickly than other nucleolar components. Studies involving time-lapse fluorescence microscopy of stable HeLa cell lines expressing fluorescent-protein-tagged nucleolar factors also showed that ribosomal proteins accumulate more quickly than other components. Photobleaching and mass-spectrometry experiments suggest that only a subset of newly synthesized ribosomal proteins are assembled into ribosomes and exported to the cytoplasm. Inhibition of the proteasome caused an accumulation of ribosomal proteins in the nucleus but not in the cytoplasm. Inhibition of rRNA transcription prior to proteasomal inhibition further increased the accumulation of ribosomal proteins in the nucleoplasm.Ribosomal proteins are expressed at high levels beyond that required for the typical rate of ribosome-subunit production and accumulate in the nucleolus more quickly than all other nucleolar components. This is balanced by continual degradation of unassembled ribosomal proteins in the nucleoplasm, thereby providing a mechanism for mammalian cells to ensure that ribosomal protein levels are never rate limiting for the efficient assembly of ribosome subunits. The dual time-lapse strategy used in this study, combining proteomics and imaging, provides a powerful approach for the quantitative analysis of the flux of newly synthesized proteins through a cell organelle. Ribosomes play a key role in gene expression, and their production is tightly linked with mechanisms for cellular-growth control -based proteomics and technologies for live-cell fluorescence imaging have revolutionized the study of protein dynamics. For example, hundreds of proteins can be identified in parallel with sensitive mass spectrometers for detection and analysis of individual peptides in complex protein mixtures. Quantitative proteomic technologies, such as SILAC In this study, we have used a dual strategy that combines the power of both quantitative MS and live-cell fluorescence imaging to investigate the dynamic behavior of nucleolar proteins in mammalian cells. Our data have uncovered a mechanism that controls the participation of rproteins in ribosome-subunit assembly.RPL27-GFP cells is predominantly in the nucleolus and cytoplasm  at its carboxy terminus (RPL27-GFP). The steady-state GFP fluorescence signal in HeLaytoplasm  online, e stages B and groe stages C, with nRPL27-GFP is detected as a single band of the expected size 45kDa; D. AdditiRPL27-GFP nuclei was photobleached, and nucleolar fluorescence was measured over a 2 hr time course  and five stably expressing FP-tagged non-ribosomal nucleolar proteins . To measure their respective rates of synthesis and nucleolar targeting, we uniformly photobleached entire cells and measured recovery of nucleolar fluorescence . RecoverRPL27-GFP cells post mitosis (ensuring that both cells were at the same cell-cycle stage) and performed photobleaching to selectively remove either the nuclear or cytoplasmic GFP fluorescence  and SILAC MS RPL27-GFP cells  fitted with an environmental chamber (Solent Scientific) so that temperature at 37°C was maintained and fitted with a CoolMax charge-coupled device camera (Roper Scientific). Detailed protocols for live-cell imaging are described in the RPL27-GFP cells were grown in SILAC medium with dialyzed serum as described 12C6 arginine and 12C6 lysine medium with pre-equilibrated 13C6 arginine and 13C6 lysine medium for 30, 60, 120, 240, 480, and 720 min. Nucleoli were isolated directly from time-course-labeled cells as described HeLa and HeLa


Hepatocyte and keratinocyte growth factors are key growth factors in the process of alveolar repair. We hypothesized that excessive alveolar destruction observed in lung emphysema involves impaired expression of hepatocyte and keratinocyte growth factors or their respective receptors, c-met and keratinocyte growth factor receptor. The aim of our study was to compare the expression of hepatocyte and keratinocyte growth factors and their receptors in lung samples from 3 groups of patients: emphysema; smokers without emphysema and non-smokers without emphysema.Hepatocyte and keratinocyte growth factor proteins were analysed by immunoassay and western blot; mRNA expression was measured by real time quantitative polymerase chain reaction.Hepatocyte and keratinocyte growth factors, c-met and keratinocyte growth factor receptor mRNA levels were similar in emphysema and non-emphysema patients. Hepatocyte growth factor mRNA correlated negatively with FEV1 and the FEV1/FVC ratio both in emphysema patients and in smokers with or without emphysema. Hepatocyte and keratinocyte growth factor protein concentrations were similar in all patients' groups.The expression of hepatocyte and keratinocyte growth factors and their receptors is preserved in patients with lung emphysema as compared to patients without emphysema. Hepatocyte growth factor mRNA correlates with the severity of airflow obstruction in smokers. Emphysema is one of the most important cause of respiratory insufficiency with increasing mortality and morbidity . Lung emThe alveolar epithelium is essential for maintenance of the integrity of the alveolar spaces. Functional restoration of the alveolar epithelium after an injury requires the proliferation and migration of type 2 pneumocytes and their differentiation into type 1 pneumocytes, a tightly regulated phenomenon. Growth factors have been shown to control both phases of the process -12. AmonWe hypothesized that a defective expression of growth factors involved in human alveolar epithelium repair such as HGF and KGF or their specific receptors might participate in the pathophysiology of lung emphysema. We therefore evaluated the expression of HGF, KGF, and their receptors c-met and KGF-R in lung biopsies from patients with emphysema and from non-emphysema patients, according to their smoking status.This study was approved by the ethics committee of Saint Germain-en-Laye hospital.Lung samples were obtained during surgery in adult patients (>18 years) from Beaujon university hospital  and from Bichat university hospital .Seventeen patients with radiographically defined emphysema (E group) were included. All patients were active or ex-smokers Table . CT-scanNormal tissue was obtained from 18 non-emphysema patients. Eight patients were smokers  and 10 were non-smokers . S patients were undergoing surgery for the resection of a localised primary lung carcinoma (n = 7) or a benign lesion (n = 1). NS patients were undergoing surgery for the resection of a localised primary lung carcinoma (n = 3), lung metastases n = 2) or a benign lesion (n = 5). Tissue samples were taken at a site distant from the pathological process and without macroscopical and microscopical evidence of emphysema. S patients had mild to moderate alterations of pulmonary functions tests (table  or a ben3) were immediately frozen in liquid N2 and stored at -80°C until RNA and protein analysis. The histopathology of biopsies was evaluated on paraffin-embedded sections to verify the features of emphysema or normal lungs. The concentration of proteins in biopsies was evaluated from 100 mg of lung samples homogenised with 0.5 ml PBS containing 200 μM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin and 1 mg/ml aprotinin. The homogenates were centrifuged at 10,000 g for 10 min at 4°C to remove tissue fragments and the supernatants were collected and stored at -80°C until measurement.Lung tissue fragments  with specific primers table  to quant®, R&D Systems; respective detection limits were 40 and 15 pg/ml for HGF, KGF) were measured in lung homogenates from 26 out of 35 patients when enough lung sample was available .The proteins HGF and KGF (QuantikineLung homogenates from 4 patients (2 patients with emphysema and 2 patients without emphysema) were examined by Western blotting as previously described .Data were analysed by Statview software  and displayed as mean ± SD. Between-group differences were first assessed by non-parametric analysis of variance . In the case of global significant difference, between two groups comparisons were assessed by the non-parametric Mann-Whitney U-test. Correlations were assessed by the Spearman's rank order test. Categorical data were analysed using the Chi-squared test. A p value < 0.05 was regarded as significant.KGF mRNA figure  and KGF KGF-R mRNA figure  was deteKGF mRNA, KGF protein and KGF-R mRNA did not correlate with age, cumulative tobacco exposure, period since smoking cessation, use of inhaled corticosteroids, pulmonary function tests and arterial blood gases.Although a considerable variability of HGF mRNA expression was observed, no difference was found between emphysema and non-emphysema groups figure . In emphHGF protein was detected in lung homogenates from all patients as assessed by immunoassay. HGF concentration was not different between groups figure . HGF proHGF-R mRNA was detected in all patients. We found no difference between groups figure . Strong The involvement of KGF and HGF in lung repair has been widely documented. Numerous studies in vitro and in vivo have demonstrated that KGF and HGF have protective effects in experimental lung injury ,23. To oThis study has some methodological limitations. A limited number of patients was studied in each group, especially in non emphysema groups which were mostly composed of lung biopsies obtained at a site distant from localised carcinoma. Furthermore, the patients could only be evaluated at one time point in the course of their disease. Inclusion of smokers without emphysema allowed the differentiation of emphysema-related and tobacco-related events. Because only one tissue sample from surgically resected material was available for examination, the expression of HGF, KGF and their receptors reflects regional disease activity and may be unrepresentative of the entire lung. Indeed, it is well known that emphysema affects different lung regions to a varying extent. Moreover, we evaluated HGF and KGF in lung homogenates only. Future studies should address the expression of HGF and KGF in a more cell-specific fashion.In our study, although HGF mRNA lung expression was similar in emphysema and non emphysema patients, a correlation was found between HGF mRNA and the deterioration of pulmonary function tests in emphysema patients. The correlation between airflow obstruction and HGF mRNA level was similarly observed when all smokers with or without emphysema were studied, suggesting that emphysema was not a main determinant of HGF mRNA level in the lung. This strong correlation between airflow obstruction and HGF mRNA in smokers suggests that the increase of HGF mRNA was not related to the presence of emphysema but rather to the degree of airflow obstruction. This observation is supported by the correlation between HGF protein in lung homogenates and the FEV1/FVC ratio in our population. These results are in agreement with the observations of Sauleda et al, who reported that HGF protein concentrations were increased in broncho-alveolar lavage of patients with chronic obstructive pulmonary disease as compared to smokers and non-smoker controls . InteresThe mechanisms underlying the correlation between airflow obstruction and HGF mRNA in smokers are unclear. Although speculative, we can propose that the mechanical constraints applied to alveolar tissue secondary to airflow obstruction may stimulate HGF production by alveolar epithelial cells, since Yamamoto et al showed that mechanical stretch induced HGF in alveolar type II cells in vitro . FurtherAs HGF and KGF are key factors in the process of alveolar repair , we suggRecently, HGF has been shown to stimulate pulmonary regeneration and to improve pulmonary function in animal models of elastase-induced lung emphysema ,19. PresThe main results of our study are that: i) KGF and HGF lung expression is preserved in emphysema patients, ii) HGF-R and KGF-R mRNA are consistently expressed in the lung of emphysema patients and are not modified by the smoking status, iii) HGF mRNA correlates with the severity of airflow obstruction in smokers.α1-AT: α1-antitrypsinE: emphysemaFEV1: forced expiratory volume in one secondFGF: fibroblast growth factorFVC: forced vital capacityHGF: hepatocyte growth factorKGF: keratinocyte growth factormRNA: messenger ribonucleic acidNS: non-smoker without emphysemaRT-PCR: reverse transcriptase-real-time polymerase chain reactionRV: residual volumeS: smoker without emphysemaSD: standard deviationTLC: total lung capacityThe author(s) declare that they have no competing interests.MB and AB equally participated in the design of the study, conducted the majority of the research experiments and drafted the manuscript.VL participated in the majority of the research experiments.MF participated in the design of the study.JM and PS conducted some experiments.GL participated in the design of the study.MD and BC conceived of the study, participated in its design, and in drafting the manuscript.All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:


Chronic arsenic exposure causes vascular diseases associated with systematic dysfunction of endogenous nitric oxide. Replacement of heavily arsenic-contaminated drinking water with low-arsenic water is a potential intervention strategy for arsenosis, although the reversibility of arsenic intoxication has not established. In the present study, we examined urinary excretion of cyclic guanosine 3′,5′-monophosphate (cGMP), a second messenger of the vasoactive effects of nitric oxide, and signs and symptoms for peripheral vascular function in 54 arsenosis patients before and after they were supplied with low-arsenic drinking water in an endemic area of chronic arsenic poisoning in Inner Mongolia, China. The arsenosis patients showed a marked decrease in urinary excretion of cGMP , and a 13-month period of consuming low-arsenic drinking water reversed this trend  and improved peripheral vascular response to cold stress. Our intervention study indicates that peripheral vascular disease in arsenosis patients can be reversed by exposure cessation and has important implications for the public health approach to arsenic exposure. The consumption of water contaminated by naturally occurring arsenic poses a serious threat to human health worldwide . Arsenicl-arginine by NO synthases is involved in many physiologic and pathophysiologic processes  were present in tube-well water from the end of the 1970s to August 1999. At this point, an alternative community water supply with a drastically lower arsenic level (37 μg/L) was installed. The investigations were conducted in August 1999, just before the new water system was installed, and again in September 2000, 13 months after the switch to less-contaminated drinking water. We obtained informed consent from all participants. Two certified dermatologists and two trained physicians performed physical examinations and administered a standardized questionnaire interview at both time points. A total of 54 volunteer residents  who participated in the two surveys in 1999 and 2000 and provided urine samples were enrolled in the present study. Some patients were excluded from this study because of unclear exposure history (14 cases) or because they provided no urine samples in 2000 (10 cases). Before arsenic remediation, 29 of the subjects were identified as having skin lesions typical of arsenosis, which include verrucous hyperkeratoses of the palms and soles of the feet and hypo- and hyperpigmentation in the trunk area . These sWe evaluated peripheral vascular response to cold stress by the difference of finger systolic blood pressure before and after ice-water immersion, which made the surface temperature of the finger decrease by 10ºC. Finger systolic blood pressure was determined by a finger blood pressure monitor . We measured skin temperature using a Tele-thermometer .We determined arsenic levels in water and biological samples by atomic absorption spectrophotometry  according to our previously reported method . The det125I-labeled cGMP radioimmunoassay kit  with a detection limit of 256 fmol/ml to measure urinary cGMP. The interassay coefficient of variation was 3.2% (n = 8). To control the differences in renal function, we divided the urinary excretion of cGMP by the urinary creatinine concentration (expressed in nanomoles cGMP per millimoles of creatinine) (We used an atinine) . Urinaryt-test. A value of p = 0.05 was considered statistically significant.Data are expressed as mean ± SEM in all cases. Comparisons between data obtained before and after the water switch were performed with a two-tailed, paired Student’s n = 510; 40–65 years of age) and 70.6 ± 3.0 in females  (As shown in  of age) .In agreement with the recovery of the arsenic-induced dysfunction of the NO/cGMP system, as indicated by the increase in urinary cGMP excretion, peripheral vascular response to cold stress, measured as the difference in finger systolic blood pressure before and after ice-water immersion, was significantly improved in male arsenosis patients . In femaReplacement of drinking water heavily contaminated with arsenic with low-arsenic water is a potential intervention strategy to minimize or reverse the adverse health effects of this toxic inorganic element. Consistent with our previous results , male anIn conclusion, a 13-month arsenic exposure reduction effectively reverses the arsenic-induced impairment of the NO/cGMP pathway in both males and females and improves peripheral vascular function in males. Additional comprehensive follow-up studies are necessary to document the long-term health benefits of arsenic exposure reduction, but the present results indicate the reduction of arsenic exposure could be an important public health strategy.


Haemophilus influenzae type B (DTP-IPV-Hib) immunization to preterm infants. The effect of the recent substitution of acellular pertussis vaccine for whole cell pertussis vaccine on the frequency of these events requires further study.Adverse cardiorespiratory events including apnea, bradycardia, and desaturations have been described following administration of the first diphtheria-tetanus-pertussis-inactivated polio-Infants with gestational age of ≤ 32 weeks who received their first DTP-IPV-Hib immunization prior to discharge from two Edmonton Neonatal Intensive Care Units January 1, 1996 to November 30, 2000 were eligible for the study. Each immunized infant was matched by gestational age to one control infant. The number of episodes of apnea, bradycardia, and/or desaturations (ABD) and the treatment required for these episodes in the 72 hours prior to and 72 hours post-immunization (for the immunized cohort) or at the same post-natal age (for controls) was recorded.Thirty-four infants who received DTP-IPV-Hib with whole cell pertussis vaccine, 90 infants who received DTP-IPV-Hib with acellular pertussis vaccine, and 124 control infants were entered in the study. Fifty-six immunized infants (45.1%) and 36 control infants (29.0%) had a resurgence of or increased ABD in the 72 hours post-immunization in the immunized infants and at the same post-natal age in the controls with an adjusted odds ratio for immunized infants of 2.41  as compared to control infants. The incidence of an increase in adverse cardiorespiratory events post-immunization was the same in infants receiving whole cell or acellular pertussis vaccine (44.1% versus 45.6%). Eighteen immunized infants (14.5%) and 51 control infants (41.1%) had a reduction in ABD in the 72 hours post- immunization or at the equivalent postnatal age in controls for an odds ratio of 0.175 . The need for therapy of ABD in the immunized infants was not statistically different from the control infants. Lower weight at the time of immunization was a risk factor for a resurgence of or increased ABD post-immunization. Birth weight, gestational age, postnatal age or sex were not risk factors.There is an increase in adverse cardiorespiratory events following the first dose of DTP-IPV-Hib in preterm infants. Lower current weight was identified as a risk factor, with the risk being equivalent for whole cell versus acellular pertussis vaccine. Although most of these events are of limited clinical significance, cardiorespiratory monitoring of infants who are sufficiently preterm that they are receiving their first immunization prior to hospital discharge should be considered for 72 hours post-immunization. Haemophilus influenzae type B (DTP-IPV-Hib) immunization of preterm infants [The current recommendation is to administer the first routine immunization to preterm infants at the usual chronological age of 8 weeks as the immunologic response to vaccines at that age is almost equivalent to that of term infants . However infants -10, with infants . Previou infants -7,9,10.A retrospective stratified cohort study was completed to determine the frequency of adverse cardiorespiratory events among premature infants following their first immunization with DTP-IPV-Hib vaccine and to compare the frequency of events following whole cell versus acellular pertussis vaccines. A further objective was to study possible risk factors for these adverse events.Approval from the Health Research Ethics Board of the University of Alberta was obtained for conducting this study.Infants with a gestational age of ≤ 32 weeks who received their first DTP-IPV-Hib immunization prior to discharge from two Neonatal Intensive Care Units (NICUs) at the University of Alberta and Royal Alexandra Hospitals in Edmonton, Alberta January 1, 1996 to November 30, 2000 were eligible for the study. A public health nurse visits each NICU on a frequent basis and administers immunizations to all infants who have reached 8 weeks of age, assuming the attending physician identifies no contraindication. Compliance with this intervention is excellent with almost all infants discharged after 8 weeks of age having received DTP-IPV-Hib. Each immunized infant was then matched by gestational age to one control selected from infants not yet discharged from NICU at an equivalent chronologic age  during the 5-year study period by using a random numbers table. The study date for controls was equivalent to the postnatal age at time of immunization of the matched immunized infant, with the date being adjusted up to seven days in either direction to avoid the control's own immunization, discharge, septic work up (as infants unlikely to be immunized within seven days of a septic work) or surgical procedure. Infants could be entered in the study once as a control and once as an immunized infant, spanning different dates each time. Immunized infants and controls were excluded if they were not on a cardiorespiratory monitor in the NICU for the entire observation period, they were still ventilated, there was concurrent administration of other vaccines, or documentation of case notes was inadequate.The frequency of ABD was recorded in two observation periods for each infant. For immunized infants, observation period #1 was the 72 hours pre-immunization and observation period #2 was the 72 hours post-immunization. For controls, observation period #1 was the 72 hours prior to the date that corresponded to the equivalent age at which the infant they were matched to was immunized and observation period #2 was that date and the 48 hours following that date. ABD were detected by cardiorespiratory monitors or by observation and recorded by nurses caring for the infants. Apnea was defined as cessation of respiration for 20 seconds or more. Bradycardia was defined as a heart rate less than 100 beats per minute for 20 seconds or more. Desaturation was defined as oxygen saturation ≤ 85%. An increase in adverse cardiorespiratory event was defined as: 1) a resurgence of ABD as indicated by greater than one ABD within observation period #2 with no ABD during observation period #1, or 2) increased ABD as indicated by a 25% increase in the number of ABD events within observation period #2 versus observation period #1. A reduction in adverse cardiorespiratory events was defined as: 1) resolution of ABD as indicated by greater than one ABD event in observation period #1 with none in observation period #2 or 2) decreased ABD as indicated by a 25% decrease in ABD events within observation period #2 versus observation period #1.Birth weight, current weight, gestational age, sex, and theophylline or doxapram use were recorded for immunized infants and for controls, and the postnatal age at the time of immunization and the vaccine type were recorded for immunized infants. The number of episodes of ABD and the treatment required for these events in observation periods #1 and #2 were recorded for immunized infants and for controls.Data were analyzed using Stata. Cellular and acellular vaccines were compared by using Chi-square. A p < 0.05 was considered statistically significant. Odds ratios were calculated using conditional logistic regression with the dependent variable being either (a) an increase in adverse cardiorespiratory events or (b) a decrease in adverse cardiorespiratory events and the independent variable being immunization. Differences were considered statistically significant if the 95% confidence interval of the odds ratio did not include the value one.One hundred ninety-two infants received their first DTP-IPV-Hib immunization in NICU during the study period. DTP-IPV-Hib with whole cell pertussis vaccine was used prior to July 1, 1997 and DTP-IPV-Hib with acellular pertussis vaccine was used starting on this date. Sixty infants had exclusion criteria and a further eight immunized infants had to be excluded as an appropriate control infant could not be identified.One hundred twenty-four immunized infants and 124 controls were evaluated. The study date for controls was adjusted up to seven days in either direction to avoid the control's own immunization (n = 35), discharge (n = 10), septic work up (n = 9), or surgical procedure (n = 1). There was no statistical difference between the immunized infants and controls in birth weight, current weight, gestational age, postnatal age or sex . Eighteen (14.5%) of the 124 infants who received their first DTP-IPV-Hib had a reduction in cardiorespiratory events in observation period #2 compared to 51 (41.1%) of the 124 control infants .Fifty-six (45.1%) of the 124 infants who received their first DTP-IPV-Hib and 36 (29.0%) of the 124 control infants had an increase in adverse cardiorespiratory events during observation period #2  had increased oxygen requirements and/or required initiation of continuous positive airway pressure (CPAP) and 9 infants (16.1%) required an increased theophylline dose. A 26-week gestation male infant who received acellular pertussis vaccine required mechanical ventilation on the third day post-immunization as he had a 36% increase in apneas post-immunization that persisted despite initiation of CPAP and theophylline. The need for therapy of adverse cardiorespiratory events in the immunized infants was not statistically different from the control group where 6 infants (16.6%) had increased oxygen requirements and/or required initiation of CPAP and 7 infants (19.4%) required an increased theophylline dose.Lower weight at the time of immunization was a risk factor for an increase in adverse cardiorespiratory events, with the odds ratio per 100 gram increase in weight being 0.83 . There was no statistically significant difference in birth weight, gestational age, postnatal age or sex in infants with and without an increase in adverse cardiorespiratory events post-immunization.Previous studies have identified a possible increase in the frequency of adverse cardiorespiratory events following the first DTP-IPV-Hib immunization in preterm infants -10. Our Despite the fact that the current study demonstrated an increase in adverse cardiorespiratory events post-immunization, most of these events were clinically insignificant and the number requiring treatment was not statistically different between the immunized and control groups. As described in the previous literature, most events resolved spontaneously or required only brief stimulation or transient low flow oxygen -10. SixtThe main reason for changing from whole cell to acellular pertussis vaccine is the decreased reactogenicity of the latter vaccine. However, in the current study there was no statistically significant difference in the frequency of adverse cardiorespiratory events post-immunization with whole cell as compared to acellular pertussis vaccines in preterm infants. This differs from a previous study in preterm infants where increased adverse cardiorespiratory events and increased systemic inflammatory markers  occurred in 30% of infants receiving whole cell pertussis vaccine but in no infants receiving acellular pertussis vaccine .Previous studies found that the risk of adverse cardiorespiratory events post- immunization was higher in infants with lower birth weight , lower cOne limitation of our study is that patients not on electronic cardiorespiratory monitors 72 hours prior to and post-immunization were excluded. These infants would presumably be more stable and may be less likely to have an adverse event precipitated by the immunization. Therefore, the frequency of apnea or bradycardia in preterm infants receiving their first DTP-IPV- Hib immunization may have been over-estimated in the current study. The study dates of the controls that corresponded to the immunization date of the immunized infants were adjusted up to 7 days in 55 patients to avoid confounding factors, which could have affected results. It would have been ideal but impractical to use a shorter window. Charting of adverse cardiorespiratory events by nursing staff can vary and it is possible that nurses were more diligent about observing infants who had recently received an immunization. However, we suspect there was limited awareness amongst nurses or physicians of the potential for immunizations to precipitate adverse cardiorespiratory events, as many infants could not be included in the study as they were not on a monitor post-immunization. It has been suggested that when looking for adverse events related to immunization, it may be inappropriate to use unimmunized children as controls as they may differ in important ways from immunized children . HoweverMore hospitalized preterm infants ≤ 32 weeks developed an increase in adverse cardiorespiratory events after their first immunization with DTP-IPV-Hib than did a control group at an equivalent chronologic age. A lower weight at the time of immunization was a risk factor for an increase in adverse cardiorespiratory events. The risk of these events does not appear to have been decreased by the use of acellular pertussis vaccine. The majority of these events were clinically insignificant and should not prompt delays in immunization. However, whenever practical, immunization should be given at least 72 hours prior to planned discharge in this fragile population to allow for cardiorespiratory monitoring post-immunization.The author(s) declare that they have no competing interests.JL and JLR designed the study and wrote the manuscript. JL collected the data. DWS performed the statistical analysis and reviewed the manuscript.The pre-publication history for this paper can be accessed here:


The implantable cardioverter defibrillator (ICD) is a life saving device for individuals with life threatening ventricular arrhythmias. There is no doubt that it is a cost effective therapy in various congenital and acquired arrhythmogenic disorders. Nevertheless, shock delivery may be painful and frightening which causes psychological distress and deterioration of perceived quality of life.A systematic meta-analysis on studies reporting quality of life in patients implanted with ICDs was done using professional databases. Related articles and references of the relevant articles were also searched for suitable studies.Thirty studies with a total of 3412 patients on implantable defibrillators were identified. Five of them were large randomised studies with a total of 1680 patients, while 25 were non-randomised studies. Medical Outcome Study 36-item Short Form health survey (SF -94 36) was the most common instrument used for assessment of quality of life. Only one of the 5 major randomised trial reported worsening of quality of life after implantation of a defibrillator. In the subgroup of patients receiving shocks, three out of the five trials reported worsening of quality of life.Most of the randomised studies showed either neutral or better quality of life in patients on implantable defibrillators. In the subset of patients receiving shocks, worsening of quality of life was found in most randomised studies. Therefore, activation of antitachycardia pacing should be performed in every ICD-patient in order to miminze painful shocks and consequent deterioration of quality of life. Implantable cardioverter - defibrillators (ICD) have proven their value as life saving devices -3. As thA systematic accumulation of available data on QOL in patients with ICDs. PubMed searches were conducted using the search words implantable defibrillator, ICD, quality of life and QOL. The retrieved results were checked to identify relevant studies. Further studies were sought by searching for the related articles and the references of the retrieved article. Reports of major ICD trials were screened for data on quality of life.Thirty studies on patients with ICDs reported data on QOL  4-33]. -33. 4-33SF - 36 was the most common instrument used for QOL assessment across the studies. It comprises of a 36-item short-form questionnaire constructed to survey health status in the Medical Outcomes Study .  SF - 3Various other scales have also been used in the studies, like Ferrans and Powers Quality of Life Index ,18,30, QOf the thirty studies available, five were large randomized trials  with 168Patients with impaired left ventricular function and positive signal averaged electrocardiogram undergoing CABG were randomly assigned to ICD versus no antiarrhythmic treatment in the CABG Patch trial . All ICDAmiodarone versus implantable cardioverter-defibrillator (AMIOVIRT) trial included one hundred three patients with non-ischemic dilated cardiomyopathy (NIDCM), left ventricular ejection fraction < or = 0.35, and asymptomatic non sustained ventricular tachycardia (NSVT) . They wePacing Fast Ventricular Tachycardia Reduces Shock Therapies (PainFREE Rx II) trial was the largest trial reporting data on QOL as well as the latest . This trCIDS  comparedThe total mortality was lower in the ICD group, but the difference did not reach statistical significance. (P=0.09). QOL evaluation was done as a secondary endpoint in the study with the Rand Corporation's 38-item Mental Health Inventory (MHI) and the Nottingham Health Profile (NHP) for 317 English-speaking participants.  Perceived QOL was found to be improved more in the ICD group. Significant improvement from baseline to the 6- and 12-month follow-up assessment was seen for 7 of the 10 variables assessed. Quality of life did not improve in those patients who received > or =5 shocks from their device. Still they were comparable to the antiarrhythmic group in QOL. There was no difference between those who received no shock and those who received 1-4 shocks from their ICD. None of the quality-of-life variables improved with time in the antiarrhythmic therapy group.AVID Trial  comparedOf the 30 studies evaluated in this report, 25 were non-randomised . SixteenThe earliest study which reported QOL data from ICD patients was by Luderitz et al . Fifty-fThe largest of the non-randomised studies actually reported on dual chamber defibrillators for restoration of sinus rhythm in atrial tachyarrhythmias. This study by Newman et al  had 267 In the next largest non-randomized study by Kamphuis et al  133 patiDistressing aspects of ICD shocks are lack of warning, multiple shocks, and progressively increased sensations with multiple shocks. Three of the major randomised studies reported worsening of QOL in those patients receiving ICD shocks, especially when  patients were receiving five or more shocks. Despite the AMIOVIRT Trial  did not With regard to a potential deterioration of perceived QOL caused by delivery of ICD shocks for fast VTs, we would recommend to program ATP attempts in all ICD patients regardless of the cycle length of the index arrhythmia.