Sequences flanking microsatellites of similar length show remarkable levels of convergent evolution, indicating shared mutational biases. These biases extend 25–50 bases either side of the microsatellite and may therefore affect more than 30% of the entire genome. To explore the extent and absolute strength of these effects, we quantified the observed convergence. We also compared homologous human and chimpanzee loci to look for evidence of changes in mutation rate around microsatellites. Most models of DNA sequence evolution assume that mutations are independent and occur randomly. Allowances may be made for sites mutating at different rates and for general mutation biases such as the faster rate of transitions over transversions. Our analysis suggests that these models may be inadequate, in that proximity to even very short microsatellites may alter the rate and distribution of mutations that occur. The elevated local mutation rate combined with sequence convergence, both of which we find evidence for, also provide a possible resolution for the apparently contradictory inferences of mutation rates in microsatellite flanking sequences.Microsatellites are a major component of the human genome, and their evolution has been much studied. However, the evolution of microsatellite flanking sequences has received less attention, with reports of both high and low mutation rates and of a tendency for microsatellites to cluster. From the human genome we generated a database of many thousands of (AC) An analysis of sequences flanking microsatellites in the human and chimpanzee genomes suggests that mutations do not occur independently and randomly, as is commonly assumed in models of DNA sequence evolution DNA base substitutions do not occur randomly . InsteadMicrosatellites are sequences of repeated 1–6-bp motifs that mutate primarily through the gain and loss of repeat units, in a process thought to depend on DNA replication slippage . PreviouTo our knowledge, no one has yet conducted a systematic study of mutational biases operating around microsatellites. The direct study of naturally occurring mutations in flanking sequences is virtually prohibited by their slow rate of accumulation, and inferences based on comparisons between homologous microsatellite loci rely on small numbers of sequences. However, an indirect approach is possible, based on comparisons among very large numbers of microsatellite flanking sequences from the finished human genome. If microsatellites have little or variable influence on their flanking regions, among-locus similarities will be minimal or absent. Conversely, if microsatellites generate similar local mutation biases, nonhomologous loci should betray evidence of convergent evolution. With the publication of large blocks of sequence from the chimpanzee genome, one can extend this approach to ask questions about rate of divergence between homologous flanking sequences.Here we use a combination of these indirect approaches to show that microsatellites appear to create regions around them in which both the rate and spectrum of mutations are modified.n, and for simplicity considered only ‘isolated’ repeats, defined as those at least 100 bp from the nearest AC repeat as small as two units in length. 47% of AC repeats on human Chromosome 1 match these criteria. From the human genomic sequence, maximum sample size was set at 5,000 randomly selected loci for length classes (AC)2 to (AC)5. For longer microsatellites, of which fewer than 5,000 could be found, all sequences encountered were included. 2+ repeats, was generated from Chromosome 1.We studied the most abundant class of human dinucleotide repeats, (AC)n and (CA)n repeats, (AC)n microsatellites were divided into subclasses, termed ‘cassettes’, according to their immediate 5′ and 3′ flanking bases such that 5′-X(AC)nY-3′ is referred to as cassette X/Y. Hence, a (CA)3 repeat would be classed as cassette C/A around (AC)2. Such a distinction may appear pedantic, but since both DNA replication fidelity and repair efficiency are known to be influenced by base order and local sequence context (n and (CA)n are equivalent. This nomenclature also helps to resolve the problem of defining microsatellite length because (CA)3 equals (AC)2. nY is viewed as comprising two dinucleotides, XA and CY. The probability of observing XA and CY jointly is then calculated from the individual frequencies of XA and CY estimated using 1 Mb of randomly sampled sequence from Chromosome 1 containing no (AC)2+ repeats.To distinguish between (AC) context , it seem2 microsatellites, the cassette frequencies are broadly similar to those expected from the frequencies of the component dinucleotides in unique sequence DNA, though eight cassettes show significant differences (Around (AC)ferences A. Howeve5, since this exhibits the strong patterns while at the same time retaining sufficient sample sizes for analyses to be conducted on the rarer cassette types. Several features are apparent. First, levels of patterning can be remarkably strong, with the probability of observing a given base at a given site varying from less than half of that in unique sequence to more than double, often at adjacent sites. Second, many of the patterns show strong dinucleotide periodicities, presumably reflecting the dinucleotide structure of the microsatellite. Third, there are several examples of clear 5′ to 3′ asymmetry, indicating that mutational patterns on one side of a microsatellite may not be the same as those on the other.We consider flanking sequences to extend 50 bases either side of a microsatellite. To compare flanking sequences, we first divided microsatellites according to (AC) repeat number and cassette type, and then calculated the frequency of each of the four nucleotides at each of the 100 possible positions. Any mutation biases present should be revealed by locally changed base composition, and this appears to be the case. For many microsatellite length–cassette combinations, the flanking sequences exhibit strong deviations from random. The observed patterns can be placed in six broad classes according to the strength of a two-base periodicity and the degree of 5′ to 3′ asymmetry. These patterns are illustrated in 9 but then declines in longer microsatellites. As well as showing variation between cassettes, flanking sequence patterning also changes with microsatellite length. For those cassettes where sample size is sufficient to show a trend, that is, T/A and to some extent C/A and A/A, both amplitude of deviation from random and the breadth of patterning tend to increase with increasing AC repeat number. We do not see dramatic changes such as a reversal in 5′ to 3′ asymmetry or the emergence of new patterns. For cassette T/A, pattern strength increases up to (AC)In view of the strong two-base periodicities seen for some base–cassette combinations, we next examined the distribution of frequencies of all 16 possible dinucleotide motifs. As expected from the single nucleotide patterning, dinucleotides also tend to show periodic patterning  and S2, From 3, then increases towards a peak in strength at (AC)9 before diminishing as AC repeat number increases further. Cassettes A/A and C/A show similar trends and together suggest that, where patterning occurs, it is apparent by (AC)6. When data were plotted without first categorizing them by cassette type, although a number of the dinucleotide patterns in As with the mononucleotide patterning, the periodicity in dinucleotide frequencies changes in amplitude and width 5′ to 3′) as repeat number increases. For most cassettes, the paucity of long microsatellites precludes study of how the patterning changes with repeat number. However, cassette T/A is sufficiently abundant for the progression to be described, and cassettes A/A and C/A, although less common, nonetheless yield meaningful results. ′ to 3′ a5. Classifying loci by cassette type, length, and whether they occurred in LINE/L1, SINE/Alu, or unique sequence DNA yielded in most classes sample sizes too low to be of use. However, where sample sizes were adequate, that is, cassette T/A, dinucleotide AT, patterning in unique sequence loci was indistinguishable from that in LINE and SINE microsatellites 5 to provide strong patterning and large sample sizes. The results of deleting all flanking sequences containing AT microsatellites with n or more repeats, where n = 2, 3, 4, and 5, are given in n allowed, suggesting that the dominant AT patterning results from (AT)n microsatellites developing immediately adjacent to the AC tract.The strongest two-base periodicities we find tend to involve motif AT. Such periodicity might arise in two main ways: either because AT motifs in phase with the microsatellite tend to expand through slippage to form (AT)n repeats. This is an important test given that our strict definition of a microsatellite restricts our analysis to pure AC repeats and allows the possibility that compound repeats, for example, (AT)n(AC)n(AT)n are included. To do this, we plotted the distribution of single AT motifs around (AC)3+ microsatellites in the subset of flanking sequences with no (AT)n microsatellites, where n > 1. Summed over all AC microsatellite lengths, single AT dinucleotides are overrepresented in 5′ sequences at odd numbered positions and at even numbered positions in 3′ sequences (We next examined whether the phase of AT dinucleotides was determined solely by their tendency to form clusters next to (AC)equences . Excesse2 or longer were assigned back to their own class 57% of the time, showing that these sequences consistently differ from those near to AC repeat tracts. Remarkably, this figure falls to almost half (32%) for sequences flanking (AC)2, indicating that, even with just two repeats, similarities to other microsatellite flanking sequences already exist. The same pattern extends to other length microsatellites, with flanking sequences tending to be preferentially assigned back to their own or to an adjacent length class. When a flanking sequence is not assigned back to its own class, it is usually assigned to one of three other classes: the ‘1’ class of random sequences, the ‘9’ class where patterning is strongest, or to the longest class, class 21 (unpublished data).To assess the level of any convergent evolution, we used a simple assignment test to determine how often individual sequences resemble others flanking unrelated microsatellites of similar length see . Figure These analyses reveal a tendency for microsatellite flanking sequences to be similar to each other, but fail to quantify the level of sequence change involved. To do this, we sought to estimate similarity among three classes of sequence: (1) blocks of 50 bp lying immediately adjacent to a microsatellite; (2) blocks of 50 bp chosen randomly to lie between 500 and 600 bases downstream from a microsatellite (the random selection aims to remove possible complications of exact position and phase with the microsatellite); and (3) blocks of 50 bp randomly selected from around the genome. Comparisons within class 3 define the average level chance similarity in the genome, here estimated at 12.77 ± 3.28 (sd) bases out of 50. Comparisons within class 1 estimate how much convergent evolution is apparent at any given repeat number, and reveal a profile that rises to a maximum of 14.31 at a length of seven repeats, followed by a gentle decline with increasing length thereafter A. SimilaThus, in all cases, sequences immediately flanking a microsatellite show greater similarity to each other, to sequences nearby, and to sequences elsewhere in the genome than randomly selected sequences do to each other, a trend that is maximal for microsatellites around 7–10 repeats in length. We believe these similarities are generated primarily by the enhanced simplicity of microsatellite flanking regions and their tendency to gain AT motifs. Such characteristics allow unusually high matches when compared with random blocks of 50 bases that have high simplicity or contain polyA tracts. Over and above this background level of elevated similarity, proximity to any microsatellite appears to increase similarity, implying that microsatellites tend to lie more generally in regions of similar base composition. This might reflect either a tendency for microsatellites to arise preferentially in certain broad sequence contexts, or modification of the local base composition by the microsatellite itself. Moreover, similarity is further enhanced when a flanking sequence is compared with a neighbouring block. This suggests a local context effect such as might arise through the isochore structure of the genome, with neighbouring blocks being located in the same isochore and hence ‘coloured’ by the same nucleotide biases.2 flanking sequences to their own class is significant up to ten bases away from the microsatellite , and only when the window reaches 24 bases from the microsatellite does the assignment level fall to the value of 4.8% expected of random sequences.To define more precisely the regions where convergent evolution is occurring, we repeated the assignment test but instead of using the full 50 bases either side we now used a symmetric pair of moving 25-base windows placed either side of the AC microsatellite . Close tftp://ftp.ncbi.nih.gov/blast/executables) to compare microsatellites identified in completed sections of the chimpanzee genome against homologous loci identified in humans. If homologous loci are identified through comparisons among their immediate flanking sequences, an element of circularity will be introduced, since loci with lower rates of evolution will be identified preferentially. To circumvent this problem, we used a region 300 bases in length and 220 bases downstream of the microsatellite to conduct each Megablast search.So far we have considered only the nature of the mutations that affect flanking sequences, and not their rate. To examine whether mutation rates are affected by the presence of a microsatellite, we used Megablast ; A total of 8218 chimpanzee loci were identified, and they yielded 5537 unique human homologues. Since microsatellite flanking sequences may contain insertions or deletions, we adopted the following approach so as to minimise problems of alignment. The chimpanzee flanking sequence was divided into 20 contiguous blocks of 20 bases, ten on each side of the microsatellite. Each block was then compared against 1,000 bases of human sequence, downstream from the region identified by Megablast, to find the best possible match. To filter sequences with major rearrangement we required both that all 20 blocks match with at least 15/20 bases, and that matched blocks all lie in the same order as their homologues. Differences between pairs of homologous flanking sequences were then quantified, at each of the 20-block positions, as the proportion of perfectly matching blocks and the average number of matches within each block .2–3 there is little apparent variation in either measure of similarity with block position apart from a small tendency for 5′ blocks to show an increasing average percentage match closer to the microsatellite. However, although the trends are by no means strong, around longer microsatellites similarity of blocks near to the microsatellite is reduced, particularly when measured using average percentage matches , and among these, approximately 77% of the blocks were identical. Around (AC) matches .Our analysis of dinucleotide frequencies around microsatellites shows patterns of similarity on a smaller scale than might be revealed with 20-base blocks of sequence. To investigate the number of changes in the immediate flanking bases, we also calculated the proportion of mismatches occurring at a given base for each 5′ and 3′ sequence in the immediate flanking blocks 9, but appears present even around sequences as short as (AC)2 and may extend as many as 50 bases either side. Some patterning is more or less symmetrical, but we also found several examples showing strong 5′ to 3′ asymmetry, implying that the two ends of a microsatellite are by no means equivalent. The net result is that sequences flanking microsatellites of a given length tend to be more similar to each other than to random sequences or to sequences flanking microsatellites of different lengths. Thus, there appears to be convergent evolution. Finally, we compared large numbers of homologous flanking sequences between humans and chimpanzees, and found evidence that mutation rates near microsatellites tend to be somewhat elevated.We have studied very large numbers of (AC)n tracts exhibit remarkable levels of patterning, with any given dinucleotide motif tending to be much more likely to occur at even numbered positions rather than odd, or vice versa. For several reasons, we believe that the patterning arises due to the structural properties of the microsatellite (see below), becoming more pronounced as repeat number increases. These reasons include the following: the consistently central placement of microsatellites within the patterning, the dependence of the strength of patterning on AC repeat number, the similarity between microsatellites in LINE and SINE elements and those elsewhere, the weakness of the patterning around (AC)2, and the strong influence of cassette type on the form of patterning. Unfortunately, it is surprisingly difficult to eliminate the alternative hypothesis, namely that the patterning arises due to some other force and that AC repeats then either form or expand more rapidly when placed centrally within the pattern. This ambiguity is particularly relevant to the question of cassette distribution, where it seems reasonable both that (AC)n tracts might cause biased interconversion between cassettes and that certain cassettes may allow slippage more than others. For example, while the structural properties of AC repeats are known to generate mutational biases in adjacent bases (Sequences surrounding (AC)nt bases  capable nt bases .2 already showing a small but significant bias in the distribution of cassette types and greater similarity to other sequences flanking AC microsatellites than to random sequences. In addition, the moving window assignment test indicates that significant convergence exists even when the ten bases closest to the microsatellite are excluded. Such a wide influence around such a common, short motif is remarkable and suggests that a high proportion of the genome may be affected by these and similar forces. To illustrate, (AC)2 is expected to occur every 250 bases, as is (GT)2. Taking the sphere of influence on each side as ten bases plus half the 25-bp window yields a value of 45. This predicts that over 30% (approximately 45 bases of every 125) of the genome will be affected by (AC)2 on one strand or the other, a figure that will only increase with inclusion of longer arrays and other microsatellite motifs.The relationship between an AC microsatellite and its flanking sequences begins surprisingly early, with (AC)5 and peaking in strength at (AC)9. Although patterning is seen in several different dinucleotide motifs, even in the human genome there are insufficient data to study any but the commonest cassette–motif combinations over a wide range of microsatellite lengths. Focusing on the motif AT, we found evidence that the strongest patterning was due to the development of AT microsatellites abutting AC tracts. However, this is not the only effect. After removal of all (AT)2 or longer microsatellites, there remains a significant tendency for single AT motifs to appear in phase with AC tracts, suggesting that mutation bias as well as slippage is involved.As AC repeat number increases, so does the strength of patterning, becoming pronounced by (AC)2 and (AC)9, it might seem logical that the pattern would become stronger and stronger as repeat number increases further. Instead, (AC)9 appears to be the peak strength, with longer microsatellites showing lower amplitude but a broader spread of patterning. It is interesting that this peak coincides with the length at which microsatellites begin to become polymorphic: a common rule of thumb for marker development in mammals is that primers are designed for loci carrying ten or more repeats (Given the increase in strength of patterning between (AC) repeats . This ma repeats . Again,  repeats .The rich patterning we find presumably arises through local mutation biases. Previous work on mutation biases has tended to reveal either generic effects such as isochors , where sn and (A)n induce local mutation biases  or A to Y (pyrimidine) transversion mutations, and also the increase in pattern strength around longer microsatellites, with interruptions in longer arrays more likely to be internal to the repeat tract and hence be excluded from the analysis. However, we suggest that this model is unlikely for two reasons. First, such a model fails to accommodate the strong asymmetry in patterning that is observed for some dinucleotides and specific cassette bases around the (AC)n repeat tract. Polarity has been noted for minisatellite mutations, with mutational processes differing between the two ends of the repeat tract at tract , but a mat tract .The patterning we describe appears to represent an important component of the forces that shape genome evolution, both in terms of its ubiquity and the absolute strength of its effect. It follows that there are many possible practical and theoretical implications. For example, even very short microsatellites appear able to cause some level of convergent sequence evolution, and hence to confound phylogenetic analyses. Similarly, microsatellites near genes may increase local mutation rates and influence the spectrum of new mutations that arise. To explore the size of these effects we designed experiments both to measure absolute convergence and to ask about evidence for changes in mutation rate.7–10 both for assignment to their own class and for similarity to each other relative to random blocks provide a clear indication that convergent sequence evolution is occurring. Interestingly, any given flanking sequence tends to be more similar to a block 500 bases away than to a similarly placed block near a different microsatellite, suggesting longer range patterning such as might arise through placement within the same isochore isochore . FurtherIn conclusion, previous studies of microsatellite flanking sequences have identified several features, including a tendency to harbour other microsatellites, a locally increased mutation rate, and, conversely, conservation over unexpectedly large tracts of evolutionary time. Our analyses support all these trends and provide a possible resolution for the apparent contradiction between faster evolution but at the same time greater sequence conservation. Although there is evidence that mutation rates near microsatellites are elevated, we also find evidence of convergent evolution. Consequently, the increased rate of change may be to some extent neutralised and perhaps even reversed by the tendency for similar changes to occur in related lineages. Furthermore, the greatest changes appear to occur in flanking sequences around microsatellites that are below the length used as markers, at least in humans. Overall, therefore, we have been able to formalise previous anecdotal evidence and hence to document a remarkably widespread source of directional change and nonrandom evolution that undoubtedly plays an important role in shaping the make-up of our genomes.n dinucleotide repeats was extracted from the human genome  using a custom macro written in Visual Basic. Only microsatellites separated by at least 100 bp from the nearest (AC)2 or longer were included in the dataset. Thus (AC)2AT(AC)10 would not be included in the dataset, whereas ACAT(AC)10 would be included as (AC)10. Flanking sequences are here defined as the 50 bases lying either side of a microsatellite. No attempt was made to translate TG repeats with complementary AC repeats on the opposite strand. Consequently, all our microsatellites are 5′-(AC)n-3′.Our dataset of (AC)n < 200 observations) or a normal approximation (n ≥ 200 observations). Bases used to define cassette type were excluded from all calculations, and expected frequencies were taken as the average frequency across all positions.For each frequency estimate, 95% confidence intervals were derived based on the binomial distribution (5 and declined to 175 for (AC)20 C)20 see .As an index of similarity, we calculated the log likelihood of observing a given sequence based on its position-specific dinucleotide motif composition:ijkf is the frequency of dinucleotide i at position j  in flanking sequences of class k. To avoid bias, when a sequence was compared with its own class, its contribution to the dinucleotide frequencies was first removed. For each sequence in turn, A was calculated for every class and the sequence was then assigned to the class that yielded the highest index value. Under convergent evolution, we expect sequences to tend to be assigned to their own or similar length classes.where Sequences were again divided into length classes 2 to 21, and each sequence contributed four 50-bp blocks of sequence, one from each side immediately adjacent to the microsatellite but excluding the cassette bases (class 1), and one from each side displaced by a randomly selected number 500–600 bases distal (class 2). In addition, we also generated a database of 5,000 non-microsatellite-associated sequences. When making comparisons within a class, nonindependence was avoided by randomising the sequence order and then comparing sequence 1 with sequence 2, 2 with 3, …, (n − 1) with n. Our index of similarity was simply a count of the number of matching bases. A few pairs of sequences (less than 0.1%) gave high similarity scores of over 30/50 matching bases, presumably because these loci have been duplicated or lie in repetitive elements. Such sequences were discarded. As with all other analyses, sequences containing base ambiguities (marked base N) were also discarded.n repeat microsatellites were extracted from the available chimpanzee finished-quality high-throughput genomic sequence (NCBI) as outlined above for humans. A 300-base region 220 bases upstream from each chimpanzee microsatellite was used by Megablast  to identify homologous human loci in the finished genome sequence. Sequences with multiple high-scoring hits were discarded, as they presumably occur because a locus is found in repetitive elements or has been duplicated. Those nonoverlapping hits with at least 280/300 matching bases and an expectation  greater than five times that of any other hit to the same sequence were thus retained, giving a dataset of 5,537 sequences.(AC)Figure S1From little structure of any kind (A) to complicated aperiodic clustering (C). Plots are as described in (1.7 MB TIF).Click here for additional data file.Figure S2Plots are as described in (3.0 MB TIF).Click here for additional data file.Figure S3Figure depicts equivalent patterns of asymmetry in AT dinucleotide frequencies for the commonest cassette type, (T/A), around microsatellites in unique sequence DNA (A), LINE/L1 elements (B), and SINE/Alu elements (C). Plotting conventions are the same as for (1.4 MB TIF).Click here for additional data file.Figure S4Block position is relative to the central microsatellite (not shown).2–3 microsatellites .(A and B) Proportion of exact matches  and average number of matches, excluding exact matches (± standard error), with block position around (AC)4+ microsatellites (n = 356). Average proportion of exact matches and number of matches, calculated separately for 5′ and 3′ blocks around (AC)2–3 microsatellites, are shown by a black line in (A) and (C), and (B) and (D), respectively. Average percentage match rather than average match is plotted in (B) and (D) because overlapping blocks were truncated to exclude overlapping regions from the analysis, with the result that not all blocks contained 20 bases.(C and D) As (A and B) but for (AC)(2.9 MB TIF).Click here for additional data file.Figure S52–3 microsatellite loci (A) and over (AC)4+ microsatellite loci (B). Shown ± standard error. The microsatellite at base position 0 is not shown. Expectation, calculated separately for 5′ and 3′ sequences around (AC)2–3 microsatellites, is shown by a black line in both plots.The proportion of mismatches occurring at a given base in a flanking sequence are averaged over (AC)(3.4 MB TIF).Click here for additional data file.


PLoS Biology, volume 1, issue 1:In The Roles of APC and Axin Derived from Experimental and Theoretical Analysis of the Wnt PathwayEthan Lee, Adrian Salic, Roland Krüger, Reinhart Heinrich, Marc W. KirschnerDOI: 10.1371/journal.pbio.0000010fluxes and flux appeared without the fl.Table 1: In the legend, the words coefficients appeared without the fi. In the table, some numbers in the section “Binding, dissociation” were marked with a ± sign that should have been a ∓.Table 3: In the legend, the word coefficients appeared without the fi.Table 4: In the legend, the word Please see the corrected legends and table below.Table 1. Numeric Values of Input Quantities of the Model for the Reference Statev12 = 0.42 nM · min−1 (rate of β-catenin synthesis), v14 = 8.2 · 10−5 · nM · min−1 (rate of axin synthesis), k4 = 0.27 min−1, k5 = 0.13 min−1, k6 = 9.1 · 10−2 nM−1 · min−1, k−6 = 0.91 · nM−1 · min−1, k9 = 210 min−1, k10 = 210 min−1, k11 · 0.42 min−1, k13 = 2.6 · 10−4 min−1, k15 = 0.17 · min−1. See Table S2, found at http://dx.doi.org/10.1371/journal.pbio.0000010.t002, for more precise numbers used in the calculations.The data are grouped into concentrations of pathway components, dissociation constants of protein complexes, concentration ratios, fluxes and flux ratios, and characteristic times of selected processes. Experimental evidence for these data is discussed in the text. From these data, the following rates and rate constants are calculated: Bold: Measured values, Italics: Estimated values.DOI: 10.1371/journal.pbio.0000010.t001Table 4. Concentration Control Coefficients for the Total Concentrations of β-Catenin and Axin Relative to Changes in the Concentrations of Pathway ComponentsThe control coefficients were obtained by numerical determination of the response to a change of total concentrations by 1%. Coefficients are given for the reference state and for the standard stimulated state.DOI: 10.1371/journal.pbio.0000010.t004The full text XML and HTML versions of the article have been corrected online. This correction note may be found at DOI: 10.1371/journal.pbio.0020089.


This article reviews 19 studies (1987–2004) on quality of life for family caregivers helping those with chronic physical illness. Here we explore the concepts of and instruments used to measure caregivers' quality of life. We were particularly interested in understanding stress-related variables and documenting factors influencing quality of life based on family stress theory. Findings show that various positive and negative terms equated with quality of life were used to measure them. Results indicate that stress-related variables as possible predictors influencing caregivers' quality of life include: patient and caregiver characteristics, stressors, stress appraisal, stress coping methods, and social support. Our recommendations touch upon applying theory for intervention, developing measurement, making operable the concepts for measuring, and the need for longitudinal and comprehensive study. Recent reforms in U.S. health care systems mean that individuals with long-term, complex health problems are being cared for at home by family members . Specifiquality of life – may be partly influenced by other existing environmental stressors, stress appraisal, coping methods and social support.Existing studies document how caring for chronically ill family members or significant others at home influences multiple aspects of caregivers' lives. These effects are physical, psychological and social and may include worsened physical health, impaired social and family life, and increased stress, anxiety and depression -9). Plac. Plac9])The importance of family stress theory in studying normative family transitions and adaptation to major life changes and illness is based on the central role that family strengths and capabilities play in understanding and explaining psychological and behavioral outcomes . In famiQuality of life is a construct that encompasses health and functioning, socioeconomic status, psychological, emotional and spiritual aspects, and family . FerrellTo address the lack of review papers on this topic as well as inconsistent results among empirical studies, we undertook a thorough review of the literature. We were particularly interested in research that reported psychosocial or QOL outcomes and accounted for family stress variables as correlates or explanatory variables. The purpose of this paper is twofold: to review caregiver QOL impacts (loosely-defined) organized around family stress theory, and to discuss the implications of findings for future research .Only published peer reviewed research articles were included in this review. Several methods were applied in searching the literature. First, articles were limited to those published in English between January 1, 1987, and January 31, 2004. Second, participants were caregivers of patients over 21 years of age with chronic physical illnesses. Third, a study was excluded if patients were receiving hospice care. Fourth, a computer search was conducted in February 2004 to review the databases of MedLine and PubMed by using the following key words: caregivers, caregiving, chronic illness, quality of life, adjustment, life satisfaction, burden, distress, and family stress theory. The terms "health," "stress," and "coping" were also used for study retrieval. Databases also were searched for review articles published during the same time period. Additional sources for empirical reports included reference lists from published studies. Of the more than 220 articles identified, there were only 16 empirical studies based on family stress theory that focused on the caregivers of patients with chronic physical illness and measured the QOL of caregivers. Two additional articles were identified from searching review articles. Finally, one more article was found in the references of these 18 articles. Thus, a total of 19 articles are included in this review (Appendix 1 See ).Reviewed studies used diverse outcomes and models to indicate caregivers' QOL. Psychosocial outcomes falling with varied dimensions of QOL and measured included adaptation, mental health, life satisfaction, stress, emotional distress, health, caregiver burden, and depression. Given the variety of terms equated with QOL, both positive and negative terms were used to measure the QOL. Three studies ,13,21) u,21 u13,2per se: the Caregiver Quality of Life Index containing items related to physical, emotional, social, and financial wellbeing )Eleven articles investigated the relationship between stressors and caregiver QOL, but they did not show consistent results. To varying degrees, caregiving demands, patient impairment, the duration and intensity of care, ADL (activities of daily living) dependency, stressor types, caregiver overload, how much assistance is given the caregiver, recurrence of illness and problem behavior in the patient were identified as primary stressors. Secondary stressors were caregiving demands, role change, responsibility, caregiver experience, and life-style interference. Seven studies  f,25 f15,2Haley et al.  found thOf the four coping strategies, Wallhagen  discoverSix articles examined the association between stress appraisal and caregiver QOL. Two ,14) demo demo14])Haley et al.  found thIn contrast, Wallhagen  did not Miaskowski, et al.  investigA majority of the articles addressed social support as a mediator and predictor variable. Six articles . ,28. 13,2For a number of reasons, we need longitudinal studies in research applying family stress theory to caregivers of the chronically ill. Otherwise we cannot understand longer-term or sustained effects. First, although a patient with chronic illness may have completed treatment, recurrence of the illness is possible. Second, coping methods and appraisal may change depending on the circumstances, because family stress theory is dynamic and influenced by both internal and external environments. A longitudinal study may see the attrition of participants over time. Due to the nature of chronic illness, many patients may have died or experienced recurrence at the follow-up evaluation. Therefore, caregivers of these patients need to be followed through the end to assess predictors and consequences of patient death and recurrence of illness.Family stress theory is very complex and comprehensive. As a result, it is difficult to conduct research using the full model. Examples of this complexity include interactions among various factors, interactions and transactions over time, individual dynamics within the family system, and the balance between family demands and the family. Considering these multidimensional aspects, it would take much hard work to test the full model of family stress theory. However, we need to try. Otherwise, we may never understand the complex relation between individual, family, and environment.Appendix 1: Review of empirical findings on quality of life of family caregivers. Author, topic, significant predictor variables, mediating variables, outcome variables, measurement, and intervention of 19 reviewed articles are shown in detail.Click here for file


Retrovirology's choice for best basic science "retrovirus paper of the year" and a perspective on challenges and responsibilities facing HIV-1 and HTLV-I research are presented.In this editorial,  Retrovirology concluded its first calendar year of publishing. In actual fact, Retrovirology launched as an Open Access journal the final week of February 2004 and has been publishing continuously for a little more than 10 months. Over that period, with the wonderful efforts from my 6 very capable Associate Editors , the journal has thrived.The beginning of a year provides an occasion to look back upon progress made over the past 52 weeks. With the end of 2004, Retrovirology is to provide a visible forum for retrovirologists so that their works can be read by all in a free and openly accessible manner. What this means is that if you are a human immunodeficiency virus (HIV)-researcher and you had published a paper in Retrovirology, a graduate student in Sri Lanka updating his/her research protocol, an AIDS activist in South Africa looking for the latest information, and even your long-lost high school sweetheart wondering what you have been doing all these years, can all find your work (i.e. through a simple Google or PubMed search) and read your most recent findings. Perhaps more relevant to the enterprise of scientific communication is that numerous academic peers in Eastern Europe, Asia, South America, Africa and elsewhere do not have funds which would permit them to read Cell, Science or Nature. Hence, while some can read your research in Cell, Science, or Nature, all colleagues, rich or poor alike, can read your Retrovirology paper.The goal that we set for Retrovirology? You bet! Our monitored statistics tell us that in 2004, the most highly accessed review article [Retrovirology was read by over 3,500 individuals while a comparably popular original research article [Retrovirology articles with great immediacy. Thus, a recent paper by Rana and colleagues [Retrovirology on December 27th, 2004; and already by December 31st, 2004, a short 4 days later, that study had been read 389 times. Just as readers are quick to read our papers, I am equally pleased by our unmatched speed in publishing authors'works. In 2004, based on all papers we published in Retrovirology, the time from submission to publication averaged 40 days. From my personal experience of publishing in other virological journals, this duration is 3 to 4 times faster than our best competitors.Are they reading  article  publisheRetrovirology will initiate the annual recognition of the best basic science "retrovirus paper of the year". The Associate Editors and I decided that in 2004, the best basic science retrovirus paper was the work from Joseph Sodroski and colleagues describing the HIV-1 restrictive property of the tripartite motif 5 (TRIM5) protein [Retrovirology Editors will find it fitting to recognize a Retrovirology paper as the "best basic science retrovirus paper" of the preceding year.Different journals/magazines recognize "Molecule of the Year", "Breakthrough of the Year", or even "Person of the Year". With this editorial, I explain to my postdoctoral fellows that challenges are those issues which you think others should solve, while responsibilities are items that you think you should tackle. As a retrovirologist depending on how you regard yourself, pressing problems are either others' challenges or your responsibilities. I study two retroviruses, HIV-1 and human T-cell leukaemia virus type 1 (HTLV-I). The start of a new year offers me a chance to review briefly my personal bias on the important research question that confronts HIV-1 and HTLV-I, respectively.For HIV-1, the "holy grail" remains the development of an effective vaccine against the virus. As we enter 2005, mortality from AIDS is staggering. It is estimated that in 2004, 3.5 million individuals perished worldwide from AIDS; or nearly 10,000 AIDS deaths each day. We can view this number in another way. The recent tsunami in South Asia is estimated to have caused 150,000 fatalities. AIDS in 2004 is then the equivalent of 23 tsunamis. Imagine, unrelentingly tsunami-like casualties every fortnight from people dying from HIV-1! While it is laudable that the World Health Organization has a goal to treat three million HIV-1 positive individuals globally using anti-retroviral (ARV) medicine over the next five years, that approach will unlikely address the full magnitude of the AIDS problem, especially in developing nations. On the other hand, 100 million infants (even those in remote regions of the world) receive basic vaccinations each year. This fact suggests that when an AIDS vaccine does become available, that vaccine could be logistically and practically effective.in vivo.Separately, statistics from the World Cancer Report for the year 2000 show that 5.3 million men and 4.7 million women developed malignancy, and altogether 6.2 million persons died from cancer worldwide. The American Cancer Society estimates that approximately 553,000 individuals succumb to cancer in the United States each year. I see HTLV-I, the etiological agent for adult T-cell leukaemia (ATL), as perhaps the best retroviral system for retrovirologists to study human cancer. Substantive progress has indeed been made in our understanding as to how HTLV-I transforms cells in tissue culture [Retrovirology, I am also optimistic that I started this project at an age that provides ample time to develop this journal into a premier research forum.I first started studying viruses in the fall of 1977 at age 19 as a MD-PhD student at the Johns Hopkins school of medicine. For me personally, 2004 marked over a quarter-century of virus-research. At age 46, the unbridled youthful optimism of 19 is now tempered by realizations of physical and career limitations . Nonetheless, I am optimistic and hopeful that, despite enormous odds, the opportunity to see a successful AIDS vaccine will come before I leave retrovirology research in 20 or some years. Regarding Retrovirology's progress in our first year.Let me conclude this writing by thanking all authors, reviewers, Editorial board members, and our wonderful staff at Biomed Central who have contributed to


Haematuria is a classical symptom of urological disease often signifying a primary bladder cancer. Rarely, however, the presence of blood in the urine can be due to secondary spread of tumours into the bladder from distant sites. Notably this has been reported to occur in breast cancer, malignant melanoma and gastric cancers. Haematuria due to spread from a primary oesophageal cancer to the bladder has never been reported. We present a case of haematuria confirmed histologically to be due to metastases from a primary oesophageal tumour. Oesophageal cancer is capable of spread to all three neighbouring compartments  and therefore has the potential to spread to unusual sites. Clinicians should always carefully regard haematuria in a patient previously treated for cancer and retain a high index of suspicion for distant metastases as being the cause. Haematuria is a commonly encountered symptom. It often represents the presence of serious disease such as a malignancy within the bladder. The majority of bladder tumours tend to be primary, and histologically these are usually transitional cell carcinomas. We present a case of haematuria which occurred due to metastases from a primary oesophageal carcinoma diagnosed 2 years prior and treated curatively.A 45-year old male presented to our unit with acute onset macroscopic haematuria. His past medical history was significant in that he had been diagnosed with adenocarcinoma of the distal oesophagus 2 years prior and had undergone curative resection after neo-adjuvant chemotherapy. At that time the tumour was found to be poorly differentiated with evidence of local nodal spread. He had been reviewed regularly by the oncologists and remained asymptomatic until the onset of frank haematuria.He subsequently underwent cystoscopy, which revealed a solid bladder tumour on the right lateral wall, which was treated with trans-urethral resection. Pathological examination confirmed a poorly differentiated mucus-secreting adenocarcinoma, identical histologically to the original oesophageal tumour Figures  and 2.A diagnosis of metastatic oesophageal adenocarcinoma to the bladder was made. A CT scan did not demonstrate any pelvic tumour outside the bladder and therefore metastasis by the trans-coelomic route was essentially excluded, indicating haematogenous spread of the primary oesophageal carcinoma. The patient was referred for further oncological therapy but unfortunately died 4 months later from disseminated carcinoma.Metastatic tumour spread to the bladder constitutes approximately 2% of all bladder neoplasms . Gross hThe bladder can be the recipient of metastatic tumour spread from a potentially large variety of primary sites. Most commonly direct invasion can occur from adjacent tumours of the lower gastrointestinal tract (33% of secondary neoplasms), prostate 19%) and female genital tract (11%) 9% and fe-5. The mDespite curative intent, surgical resection of oesophageal adenocarcinoma is associated with an overall tumour recurrence rate of 66% at 5 years .In autopsy studies, isolated lymph node metastases were found in approximately one half of patients with end-stage oesophageal carcinoma, with a similar proportion having combined lymph node and visceral metastases. Isolated visceral spread however is rare, accounting for only a handful of cases of primary oesophageal tumour spread .Notable sites of haematogenous dissemination of primary oesophageal carcinoma to distant organs include bone, liver, skin, lungs, adrenals, brain and peritoneum in descending order of frequency . A few aInterestingly, rare synchronous primary tumours of the bladder and oesophagus have been described ,16 but hHaematuria may be the only clinically apparent symptom of metastatic tumour spread to the bladder from a potentially large number of primary sites and therefore should be considered by all clinicians irrespective of specialty.The author(s) declare that they have no competing interests.


C677T, PTHRA20210G, Factor V Leiden polimorphisms and/or inhAfter this screening we selected 39erited protein C, protein S, AT III deficiency) or acquired thrombophilia . However, because its high predictive negative value in case of suspected thrombosis, increased d-dimer has been often associated to subclinical thrombophilia. The aim of this study is to investigate the role of d-dimer as first marker of thrombophilia in women affected by unexplained infertility and subsequently to search the cause of increased d-dimer, such as inherited and/or acquired thrombophilia.D-dimer is considered a marker of hypercoagulable state and of endogenous fibrinolysis, so increased d-dimer is detectable in patients affected by thrombosis. Yet, several studies showed that also infertility, in particular secondary infertility due to recurrent fetal losses, has been often related to thrombotic events, in particular in women carrying thrombotic risk factors such as inherited thrombophilia  showed increased plasma d-dimer, in group B were included 14 patients with normal plasma level of d-dimer. After this step all 39 patients were screened for MTHFR2 test, differences were considered to be significant if p < 0.05.Statistical analysis was based on χD-dimer was increased in 25/39 and 20/25 showed inherited/acquired thrombophilia while patients with normal d-dimer showed inherited/acquired thrombophilia in 7/14 .D-dimer is a well known marker of hypercoagulable state, in particular its high predictive negative value in case of suspected thrombosis has been recognised by several reports. Yet, increased d-dimer has been identified also for subclinical thrombophilia besides for vascular thrombosis. Our data, in fact, for the first time suggest an interesting role of d-dimer to identify women affected by unexplained primary or secondary infertility and thrombophilia. So, probably there is a role for d-dimer in these subjects for its predictive positive value. Of course, further data on large based population are needed to confirm our results, because these findings may speed up a diagnostic screening in these patients also for a good cost/effectiveness of this test. D-dimer is considered a marker of hypercoagulable state besides of endogenous fibrinolysis, so increased d-dimer is detectable in patients affected by arterial and/or venous thrombosis . Yet, seWe selected 79 women affected by primary or secondary sterility  referred to our sterility center. We excluded 40 patients affected by hydrosalpinx, uterine fibroids, uterine malformations, luteal insufficiency, anovulation, cytogenetical alterations, infectious diseases, endocrinological diseases , and by immunological diseases .After this screening we selected 39 patients . These 39 patients were tested for d-dimer. D-dimer was measured by several methods ; d-dimerC677T), Factor V Leiden gene polimorphism (FVL), prothrombin A20210G gene polimorphism (PTHRA20210G), protein S deficiency, protein C deficiency, antithrombin III deficiency (AT III), lupus anticoagulant, IgM and/or IgG anticardiolipin autoantibodies   affected by sterility with increased d-dimer levels, showed inherited and/or acquired thrombophilia [, as reported in table 2  showed inherited and/or acquired thrombophilia . Data of patients of group C are summarised in table 2 ('see Five patients of group C (i.e. control group) (33.3%) showed increased d-dimer as molecular markers of ongoing proximal DVT confirmed by ultrasound vascular examination associated to CUS; moreover, all five patients revealed an underlying inherited and/or acquired thrombophilia  was not discovered.So, as showed in table 3 'see ', increaIn this report for the first time the role of d-dimer was investigated in diagnostic screening of patients affected by sterility and this is a really innovative data available in this clinical setting.D-dimer is a fibrin degradation product which usually is extensively screened in patients with suspected thrombosis and/or pulmonary embolism . An incrSo, our data showed that patients of group A, carrying increased d-dimer, has been extensively screened for inherited and/or acquired thrombophilia and 80% of them revealed a well known molecular condition associated to hypercoagulable state which may explain increased d-dimer levels  should be performed in order to avoid a misinterpretation.Also group B, with normal d-dimer levels, showed an increased rate of thrombophilia . However, this aspect seems to be in agreement with other reports in which other thrombophilic conditions were more frequent than clotting inhibitor deficiencies  [A clear relationship between thrombophilia and recurrent foetal loss has been reported for inherited deficiency of clotting inhibitors  ,36, but d so on) -20.FVL gene polymorphism has been frequently found in women affected by recurrent fetal loss, although the frequency of FVL differs in each study ,24. ThesC677T homozygosity has been found in our study population (six cases in group A and one case in group B), so confirming a clear role of homocysteine metabolism and of the related hypercoagulable state in sterility pathophysiology [An increased MTHFRysiology -40. Of cysiology .A20210G in women affected by sterility (five cases in group A and five cases in group B), and these data seem to be different from data reported by Pickering et al [We found also an increased frequency of PTHRng et al  and Deitng et al  and in ang et al ,25. As wng et al ,45.A really interesting data is the absence of APS from our study population and this data differs from data of the Literature. A possible explanation could be offered by different selection criteria: we exclude, in fact, women with immunopathological diseases , so excluding the most common causes of secondary APS and so searching only primary APS that is more rare than primary .In conclusion, in this investigation both groups of women affected by sterility, group A and B, showed increased incidence of thrombophilia compared to control group . This approach may play a role not only in differential diagnosis of sterility but also in the early diagnosis of sterility due to thrombophilia. After the first step in which d-dimer may be evaluated, causes of increased d-dimer should be subsequently identified in order to start a possible antithrombotic treatment soon.Nevertheless thrombophilia may be present in few cases also in subjects with normal d-dimer, it should be investigated always if other causes of sterility are not present.So, we strongly suggest to test d-dimer in patients affected by sterility as first step of a possible underlying thrombophilia in order to early identify the cause of thrombophilia and its prompt treatment but other data should be confirmed by further investigations on large based population.Thrombophilia frequency in studied groupsClick here for fileThrombophilia in studied groupsClick here for file2 methodstatistical analysis according with χClick here for file


Self-reported information has commonly been used to monitor mammography utilization across populations and time periods. However, longitudinal investigations regarding the prevalence and determinants of inconsistent responses over time and the impact of such responses on population screening estimates are lacking.Based on longitudinal panel data for a representative cohort of Canadian women aged 40+ years  assessed in the 1994–95 (baseline) and 1996–97 (follow-up) National Population Health Survey (NPHS), we examined the prevalence of inconsistent self-reports of mammography utilization. Logistic regression models were used to estimate the associations between women's baseline sociodemographic and health characteristics and 2 types of inconsistent responses: (i) baseline reports of ever use which were subsequently contradicted by follow-up reports of never use; and (ii) baseline reports of never use which were contradicted by follow-up reports of use prior to 1994–95.Among women who reported having a mammogram at baseline, 5.9% : 4.6–7.3%) reported at follow-up that they had never had one. Multivariate logistic regression analyses showed that women with such inconsistent responses were more often outside target age groups, from low income households and less likely to report hormone replacement therapy and Pap smear use. Among women reporting never use at baseline and ever use at follow-up, 17.4% (95%CI: 11.7–23.1%) reported their most recent mammogram as occurring prior to 1994–95 (baseline) and such responses were more common among women aged 70+ years and those in poorer health.Women with inconsistent responses of type (i), i.e., ever users at baseline but never users at follow-up, appeared to exhibit characteristics typical of never users of mammography screening. Although limited by sample size, our preliminary analyses suggest that type (ii) responses are more likely to be the result of recall bias due to competing morbidity and age. Inconsistent responses, if removed from the analyses, may be a greater source of loss to follow-up than deaths/institutionalization or item non-response. In the absence of organized screening, self-report is often the only means to monitor mammography utilization and to investigate trends in uptake at the population level . In CanaFew evaluations of the reliability of mammography self-report are reported in the literature. Excellent test-retest reliability for having ever had a mammogram was reported in interviews conducted 1 week, 6–30 days, or 6–8 months after an initial interview, while reliability within the past year varied from excellent to good ,23,24. IBased on data from the longitudinal panel of the National Population Health Survey (NPHS), the present study examines the prevalence and determinants of inconsistent self-reports of mammography utilization among Canadian women aged 40 years and older and quantifies the extent that inconsistent self-reports of mammography use contribute to biased estimates of mammography utilization and uptake. To our knowledge, this is one of the first studies of mammography utilization to provide specific longitudinal data on the determinants of inconsistent responses over time and the impact of such responses on population screening estimates.The National Population Health Survey (NPHS) is a survey of the Canadian household population. Initiated in 1994–95 and repeated biennially, it is a split panel survey, combining repeated cross-sectional components with the longitudinal follow-up of a panel of respondents. A representative sample of Canadian household residents aged 12 and older from all ten provinces was sampled using a multistage probability design with stratification and clustering at various stages. The overall response rate for the baseline 1994–95 survey was 89 percent with a 96 percent response rate for the selected panel respondent . On follThis study evaluated data from longitudinal panel respondents of the 1994–95 (baseline) and 1996–97 (follow-up) waves of the NPHS to examine inconsistencies in reported mammography utilization among women aged 40+ years at first contact. Questions about mammography use were administered to female respondents through a personal interview conducted in 1994–95 and repeated by telephone approximately two years later. In both survey years, women were asked the identical question: "Have you ever had a mammogram, that is, a breast x-ray?". Those with positive responses were further probed for the time and reason of their most recent mammogram. All women provided their own health-related information; no proxy responses were allowed.Analyses were restricted to women aged 40 and older (at baseline) who participated in the first two waves of the NPHS and consented to share their information with federal and provincial governments. Two types of inconsistent responses were assessed: (i) baseline reports of ever use which were contradicted by follow-up reports of never use; and (ii) baseline reports of never use which were contradicted by follow-up reports of use prior to 1994–95. Multivariate logistic regression techniques were used to evaluate the associations between women's baseline sociodemographic and health characteristics and type (i) inconsistent responses. Variables significant at p ≤ .05 in age-adjusted analyses were eligible for entry in the multivariate logistic models. Sample size constraints permitted only simple bivariate, rather than multivariate exploration of factors associated with reports reflecting inconsistent timing of most recent use at follow-up (type (ii) response). Estimates were weighted to reflect baseline population characteristics. To account for stratification and clustering in the NPHS sampling design, 95% confidence intervals for parameter estimates were calculated using exact standard errors generated through bootstrap re-sampling methods . All staweighted according to 1994/95 population characteristics whereas the frequency data represent actual numbers of women surveyed.Of the 3,535 women aged 40+ years who responded to the ever/never mammography question in both survey waves Figure . Among wTable Follow-up interviews were completed, on average, 1.98 years from the baseline survey (range 1.19–3.01 years). Of the 293 women who reported never use at baseline and ever use at follow-up, 17.4 percent (95%CI: 11.7–23.1) reported a time for their most recent mammogram at follow-up that was inconsistent with never use at baseline. Despite baseline reports that they had never had a mammogram, approximately half of these women reported having had a mammogram at least 5 years ago. Although limited by small numbers, determinants of such inconsistent responses were assessed with simple bivariate analyses. Inconsistencies in timing occurred more often in older women. Compared to women aged 50–69, those 70 and older were more likely to report (at follow-up) that their most recent mammogram had occurred prior to 1994–95, despite a report of never use at baseline . Women reporting fair or poor self-rated health were also more likely to report a time for their most recent mammogram at follow-up that was inconsistent with never use at baseline .Depending on how inconsistent responses are handled, different measures of use and uptake of mammography may be obtained. The lack of a gold standard such as a medical chart for validation makes the choice of a corrective measure unclear. If inconsistent ever/never responses are included in the analysis unchanged, 67.3 percent (95% CI: 65.1–69.5%) of women would be classified as ever having had a mammogram in 1994–95 while 71.7 percent (95% CI: 69.6–73.7%) would be classified as ever users in 1996–97. Conversely, if it is assumed that inconsistent ever/never responses represent false-positive responses at baseline (an assumption supported by our study findings), the 1994–95 prevalence estimate becomes 63.3 percent (95% CI: 61.0–65.6%), demonstrating an absolute increase in mammography use of 8.4 percent (95% CI: 7.1–9.6%) by this cohort of women by 1996–97.Although a limited number of studies have assessed the reliability of mammography self-report -25, detaAlthough direct comparison with our sample was not possible due to the expectation that behavior might have changed in a 2-year time span, earlier findings from longitudinal studies of fairly affluent  and low-Our analyses of factors associated with inconsistent ever/never responses indicate that women reporting ever having had a mammogram at baseline but never use at follow-up exhibited many of the sociodemographic and health behaviour characteristics  commonly observed among non-participants in mammography screening in previous studies -38. SuchValidation studies also provide support for our assumption that inconsistent ever/never responses  are most likely to be explained by false-positive responses at baseline. Women are more likely to falsely claim having had a mammogram, than not having one ,17,39,40Among women who reported never use at baseline and ever use at follow-up, approximately 17.4 percent reported a time for their most recent mammogram (at follow-up) that was inconsistent with never use at baseline. If respondents truly initiated mammography use subsequent to the baseline interview, they overestimated the time elapsed since their mammogram. Such a finding is relatively inconsistent with previous studies that have generally found that women tend to underestimate the time since their last mammogram -13,15,17Unfortunately, no gold standard was available to assess the validity of the responses women generated. Thus, the proportion of consistent responses that may actually represent invalid responses is unclear. Nor was it possible to distinguish errors in recall of timing from false reports of mammography utilization. Further, the reasons for providing inconsistent responses can only be inferred. The possibility of data entry errors is remote. Although data entry checks for consistency between survey cycles were not included among the comprehensive quality control strategies implemented by Statistics Canada, computer assisted interviewing was used by highly trained interviewers. Also, data entry errors would be expected to occur randomly and not disproportionately among women outside of the target age range or with relatively lower socioeconomic status, as observed in the present study. However, several plausible explanations for the inconsistencies exist, including survey methodology changes and deliberate or inadvertent provision of inaccurate responses by the respondent.We cannot exclude the possibility that interview changes  prompted women who reported ever use in 1994/95 to alter their response in 1996–97. The NPHS used an initial personal interview to foster a good long-term relationship with the panel representative, but the cost and logistics of traveling to different regions was prohibitive. Therefore, unless the respondent objected or had no phone, future interviews (including the 1996–97 cycle) were conducted by telephone . The neeCognitive research has implicated comprehension as a barrier to providing valid, reliable survey responses. Several researchers employ the lead-in question 'Have you ever heard of a mammogram, that is a breast x-ray?' to identify comprehension difficulties ,16,21,42There are several possible explanations for the higher rate of inconsistent responses observed in the present study. The focus of the NPHS questionnaire was not limited to preventive practices nor was it designed for reliability testing. The length and comprehensiveness of the NPHS may have contributed to greater respondent fatigue. Also, the longer time interval between surveys, relative to that observed in other studies, may have contributed to instability in responses. A more favourable level of concordance among responses may be obtained by studies that apply eligibility criteria that ensure more accurate responses  ,10,15. FThe inconsistent data reported here, if removed from longitudinal analyses, could yield losses to follow-up equivalent to or greater than other sources of attrition  over the planned 20 year course of the NPHS. The 1998–99 NPHS included probing questions to reduce inconsistencies and it should alleviate many of the problems evident here . HoweverIn summary, inconsistent responses represent a challenge to longitudinal, population-based evaluations of breast screening practices. Losses from inconsistent data regarding mammography participation are not negligible and may contribute to inaccurate estimates of mammography uptake. Women reporting inconsistent ever/never use in the present study displayed characteristics typical of never users, favoring treatment of women's baseline responses as false-positive. Inconsistent responses regarding the timing of recent mammography practices, however, may be primarily related to the impact of age and competing morbidity on recall.The authors declare that they have no competing interests.CB and CM contributed to the initial and revised analyses of the NPHS panel data, the interpretation of the results and the initial drafting/editing of the manuscript. CB and JS were responsible for the data linkage with Statistics Canada (for the NPHS Share File) and for the revised bootstrap analyses. JS also contributed to the interpretation of the results and editing of the manuscript.The pre-publication history for this paper can be accessed here:


The cytokine leukemia inhibitory factor (LIF) mediates its biological effects through binding to its high affinity receptor made of the low-affinity LIF receptor subunit gp190 (LIF-R) and the gp130 subunit. LIF exerts several important effects in the liver, however, data on liver expression of LIF are scarce. The aim of this study was to examine the expression of LIF and LIF-R in human liver.LIF expression, analyzed by immunohistochemistry, was barely detectable in normal liver but was strong within cirrhotic fibrous septa and was found in spindle-shaped cells compatible with myofibroblasts. Accordingly, cultured human liver myofibroblasts expressed high levels of LIF as shown by ELISA and Northern blot. Biological assay demonstrated that myofibroblast-derived LIF was fully active. RT-PCR showed expression of the LIF-D and M isoforms, and also of low levels of new variants of LIF-D and LIF-M resulting from deletion of exon 2 through alternative splicing. LIF receptor expression was detected mainly as a continuous sinusoidal staining that was enhanced in cirrhotic liver, suggestive of endothelial cell and/or hepatocyte labeling. Immunohistochemistry, flow cytometry and STAT-3 phosphorylation assays did not provide evidence for LIF receptor expression by myofibroblasts themselves. LIF secretion by cultured myofibroblasts was down regulated by the addition of interleukin-4.We show for the first time the expression of LIF in human liver myofibroblasts, as well as of two new isoforms of LIF mRNA. Expression of LIF by myofibroblasts and of its receptor by adjacent cells suggests a potential LIF paracrine loop in human liver that may play a role in the regulation of intra-hepatic inflammation. Leukemia inhibitory factor (LIF) belongs to the interleukin (IL)-6 family of cytokines, together with IL-11, ciliary neurotrophic factor, cardiotrophin-1, oncostatin M and neurotrophin-1/B cell stimulating factor-3. LIF is widely expressed in tissues and in many isolated cells. LIF expression is commonly up-regulated during inflammation. Nevertheless, its role seems to be complex as both pro- and anti-inflammatory properties have been described for that cytokine. Although LIF, like IL-6, is able to drive a significant acute-phase reaction in non-human primates . LIF exeSeveral isoforms of LIF consecutive to alternative splicing have been described. The second and third exons are common to all isoforms, whereas there are 3 alternate first exons – D, M, and T. The fate of the mature LIF molecule is highly dependent on exon 1 usage; thus, the human LIF-D transcript encodes a secreted protein that is biologically active and can signalize via the LIF receptor. The human LIF-M transcript does not contain any in-frame AUG, but it is known to be translated into both secreted and intracellular proteins . FinallyAs outlined above, LIF is potentially involved in liver physiology and pathophysiology; however, data on liver expression of LIF are scarce. LIF expression was not detected in normal rat liver but it was highly induced following partial hepatectomy, mainly in non- parenchymal cells , suggestTherefore, the aim of this study was to examine the expression of LIF and of its specific receptor gp190 in human liver. Results obtained with immunostaining of liver sections led us to examine LIF expression by cultured liver myofibroblasts, cells that play a major role in liver fibrogenesis.Human liver tissues were examined for LIF expression by immunohistochemistry. In normal liver, a faint but consistent LIF expression was detected in the stroma of portal tracts Fig. . No signAnalysis of total RNA from cultured human liver myofibroblasts by Northern blot revealed a single 4.5 kb transcript Fig. . RT-PCR in vivo.As shown in Figure In order to test whether myofibroblasts transcribed all the alternatively spliced D, M or T first exons, a first set of RT-PCR experiments was carried out using the forward primers chosen in the alternative D, M or T first exons , and a common reverse primer chosen in exon 3 (hLIF-N4) Table . SimilarThe sizes of the additional products obtained with the hLIF-N4 primer were shorter by about 200 bp, which is the exact size of exon 2, raising therefore the possibility that the shorter PCR products were derived from a hitherto not described mRNA species where exon 2 was deleted through alternative splicing. In order to investigate this possibility, the short D and M fragments were cloned into a plasmid and sequenced. Sequencing indeed revealed that the first exon (either D or M) was directly spliced to the third one resulting in new, short transcripts that we have designated s-LIF-D and s-LIF-M.The existence of these alternate transcripts could be observed in several hepatocellular carcinoma cell lines  and in the HEK293 cell line, derived from embryonic human kidney Fig. . They weThe relative abundance of the variant transcripts relative to the classical transcripts was studied using a semi-quantitative RT-PCR method, where PCR was carried out for varying cycles numbers. As can be seen in Fig. The expression of the gp190 subunit by liver cells was then examined by immunohistochemistry. Five different antibodies, directed against separate epitopes, were used and yielded similar results. In normal liver tissue, LIF receptor (LIF-R) expression was detected as a continuous sinusoidal staining and in the stroma of portal tracts Fig. . In the In a subsequent step, cultured human liver myofibroblasts were examined for their membrane expression of gp190 using flow cytometry. Adherent cells were released by action of EDTA and subjected to anti-gp190 labeling. No detectable levels of gp190 were observed with any of the 5 antibodies, although gp130 expression could be detected with the B-R3 antibody. In order to detect a low-level expression of functional LIF-R, myofibroblasts were exposed for 15 minutes to 10 ng/ml recombinant LIF; then, STAT-3 phosphorylation was examined by Western blot. No consistent effects were seen in 7 separate experiments. When a very weak signal was occasionally seen, it was not inhibited by 2 separate blocking antibodies to LIF-R (data not shown). Finally, production of soluble receptor was never detected in myofibroblast supernatants either.6 cells/48 h. These levels are similar to those produced by activated lymphocytes, a classic source of LIF, and suggest that liver myofibroblasts may be a major source of LIF during chronic liver diseases. Our results are in agreement with data obtained in the rat showing that non-parenchymal cells, possibly activated stellate cells , express LIF dCTP, Hybond N+ membrane, ECL reagent, and the Ready-to-go DNA labeling kit were from Amersham . Ultrahyb solution was from Ambion . Recombinant human IL-4 was a gift from Schering-Plough . Anti-gp130 mAb B-R3 was from Diaclone , anti-gp80 mAb M91 was from Coulter-Immunotech , anti-phospho-STAT-3 (Tyr705) was from Cell Signaling Technology . All other chemicals were from Sigma .Culture medium and additives, recombinant human epidermal growth factor (EGF) and Moloney Murine Leukemia Virus reverse transcriptase were from Gibco-BRL . Taq polymerase and the pGEM-Teasy plasmid were from Promega . The Qiagen RNeasy minikit was from Qiagen . The dCTP, by random priming using the Ready-to-go kit. Hybridization was performed using the Ultrahyb solution. The blots were washed in stringent conditions .Total RNA was isolated using the Qiagen RNeasy minikit. For Northern blot, 2 μg RNA were separated on a 1.0% agarose gel containing ethidium bromide in MOPS buffer. Running buffer and gel contained 0.2 M formaldehyde. The RNAs were transferred onto a Hybond Numan LIF . Probes 2, 0.4 mM dNTPs, 0.2 mM primers, and 1.25 units of Taq polymerase. Then, an aliquot of the reaction was analyzed by agarose gel electrophoresis. The primers used are listed in Table One μg of total RNA was reverse-transcribed using MMLV-RT. An aliquot was used for PCR. Thirty five cycles were performed, each consisting of 94°C, 30 s; 60°C, 30 s; and 72°C, 30 s. PCR was performed in 50 μl of a reaction buffer containing 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 1% Triton X-100, 1.5 mM MgCl+ system HRP detection and revealed with liquid diaminobenzidine (Dako). As negative control, we used either a clarified mouse myeloma ascites  or a rabbit non-immune immunoglobulin fraction (Dako), at the same concentration as the respective antibodies. Sections were examined with a Zeiss Axioplan 2 microscope . Images were acquired with an AxioCam camera  by means of the AxioVision image processing and analysis system (Carl Zeiss Vision).A commercially available polyclonal antibody against human LIF , and different monoclonal antibodies against gp190, previously described , were us5 cells were incubated for 30 min at 4°C with saturating concentrations (10 μg/ml) of the indicated antibody in 0.1 ml of PBS supplemented with 1 % bovine serum albumin (BSA) and 0.1 % human polyclonal IgG . Then, cells were washed twice with the same buffer and incubated for 30 min at 4°C with the FITC-conjugated goat anti-mouse IgG. After washing with PBS, the cells were resuspended in 0.14 ml of PBS containing 1% formaldehyde (v/v) and analysed by flow cytometry with a three color FACScalibur flow cytometer  equipped with the CellQuest software. Control stainings used the second antibody only.For each staining, 2 × 10The sandwich ELISA assay for soluble gp190 measurement has already been described . It usesConfluent cultures of myofibroblasts were left for 2 days in serum-free medium, and subsequently exposed to recombinant human LIF for 15 minutes . Then, cTH performed most of the cell culture and RT-PCR experiments and cloned the new LIF variants. AD and NS performed the immunohistochemistry experiments and prepared the corresponding figures. JLT provided the monoclonal antibodies to LIF-R and participated in the design of the experiments showing the secretion of active LIF. SD performed the LIF ELISA assays, the biological activity testing and flow cytometry experiments. VN performed the experiments looking at STAT-3 phosphorylation. JFB provided the human liver samples. JFM was involved in the coordination of the project and in the critical reading of the manuscript. JR conceived the study and was the main coordinator and responsible for the redaction of the manuscript. All authors read and approved the final manuscript.


The cycle stage was verified by determination of progesterone and LH concentrations in the peripheral blood samples collected prior to euthanasia.During the preovulatory surge of gonadotropin-releasing hormone (GnRH), a very large amount of the peptide is released in the hypothalamo-hypophyseal portal blood for 24-36H00. To study whether this release is linked to a modification of the morphological organization of the GnRH-containing neurons, The distribution of GnRH-containing neurons throughout the preoptic area around the vascular organ of the lamina terminalis was studied following visualisation using immunohistochemistry. No difference was observed in the staining intensity for GnRH between the different groups. Clusters of GnRH-containing neurons (defined as 2 or more neurons being observed in close contact) were more numerous during the late follicular phase (43 ± 7) than during the luteal phase (25 ± 6), and the percentage of clusters was higher during the beginning of the follicular phase than during the luteal phase. There was no difference in the number of labelled neurons in each group.These results indicate that the morphological organization of the GnRH-containing neurons in ewes is modified during the follicular phase. This transitory re-organization may contribute to the putative synchronization of these neurons during the surge. The molecular signal inducing this plasticity has not yet been identified, but oestradiol might play an important role, since in sheep it is the only signal which initiates the GnRH preovulatory surge. Gonadotropin-releasing hormone (GnRH), the peptide responsible for the regulation of secretory activity of the pituitary gonadotropes, is found in a diffuse neuronal system situated in the preoptic area and anterior hypothalamic area . The neuThe pulsatile nature of GnRH secretion, as well as the large amount of peptide released during the preovulatory surge, indicate that the activity of GnRH-containing neurons can be synchronized, and it has been demonstrated that the GnRH neurons of the preoptic area receive synaptic contacts from GnRH neurons -7. OestrThe aim of this work was to study the number, distribution and potential contacts present between GnRH-containing cells in intact ewes under natural conditions, focusing particularly on the neuroanatomical organization of these neurons around the preovulatory surge when a high level of oestradiol is released by the growing follicles.Because there are some individual variations in the response to the induction of ovulation treatment , an analysis of plasma hormone concentration was carried out to confirm the allocation of animals to experimental groups according to the characteristic hormonal level previously described . In grouFour of the eight animals in experiment 2 were killed during the LH surge (2 during the ascending and 2 during the descending part). The 4 remaining animals were killed 4–6 hours after the end of the LH surge and were discarded from the study.GnRH-containing neurons were distributed in the preoptic area fig  as previThere was no difference in the total number of GnRH-ir neurons in the experimental groups  fig . HoweverThe percentage of clusters was higher in group 1 (15.1 ± 1.2) than in groups 2 (11.8 ± 1.0), 3 (8.3 ± 0.4) and 4 (10.7 ± 0.9) fig .In the second experiment, four additional animals were studied during the GnRH surge, and the data compared with those of the group 2 ewes in the first experiment. As shown in table Our observations indicate that the organization of GnRH-ir neurons was modified during the oestrous cycle, since more clusters were observed during the follicular phase. However, we did not observe any difference between the ascending and descending phases of LH release in group 2. Therefore, our data support the hypothesis that there are changes in communication between GnRH cell bodies prior to the initiation of the preovulatory GnRH surge.The increase in the number of clusters was not mirrored by a parallel change in the total number of GnRH cell bodies. However, as the number of clusters was relatively small compared to the GnRH cell population (constituting 10% to 15%), the putative increase in the number of GnRH-ir neurons could have been masked due to the large variations in the number of labelled neurons between animals. An alternative hypothesis is that the number of GnRH neurons remains constant, and that the variations in the number of clusters is linked to variations in the peptide level, which are consistent with the variations in the staining intensity observed in all animals. In this case, the level of GnRH would be higher in clustered neurons than in isolated neurons at the moment of the preovulatory surge, and therefore neuronal communication would be more efficient in clustered neurons. The percentage of clusters increased only in group 1, while the difference between the number of clusters in groups 2, 3 and 4 was not statistically significant. This special morphological organization may constitute an anatomical support for the synchronization of GnRH neuronal activity needed to induce the GnRH preovulatory surge.Clusters of GnRH-ir cell bodies have been previously described in sheep  . Animals were fed daily with hay, straw and corn, and water was available ad libitum. All of the ewes had lambed at least once.Intact adult Ile de France ewes (n = 28) from the laboratory flock, maintained under natural photoperiod were used. Two experiments were performed (one year apart) during the breeding season  were killed at specific time points in the oestrous cycle. In order to synchronize oestrous cycles, animals were treated with an intravaginal progesterone-releasing device  for 14 days. Following removal of the progesterone device, animals were treated (i.m.) with 500 UI of Pregnant Mare Serum Gonadotropin (PMSG), a treatment known to induce the LH surge and ovulation in sheep (for review see ). It hasGroup 1: Animals killed 1–2 hours after oestrus, i.e. before the LH surge onset, (n = 5)Group 2: Animals killed 8–12 hours after oestrus, i.e. during the preovulatory LH surge, (n = 5)Group 3: Animals killed 8 days after the preovulatory LH surge, (n = 5)Group 4: Animals killed 15 days after the preovulatory LH surge, (n = 5)Analysis of LH secretion (see paragraph below) revealed that animals in group 2 in experiment 1 were killed either in the ascending (n = 2) or descending (n = 2) phase of the LH surge. Therefore, in order to increase the N for each sub-group, 8 animals were synchronised as previously described  and killed 12 and 20 hours (n = 4) after oestrus.For both experiments, blood samples were collected by venepuncture every 2 hours from 18 hours after PMSG administration until sacrifice (groups 1 and 2) and daily thereafter (groups 3 and 4).Plasma samples were assayed in duplicate for LH with 100 μl aliquots of plasma using a previously described RIA method . All samiv 10 min before decapitation. Both carotids were catheterised and the head was perfused with 2 litres of 1% sodium nitrite followed by 4 litres of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After perfusion, the brain was quickly removed and a block containing the whole preoptic area was isolated and post-fixed for 2 hours at +4°C in the same fixative. The blocks were immersed in 30% sucrose containing 0.1% sodium azide at +4°C for 5 days.A solution of heparin  was injected Serial 40 μm coronal sections were obtained using a freezing microtome and stored at +4°C in PBS containing 0.1% sodium azide. Every fifth section was stained for GnRH immunoreactivity.2O2 and then preincubated for 30 min at room temperature in blocking solution . Sections were exposed to GnRH primary antiserum at a dilution of 1/10,000 in PBS containing 0.3% triton, 0.2% HSA, 0.1% sodium azide for 4 days at +4°C. After washing in PBS, sections were incubated for 3 hours at room temperature in the secondary antibody (1/500 with PBS containing 0.2% BSA). After washing in PBS, sections were incubated in rabbit peroxidase-antiperoxidase complex diluted 1/10,000 in PBS containing 0.2% BSA overnight at +4°C. After washing twice in PBS and in Tris buffer (0.05 M pH 7.6), the sections were reacted with 3,3-diaminobenzidine tetrahydrochloride (0.02%) and nickel ammonium sulfate (0.25%) in the same Tris buffer containing 0.0025% H2O2 for 20 min. The reactions were stopped by rinsing the sections with Tris buffer. Sections were mounted onto gelatine-coated slides, dried, counterstained with a solution of neutral red for 5 min and mounted with a cover-slide using Depex.Sections were incubated for 2 hours at +4°C in PBS containing 0.3% triton X100 and 1% HThe characteristics and the specificity of the GnRH antibody were as previously described .i.e. about twice the maximum value of a pulse during the follicular phase for this breed and for this LH assay. For hormone values, a global comparison was carried out using a non-parametric ANOVA with exact general score test, and comparison between groups was carried out using the non-parametric exact permutation test for independent samples .The LH surge was assumed to start when LH concentration exceeded 6 ng/ml of plasma, The first observation of the sections allowed the immunoreactive area surrounding the OVLT to be determined fig. . We seleMB performed the immunohistochemical reaction and counted the neurones, AC and YT conceived the study, and participated in its design, coordination and in the collection of biological samples. BM participated in the final statistical analysis of results and corrected the manuscript.


Pattern Recognition, the protagonist posits a theory of jet lag: “Souls can't move that quickly, and are left behind, and must be awaited, upon arrival, like lost luggage.”In William Gibson's novel Science has yet to address the issue of a spiritual speed limit, but it is generally accepted that jet lag actually results from the upset of the body's circadian clock, a biochemical pacemaker that dictates daily rhythms in sleep and wakefulness as well as body temperature and metabolic activity. In humans, the circadian rhythm responds to many factors, but daytime–nighttime  cycles are one major regulator. It is possible to gradually reset an upset circadian clock; if travelers remain in the same place for long enough, their circadian rhythm will eventually adjust to the new time zone and ambient light patterns, and the symptoms of jet lag will disappear.Drosophila melanogaster. Two circadian proteins, Clock and Cycle, cooperate to induce expression of two other proteins, Per and Tim, and when levels of Per and Tim are high enough, they cooperate to shut off their own expression. This negative feedback loop leads to periodic fluctuations in the level of Per and underlies the circadian rhythm in flies. However, until recently, not much was known about the mechanics of the circadian clock in vertebrates.The more scientists know about the workings of the circadian clock, the closer they can come to manipulating it. Much is known about the molecular machinery of the circadian clock in the fruitfly, luciferase gene under the control of the promoter of the zebrafish circadian gene period3 (per3). Each cell of the transgenic fish has one normal copy of the per3 gene and one copy of the period3-luciferase fusion gene (per3-luc). Therefore, whenever per3 expression is normally turned on in a cell, the cell produces Per3 protein and also produces the luciferase protein.Maki Kaneko and Gregory Cahill have created a new tool for investigating the components of the circadian clock in vertebrates: a zebrafish that luminesces (glows) in sync with the periodicity of its circadian clock. To do this, the researchers created a transgene that places expression of the firefly per3 periodicity is not hardwired into zebrafish embryos; instead, per3 periodicity is entrained by alternating light–dark cycles, which must occur at specific stages in early development. Also, other external factors such as ambient temperature can influence both the level of per3 mRNA expressed in the animal and the magnitude of its protein-level oscillations. Because the establishment of circadian rhythms in the adult animal can be so strongly influenced by conditions experienced by the embryos, the authors suggest using a standardized set of conditions for the culture of transgenic embryos in future experiments involving adult fish.While characterizing their transgenic zebrafish, the authors made some interesting findings. First, contrary to earlier studies, the authors found that per3-luc transgenic zebrafish in forward genetic screens . In this case, mutagenized zebrafish could be examined for disruptions of per3-luc periodicity or expression. What is more, luminescence can be measured quickly and noninvasively, making this animal an ideal candidate for high-throughput screening aimed at identifying components of the circadian clock in the zebrafish. Thanks to luminescent fish, scientists may someday gain enough insight to make jet lag a thing of the past.Under these controlled conditions, Kaneko and Cahill anticipate that these transgenic zebrafish will be quite useful in examining the molecular machinery of the vertebrate circadian clock. For example, researchers can use the