C677T, PTHRA20210G, Factor V Leiden polimorphisms and/or inhAfter this screening we selected 39erited protein C, protein S, AT III deficiency) or acquired thrombophilia (primary antiphospholipid syndrome, acquired protein C, protein S, AT III deficiency, drugs induced 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 (p: < 0.05, s).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 (ie diabetes, subpituitarism), and by immunological diseases (inherited and/or acquired immunodeficiency, rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, vasculitis).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 [Subsequently d-dimer evaluation, in order to identify a possible inherited and/or acquired thrombophilia, all patients were screened for methilene-tetra-hydro-folate-reductase C677T gene polimorphism (MTHFRtibodies ,27. Moretibodies ,9-11.Furthermore, patients with increased d-dimer (group A, 25 patients) and patients with normal d-dimer (group B, 14 patients) were compared also for possible differences in molecular markers of inherited and/or acquired thrombophilia.2 test, differences were considered to be significant if p < 0.05.Statistical analysis was based on χWe found thrombophilia in group A, 80%, and in group B, 50%, so thrombophilia rate in all 39 selected was 65% if we consider together group A (i.e. women affected by sterility and showing increased d-dimer) and group B  (table 1, 'see C677T homozygosity, four FVL heterozygosity, five PTHRA20210G heterozygosity, three inherited Protein S deficiency, two showing combined defects (one MTHFRC677T homozygosity associated to protein S deficiency and one MTHFRC677T homozygosity associated to FVL heterozygosity), none protein C deficiency or AT III deficiency, none positive for the presence of lupus anticoagulant, none with increased anticardiolipin autoantibodies IgM and/or increased anticardiolipin autoantibodies IgG)]  affected by sterility with increased d-dimer levels, showed inherited and/or acquired thrombophilia [(six MTHFRe 2 'see '.C677T homozygosity, one FVL heterozygosity, five PTHRA20210G heterozygosity, none inherited Protein S deficiency, protein C deficiency, AT III deficiency, none presence of lupus anticoagulant, none with increased anticardiolipin autoantibodies IgM and/or IgG), as reported in table 2  showed inherited and/or acquired thrombophilia (one MTHFRe 2 'see '. All reC677T homozygosity, one protein S deficiency, one combined thrombophilia: FVL heterozygosity associated to protein S deficiency). 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 (e.g. FVL, MTHFRC677T homozygosity, antiphospholipid syndrome and so on) [A clear relationship between thrombophilia and recurrent foetal loss has been reported for inherited deficiency of clotting inhibitors (i.e. protein C deficiency, protein S deficiency, AT III deficiency) ,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 (e.g. rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, vasculitis), 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 (n = 3,537) 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 (OR = 6.96, 95%CI: 2.42–20.0). 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 (OR = 2.44, 95% CI: 0.99–6.05).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 (e.g., lower income, outside age groups targeted for screening, non-users of Pap screening and hormone replacement therapy) 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 (hLIF-D3, hLIF-M3 and hLIF-T5), 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 (HepG2, HuH7, Hep3B) 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 (i.e., myofibroblasts), express LIF [In this study, we demonstrate for the first time that LIF is expressed at low levels in normal human liver, whereas it is greatly increased in fibrotic liver, in a localization consistent with that of activated myofibroblasts. The slightly diffuse staining is suggestive of extracellular matrix deposition consistent with the expression of the M-type isoform of LIF. Experiments using cultured human liver myofibroblasts confirmed that these cells secreted extremely high levels of LIF in the range of 0.1–1 μg/10ress LIF ; this loOn the other hand, and in contrast with mesangial cells , liver mNonetheless, LIF receptor expression was detected by immunohistochemistry in human liver, in a peri-sinusoidal location. Similar results were obtained with 5 different antibodies directed to several epitopes of gp190. The pattern of continuous sinusoidal staining and the colocalization experiments are in favor of an expression in sinusoidal endothelial cells. However, we can not exclude staining of the sinusoidal domain of hepatocytes. In any case, these data indicate that cells close to LIF-producing myofibroblasts express LIF receptors and could thus respond to LIF in a paracrine fashion.th amino-acid (aa) and a termination at aa 88, the resulting protein bearing no homology with LIF. There are, however, several in-frame CUG codons within exon 3. Initiation at CUG 113 would result in the synthesis of a 125 aa polypeptide, recapitulating the sequence of the C-terminal part of LIF. Similar considerations apply to s-LIF-M that, in any case, does not contain an initiating AUG in exon 1. It should be emphasized that the lack of an AUG codon does not preclude the translation of the classical forms of LIF-M or LIF-T [This study led to the discovery of new LIF transcripts resulting from a direct splicing of exon 1 to exon 3. This was observed for both LIF-D and LIF-M. Those transcripts were present at much lower levels than full-length transcripts, as suggested by RT-PCR and by the fact that they do not appear on Northern blot; thus, their biological relevance can be questioned. Whether s-LIF-D or s-LIF-M transcripts are translated also remains hypothetical. In the case of s-LIF-D, initiation at the AUG within exon D would result in a reading-frame shift following the 6LIF secretion was dose-dependently decreased by IL-4, a known inhibitor of LIF secretion in other cell types ,12. IL-4For the first time, we show the expression of LIF in human liver myofibroblasts, as well as of two new isoforms of mRNA. Hepatic stellate cells and activated myofibroblasts have already been shown to synthesize a number of mediators involved in the control of inflammation, such as monocyte chemotactic-1 protein , or platHistologically normal/subnormal liver samples were obtained from macroscopically normal location in hepatectomy specimens, taken at a distance from a focal nodular hyperplasia (n = 5); a hemangioma (n = 1); or a colon cancer metastasis (n = 1). Cirrhotic specimens (n = 11) were obtained from patients undergoing liver transplantation for cirrhosis with associated hepatocellular carcinoma. In 10 out of 11 cases, the patients underwent liver transplantation. The cirrhosis etiologies were viral hepatitis C (n = 4); viral hepatitis B + D (n = 2); alcoholic (n = 4); or a combination of viral hepatitis B + C + alcoholic (n = 1).A portion of fresh tissue samples was routinely formalin-fixed and paraffin-embedded for diagnosis and a portion immediately frozen in liquid nitrogen-cooled isopentane and stored at -80°C. Five μm-thick serial frozen sections of each sample were air-dried on Super Frost/Plus slides (Menzel Glaser, Germany) and processed for immunohistochemistry. The procedures were in accordance with the European guidelines for the use of human tissues.32P]dCTP, Hybond N+ membrane, ECL reagent, and the Ready-to-go DNA labeling kit were from Amersham (Les Ulis, France). Ultrahyb solution was from Ambion (Austin, TX). Recombinant human IL-4 was a gift from Schering-Plough (Kenilworth, NJ). Anti-gp130 mAb B-R3 was from Diaclone (Besançon, France), anti-gp80 mAb M91 was from Coulter-Immunotech (Marseille, France), anti-phospho-STAT-3 (Tyr705) was from Cell Signaling Technology (Beverly, MA). 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 (Life Technologies, Cergy-Pontoise, France). Taq polymerase and the pGEM-Teasy plasmid were from Promega (Madison, WI). The Qiagen RNeasy minikit was from Qiagen (Courtaboeuf, France). The [αHuman hepatic myofibroblasts were obtained from explants of non-tumoral liver resected during partial hepatectomy and characterized as previously described ,23. MyofHuman LIF was measured using an ELISA based on two specific monoclonal antibodies, exactly as described previously . A stand3 per well, in 50 μl, in duplicates) were incubated in the presence of 50 μl of three-fold dilutions of cytokines or supernatant, as indicated. After three days at 37°C, 0.015 ml of a 5 mg/ml solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide , in PBS, was added to each well. After 4 hours at 37°C, 0.11 ml of a mixture of 95 % isopropanol + 5 % formic acid was added to the wells, and the absorbance values were read at 570 nm, in a Titertek Multiskan microplate reader (Labsystems, Les Ullis, France). The blank consisted of eight wells containing the cells incubated with the Ba/F3 culture medium without any added cytokine.The Ba/F3 proliferation assays were performed, as described previously , using t+ membrane by downward capillary transfer in running buffer. Examination of the stained membrane under UV light was used to confirm the quality of loading and transfer. The probe used was a 730 bp cDNA containing the whole coding sequence of human LIF [32P]dCTP, by random priming using the Ready-to-go kit. Hybridization was performed using the Ultrahyb solution. The blots were washed in stringent conditions (0.1X SSC, 0.1% SDS at 65°C).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 (Cappel Research Products, Durham, USA) 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 (Carl Zeiss Microscopy, Jena, Germany). 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 (R&D Systems, Minneapolis, Minnesota, USA), 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 (w/v, both from Sigma). 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 (Becton-Dickinson, Mountain View, CA) 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 (oestrus onset, preovulatory LH surge onset, etc.), 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 (group 1: 291 ± 38, n = 5, group 2: 347 ± 41 n = 4, group 3: 345 ± 38 n = 5, group 4: 241 ± 75 n = 4) 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 (one year later, during the breeding season) 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 (25,000 units) 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 (Stat Exact Software, Cytel Software Corporation, Cambridge, MA, USA).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 (or, more precisely, light–dark) 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


Eradication of the smallpox virus through extensive global vaccination efforts has resulted in one of the most important breakthroughs in medical history, saving countless lives from the severe morbidity and mortality that is associated with this disease. Although smallpox is now extinct in nature, laboratory stocks of this virus still remain and the subject of smallpox vaccination has gained renewed attention due to the potential risk that smallpox may be used as a biological weapon by terrorists or rogue states. Despite having the longest history of any modern vaccine, there is still much to be learned about smallpox vaccination and the correlates of protection remain to be formally defined. This Commentary will discuss the strengths and weaknesses of traditional smallpox vaccination in comparison with immunization using modified vaccinia virus Ankura (MVA), a non-replicating virus with a strong safety record but weakened immunogenicity. Smallpox (Variola major) is a virus that no longer exists in the wild, but during its reign it caused 20–30% mortality in previously unvaccinated individuals and often left survivors with deeply pitted scars for life . The las"I have purposely selected several cases in which the disease [i.e. cowpox] had appeared at a very distant period previous to the experiments made with variolous matter, to show that the change produced in the constitution is not affected by time." [The first line of defense against smallpox is vaccination. Smallpox vaccination is highly effective at protecting against lethal infection and even if only partial protective immunity is attained, this often still results in survival and decreased viral spread to others  with an inoculum containing vaccinia virus. After the infection has reached a point in which a great deal of exudate was observed, the purulent lymph material was scraped from the infected cow, clarified, and lyophilized in the presence of antibiotics. The inoculum is later reconstituted with diluent containing 0.25% phenol to further decrease bacterial contamination . Unlike most other vaccines that are administered by subcutaneous or intramuscular injection, vaccinia virus replicates poorly under these conditions and optimal vaccination occurs by scarification of the virus inoculum onto the skin surface . Viral r6 doses), generalized vaccinia (242 cases/106 doses), eczema vaccinatum (39 cases/106 doses), vaccinia necrosum (1.5 cases/106 doses), encephalitis (12 cases/106 doses), or death (~1 death/106 doses) [6 doses) whereas the mortality rate for older children and adolescents aged 1–4 or 5–19 is approximately 0.5 deaths/106 doses  . The morsee also  and Dryvsee also .6 smallpox vaccinations [Myopericarditis is a recently identified adverse event that occurs at a rate of ~124 cases/10inations ,15. In 2inations . A retroinations . FollowiTo overcome some of the problems associated with the current calf lymph smallpox vaccine (Dryvax), an improved tissue culture-derived vaccine has been recently developed . Unlike Both Dryvax and ACAM1000 represent replication-competent smallpox vaccines and since these vaccines are based on the use of live viruses, there is always an inherent risk of severe adverse events or death  in vaccinees that have unknown or undisclosed immunodeficiencies at the time of vaccination. To overcome the hazards of replicating viruses, a highly attenuated strain of vaccinia, designated Modified Vaccinia virus Ankara (MVA) was developed by growing the virus for >500 passages on chicken embryo fibroblasts (CEF) and following the loss of about 15% of its parental genome, it no longer was capable of replicating in most mammalian cells, including human cells . Other sEdward Jenner developed the first test for vaccine efficacy when he immunized subjects with cowpox and then later challenged them with smallpox by inoculation. If a subject showed no secondary smallpox lesions indicative of systemic spread, then the individual was believed to have protective immunity. He noted that cowpox-vaccinated individuals who developed a pox-like lesion at the vaccination site were fully protected against smallpox challenge. To this day, most studies still use the identification of a "take" (vesicular or pustular lesion) as evidence of successful vaccination. However, there are rare cases of revaccinated subjects who present with vesicle formation after smallpox vaccination, but show no detectably boosted cellular or humoral immunity  and M.K.ex vivo including vaccinia-specific IFNγ ELISPOT assays [+ T cells can be quantitated regardless of their cytokine profiles, but the main disadvantages with this approach include the lack of identified CD4+ T cell/MHC Class II epitopes, the requirement for knowledge of the MHC haplotype of the subject, and the inability to measure the total antiviral T cell response, which may be directed against any number of immunodominant and subdominant peptide epitopes.Quantitative immunology is beginning to gain acceptance as a measurement of vaccine efficacy, although the Jennerian vesicle at the site of smallpox vaccination still remains the primary endpoint of successful vaccination in most studies ,23-25. OT assays ,23,26-28T assays -33, or pT assays . Each ofet al. [Direct quantitation of the antiviral immune response induced by smallpox vaccination has been critical for several recent advances in orthopoxvirus immunobiology – especially since smallpox has been eradicated and other human orthopoxvirus outbreaks are too small and sporadic  model of monkeypox infection. Using ELISA assays and neutralizing assays to measure vaccinia-specific antibody responses, and ICCS to measure antiviral T cell responses, the authors showed that antiviral immunity appeared similar between these three groups. Likewise, each of these groups were protected against lethal monkeypox challenge, although primates that only received MVA plus an MVA booster showed partial protection with 6/6 animals presenting with 1–36 monkeypox lesions (compared to >500 monkeypox lesions in the unvaccinated controls). This indicates that two MVA vaccinations are required to induce partial immunity whereas MVA followed by Dryvax immunization or a single Dryvax immunization each provides full immunity. The protective efficacy of a single dose of MVA is unknown, but based on the results of the Earl et al. study [Another study by Earl et al. , used qul. study  wherein l. study . This isl. study  includinl. study ,32,40. Al. study ,32 and tl. study . In thisl. study . Oddly, It is difficult to speculate the efficacy of MVA vaccination of humans as a method of protection against smallpox. It is possible that booster doses of MVA would provide strong enough immunity to at least protect against lethal smallpox, similar to its ability to protect primates from lethal monkeypox . On the per se. Further analysis indicated that approximately 10% of previously vaccinated household contacts of smallpox patients were actually infected with smallpox as demonstrated by isolation of infectious virus from pharyngeal mucosa, but only 4/34 (12%) of these subjects developed the clinical symptoms of smallpox [There is more than one definition of protective immunity against smallpox. There is protection against infection, protection against disease, and protection against death. Of these, one might argue that protection against lethal infection is the ultimate definition of protective immunity. However, protection against infection and protection against disease not only reduce the morbidity of an outbreak, but these high levels of protective immunity are also associated with reduced virus spread to others  have indicated that, as long as there is strong humoral immunity against vaccinia, T cell memory is dispensable for protective immunity [A major issue in the smallpox field is that there is no consensus on what is exactly required for protective immunity against this disease. In the age of quantitative immunology, we are beginning to find clues that might help answer this age-old question. In a study of >300 subjects, the levels of vaccinia-specific serum antibody and antiviral T cell responses were determined from 30 days to up to 75 years after smallpox vaccination . Antivirterature ,26,40. Ml memory . Based oimmunity , and it immunity -46. This10 ≥ 0.7 neutralizing titer) was also established in non-human primates by simply vaccinating groups of animals with different doses of the yellow fever vaccine, quantitating vaccine-induced antibody levels, and then challenging them with a lethal dose of yellow fever virus [Based on the overlapping roles for T cell and B cell memory on protective immunity, is there any chance that a consensus can be reached in regard to defining an immunological correlate of immunity? One way to address this issue is to examine historical studies in which an immunological correlate was identified. In this regard, there are two independent studies in which investigators showed that subjects with vaccinia-specific neutralizing antibody titers of >1:20  or ≥ 1:3er virus . This seTraditional smallpox vaccination has lead to the global eradication of smallpox but continues to be used today in an effort to thwart the potential use of smallpox as a biological weapon. Since this vaccine employs the use of a live virus, there is an inherent risk of adverse events, although these are generally quite rare. New generation smallpox vaccine candidates include MVA and other non-replicating poxviruses and although they demonstrate a high degree of safety, their immunogenicity appears to be substantially lower than traditional smallpox vaccination with live vaccinia virus. The role of MVA and traditional smallpox vaccination (or a combination thereof) in future vaccination campaigns has yet to be determined. However, developing a consensus on the definition of what is required for protective immunity and defining an immunological correlate of immunity would aid in the evaluation of current and future vaccine approaches.MVA modified vaccinia virus AnkuraGMP good manufacturing practicesHIV human immunodeficiency virusCEF chicken embryo fibroblastsHPV human papilloma virusFDA Federal Drug AdministrationJEV Japanese Encephalitis VirusIMV Intracellular mature virusEEV Extracellular enveloped virusELISA Enzyme-linked immunosorbent assayIFNγ Interferon-gammaELISPOT Enzyme-linked immunosorbent Spot assayICCS Intracellular cytokine stainingMHC Major histocompatibility complexVIG Vaccinia immune globulinWHO World Health OrganizationNIBSC National Institute of Biological Standards and ControlEU Elisa UnitsIU International UnitsOHSU and Dr. Slifka have a financial interest in Najít Technologies, Inc., a company that may have a commercial interest in the results of this research and technology. This potential conflict was disclosed to the OHSU Conflict of Interest in Research Committee and an approved management plan was implemented.


It took nearly 200 years for biologists to redefine the plant/animal dichotomy set up by Linnaeus in 1758. Among the defining traits used in the new five kingdom model of the 20th century was the presence of a nucleus. Possession of a nucleus is one of the chief characteristics that earns an organism, even a single-celled organism, the name of eukaryote. Those not similarly blessed are prokaryotes. Biologists today classify life into three domains (of which microbes lay claim to two), yet the evolution of many fundamental features of eukaryotic biology remains a mystery.A pivotal moment in the evolution of early eukaryotes was the emergence of elaborate, interconnected membrane-bound compartments that make up the Golgi apparatus, endoplasmic reticulum, and nuclear envelope. The nuclear envelope, with its inner and outer membrane, forms a barrier between the cytoplasm and nucleus. Embedded in this envelope are nuclear pore complexes (NPCs), massive (over 400 subunits), cylindrically shaped protein assemblies that connect the outer and inner nuclear membranes via sharply curved sections of pore membranes. The NPC's central ring-like structure is sandwiched between a cytoplasmic ring, with fibrils extending into the cytoplasm, and a nuclear ring, with a “basket” extending into the nucleoplasm. NPCs police traffic flow between the nucleus and cytoplasm, routinely allowing entry to small molecules while providing only selective passage to macromolecules.How eukaryotes evolved complex membrane-mediated trafficking systems from their stripped down prokaryotic contemporaries is a fundamental question in biology. Michael Rout's team investigates one aspect of eukaryotic evolution—the origin and evolution of NPC proteins (nups)—by examining the structure of nups. In a new study, Rout and colleagues report the structure of a core building block of the NPC in yeast, and propose how the complex could have evolved from organisms with no such system.The researchers first tackled the structures of the seven protein components of a core NPC subcomplex, called the yNup84 subcomplex in yeast (and the vNup107-160 subcomplex in vertebrates). Rout and colleagues used algorithms that predict secondary structures to generate three-dimensional models of the component nups. Each nup, they found, consists mostly of either repeating alpha helixes , zigzagging beta sheets (in a beta-propeller fold), or a distinctive arrangement of an amino-terminal beta-propeller followed by a long stretch of alpha-solenoid. Next, the authors compared the structural conformations of the homologous nups found in humans and plants, and showed that the overall architecture of the subcomplex has been conserved throughout eukaryotic evolution.A search for evidence of the distinctive propeller/solenoid arrangement in other organisms shed light on the function and origin of the yNup87/vNup107-160 subcomplex. Neither bacterial nor archaebacterial proteins contain such an arrangement; it appears to exist only in eukaryotes. Moreover, proteins containing this arrangement function only as components of the coated vesicle complexes that operate in intracellular vesicular transport systems or as part of the NPC. That these complexes are linked by common architecture, the authors argue, suggests an “intimate connection between vesicle coating complexes and the yNup87/vNup107-160 subcomplex.” It's likely that both complexes function in curving membranes: when components of this subcomplex are disrupted in yeast, NPCs form abnormal clusters that impair nuclear membrane interactions.How did this shared molecular architecture evolve? Rout and colleagues propose that both nups and vesicle coating complexes developed from a common early eukaryotic ancestor—a primitive coating component with a simplified version of the repetitive folds described here. This molecular carpenter specialized in carving and remodeling membranes, and was repurposed to support the many specialized functions that facilitate molecular transport through the elaborately connected, highly specialized internal membrane systems of the modern eukaryote.


The exploitation of nature's machinery at length scales below the dimensions of a cell is an exciting challenge for biologists, chemists and physicists, while advances in our understanding of these biological motifs are now providing an opportunity to develop real single molecule devices for technological applications. Single molecule studies are already well advanced and biological molecular motors are being used to guide the design of nano-scale machines. However, controlling the specific functions of these devices in biological systems under changing conditions is difficult. In this review we describe the principles underlying the development of a molecular motor with numerous potential applications in nanotechnology and the use of specific synthetic polymers as prototypic molecular switches for control of the motor function. The molecular motor is a derivative of a TypeI Restriction-Modification (R-M) enzyme and the synthetic polymer is drawn from the class of materials that exhibit a temperature-dependent phase transition.The potential exploitation of single molecules as functional devices has been heralded as the dawn of new era in biotechnology and medicine. It is not surprising, therefore, that the efforts of numerous multidisciplinary teams ,2. have  Type I R-M enzymes are multifunctional, multisubunit enzymes that provide bacteria with protection against infection by DNA-based bacteriophage  They acc2M2S [et al. also showed that the EcoR124I R-M holoenzyme exists in equilibrium with a sub-assembly complex of stoichiometry R1M2S [2S) [Type I R-M enzymes fall into families based on complementation grouping, protein sequence similarities, gene order and related biochemical characteristics -8. Withi2M2S ,11,  ,14. HsdRM2S [2S) -18. HoweM2S [2S) .et al. [cis fashion indicating that the motor component of the HsdR subunit is able to 'grasp' adjacent DNA and pull this DNA through the enzyme-DNA-bound complex. According to the Studier model [The motor activity of Type I R-M enzymes is the mechanism through which random DNA cleavage is accomplished. Szczelkun et al.  showed ter model  cleavageer model ,22.DNA translocation has been assayed in bulk solution using protein-directed displacement of a DNA triplex and the kinetics of one-dimensional motion determined. The data shows processive DNA translocation followed by collision with the triplex and oligonucleotide displacement. A linear relationship between lag duration and inter-site distance gives a translocation velocity of 400 ± 32 bp/s at 20°C. Furthermore, this can only be explained by bi-directional translocation. An endonuclease with only one of the two HsdR subunits responsible for motion could still catalyse translocation. The reaction is less processive, but can 'reset' in either direction whenever the DNA is released  and HsdR(prrI), which provides a stable R1-complex, also gives a useful molecular motor that will not cleave the DNA that it translocates.As previously mentioned, the final step of the subunit assembly pathway of the Type I Restriction-Modification enzyme EcoR124I produces a weak endonuclease complex of stoichiometry R EcoprrI -25 Type et al. [via the HsdS subunit and that for some enzymes this may involve DNA [As can be seen from the above, DNA cleavage by Type I restriction enzymes occurs by means of a very unusual, and highly energy-dependent, mechanism. Therefore, these enzymes are believed to be involved not only as a defence mechanism for the bacterial cell, but also in some types of specialised recombination system controlling the flow of genes between bacterial strains ,27. A peet al.  detectedolve DNA .One of the major obstacles for the practical application of single molecule devices is the absence of control methods in biological media, where substrates or energy sources (such as ATP) are ubiquitous. Synthetic polymers offer a robust and highly flexible means by which devices based on single biological molecules can be controlled. They can also be used to link individual biomacromolecules to surfaces, package them or to control their specific functions, thus expanding the applicability of the natural molecules outside conventional biological environments.Moreover, a number of synthetic polymers have been recently developed that can potentially perform nanoscale operations in a manner identical to natural and engineered biopolymers. A key property of these materials is 'smart' behaviour, especially the ability to undergo conformational or phase changes in response to variations in temperature and/or pH. Synthetic polymers with these properties are being developed for applications ranging from microfluidic device formation,  through In the context of bio-nanotechnology we focus here on the uses of one particular subclass of smart materials, i.e. substituted polyacrylamides, but it should be noted that there are many more examples of synthetic polymers and engineered/modified biopolymers that exhibit responsive behaviour and new types and applications of smart materials are constantly being reported.Poly(N-isopropylacrylamide) (PNIPAm) is the prototypical smart polymer and is both readily available and of well-understood properties . PNIPAm Covalent attachment of single or multiple responsive polymer chains to biopolymers offers the possibility of exerting control over their biological activity as, in theory at least, the properties of the resultant polymer-biopolymer conjugate should be a simple additive function of those of the individual components. This principle is now being widely exploited in pharmaceutical development, as covalent attachment of, for example, PEG chains to therapeutic proteins has been shown to stabilize the proteins without losing their biological function -48. Polyvia site-directed mutagenesis was accessible for reaction with maleimide end-functionalised PNIPAm [Pioneering work in this area has been carried out by Hoffman, Stayton and co-workers, who engineered a mutant of cytochrome b5 such that a single cysteine introduced d PNIPAm . Since td PNIPAm ,51,50,51d PNIPAm .We are currently developing responsive polymers as a switch to control the EcoR124I motor function and are investigating this polymer-motor conjugate as part of an active drug delivery system. We aim for the practical demonstration of a nano-scale DNA packaging/separation and delivery system uniting the optimal features of both natural and synthetic molecules. In essence, we assemble a supramolecular device containing the molecular motor capable of binding and directionally translocating DNA through an impermeable barrier. To control the process of translocation in biological systems, where a constant supply of ATP is present, we have added to the motor subunit of EcoR124I the thermoresponsive poly(N-isopropylacrylamide) (PNIPAm), which, through its coil-globule transition, acts as a temperature-dependent switch controlling motor activity.via coupling of a maleimide-tipped linker on the synthetic polymer terminus to a cysteine residue. This residue has been selected, as it is both accessible and located close to the active centre on the R subunit of the motor. The protein-polymer conjugates are stable to extensive purification and, when combined with M2S complex, the activity of this conjugate motor system is similar to the native counterpart, but can be switched on or off as a result of thermally induced polymer phase transitions [PNIPAm copolymers with reactive end-groups are being attached to a preformed R subunit of the motor nsitions ,54.Thus the conjugation of the responsive polymer to the molecular motor generates a nano-scale, switchable device Fig , which cThe conjugation of the motor with synthetic polymers brings additional advantages. One such benefit arises from the ability to functionalise the polymer side chains or terminus in a way that allows attachment of the entire complex to surfaces for sensing and device applications.Therefore, although our hybrid polymer-protein conjugate was originally aimed at gene targeting  this system may also be used in building automated nano-chip sensors, therapeutic and diagnostic devices, where DNA itself would be a target, or where DNA might be used as a 'conveyor-belt' for attached molecules. The strength of the molecular motor has proven sufficient to disrupt most protein-DNA interactions and thus numerous processes and applications where highly localised force is required can also be envisaged.in vitro and especially in vivo opens up a whole host of possibilities in medicine, including drug or DNA delivery (e.g. gene therapy), but also where protection of a therapeutic is required under one biological regime and release in another (e.g. prodrugs conjugated to DNA which can be released by nuclease-mediated degradation at the site of action). In addition, this system may allow the generation of switchable nanodevices and actuators, controllable by changes in the synthetic copolymer structure as well as ATP-mediated DNA motion and may pave the way for biofeedback-responsive nanosystems. It can be used for nano-scale isolation of various biochemical processes in separate compartments connected via a tightly controlled shuttle device.The use of synthetic polymers offers a number of possibilities, which otherwise could not be exploited or would be difficult to take advantage of, if purely biological systems were used. Moreover, the combination of the properties of molecular motors with "smart" polymers has hitherto been unexplored and represents a novel concept in nanotechnology, which could ultimately lead to a wholly new class of molecular devices. Nanoscale control of molecular transport In essence, this concept bridges the disciplines of chemistry and biology by using a biological motor to control chemistry and a synthetic polymer to regulate biological processes.KF conceived the idea of using the modified R-M enzyme as a molecular motor and carried out, with co-workers, the molecular studies of the motor components, SSP carried out the polymer synthesis, polymer-motor conjugations and functional studies, CA designed and participated in the synthesis of smart polymers and DCG conceived of the study. All authors participated in study design and coordination as well as the reading and approval of the final manuscript


The hit criterion is a key component of heuristic local alignment algorithms. It specifies a class of patterns assumed to witness a potential similarity, and this choice is decisive for the selectivity and sensitivity of the whole method.group criterion combining the advantages of the single-seed and double-seed approaches used in existing algorithms. Second, we introduce transition-constrained seeds that extend spaced seeds by the possibility of distinguishing transition and transversion mismatches. We provide analytical data as well as experimental results, obtained with the YASS software, supporting both improvements.In this paper, we propose two ways to improve the hit criterion. First, we define the .Proposed algorithmic ideas allow to obtain a significant gain in sensitivity of similarity search without increase in execution time. The method has been implemented in YASS software available at  The central problem is therefore to improve the trade-off between those opposite requirements.Sequence alignment is a fundamental problem in Bioinformatics. Despite of a big amount of efforts spent by researchers on designing efficient alignment methods, improving the alignment efficiency remains of primary importance. This is due to the continuously increasing amount of nucleotide sequence data, such as EST and newly sequenced genomic sequences, that need to be compared in order to detect similar regions occurring in them. Those comparisons are done routinely, and therefore need to be done very fast, preferably instantaneously on commonly used computers. On the other hand, they need to be precise, i.e. should report all, or at least a vast majority of interesting similarities that could be relevant in the underlying biological study. The latter requirement for the alignment method, called the hit) a potential similarity. Those patterns are formed by seeds which are small strings  that appear in both sequences. FASTA [The Smith-Waterman algorithm  providess. FASTA ,4 are wehit criteria that define which pattern shared by two sequences is assumed to witness a potential alignment. Two types of improvements can be distinguished. On the one hand, using two or more closely located smaller seeds instead of one larger seed has been shown to improve the sensitivity/selectivity trade-off [More recently, several new alignment methods have been proposed, such as BLAT , Patternrade-off -6, especrade-off , or vectrade-off .Group hit criterion), we propose a new flexible and efficiently computable hit criterion, called group criterion, combining the advantages of the single-seed , we propose a new more expressive seed model which extends the spaced seed model of PatternHunter [In this paper, we propose further improvements in both those directions. In the first part (Section le-seed  and mulle-seed ,4-6) cri-6 criGrornHunter , used inseeds occurring in the input sequences. In this section, we assume that a seed of weight k is a word consisting of k contiguous nucleotides (k-word), more general notions of seed will be considered in the next Section.The first preparatory step of most heuristic alignment algorithms consists of constructing a hash table of all hit of a potential alignment. It triggers the X-drop algorithm trying to extend the seed to a so-called High-scoring Segment Pair (HSP), used then to obtain a larger final alignment. Gapped BLAST [double-seed criterion that defines a hit as two non-overlapping seeds occurring at the same dotplot diagonal within a fixed-size window. This allows to considerably increase the selectivity with respect to the single-seed approach, and at the same time to preserve, and even to improve, the sensitivity on large similarities. On the other hand, Gapped BLAST is less sensitive on short and middle-size similarities of weak score.  Most of the existing alignment programs [In the simplest case, implemented in the early version of BLAST , an indied BLAST  proposesprograms ,6 use eiHere we propose a new flexible hit criterion defining a hit as a group containing an arbitrary number of possibly overlapping seeds, with an additional constraint on the minimal number of matching nucleotides. The seeds of the same group are assumed to belong to the same similarity, and therefore should be proximate to each other. In contrast to other multi-seed hit criteria -6, we doselective enough to avoid spurious extensions and, on the other hand, should be sensitive to detect as many relevant similarities as possible.A hit criterion defines a pattern which is considered as an evidence of a potential similarity. Every time this pattern is found, its extension is triggered to compute a potential larger alignment. The extension is usually done via a dynamic programming algorithm and is a costly step. The hit criterion should be group of seeds verifying conditions (1), (2) (see Section Methods). By the considered statistical analysis, this ensures a good sensitivity. However, many groups will contain a single seed or two strongly overlapping seeds, that either belong to a similarity with a low score, or do not belong to any similarity at all . To cope with this problem, we introduce an additional criterion that selects groups that will be actually extended. The criterion, called group criterion, is based on the group size defined as the minimal number of matching individual nucleotides in all seeds of the group. The group size can be seen as a parameter specifying the maximal overlap of the seeds of a group. For example, if the group size is k + 1, then no constraint on the overlap is imposed, i.e. any group containing two distinct seeds forms a hit. If the group size is 2k, then the group must contain at least two non-overlapping seeds, etc.The hit criterion we propose is based on a Allowing overlapping seeds is an important point that brings a flexibility to our method. Note that other popular multi-seed methods ,5 considgapless alignments of a fixed score. Moreover, to make this model yet more close to reality, only homogeneous alignments are considered, i.e. alignments that don't contain proper sub-alignments of bigger score . The main assumption is that the sensitivity is estimated as the probability of hitting a random ore (see ). For a In this setting, we computed the hit probability of a single-seed criterion with seed weight 11 (default for BLAST) and compared it with multi-seed criteria of Gapped BLAST and YASS for seed weight 9 (default for Gapped BLAST). For YASS, the group size was set to 13. Figure Comparing BLAST and Gapped BLAST, the former is more sensitive on short similarities (having higher identity rate), while the latter is more sensitive on longer similarities, in which two close non-overlapping runs of 9 matches are more likely to occur than one run of 11 matches. The YASS group criterion combines the advantages of both: it is more sensitive than the single-seed criterion even for short similarities, and than the non-overlapping double-seed criterion for large similarities  by more than ten. For Gapped BLAST, this probability is 7.3·10-9. To make an accurate sensitivity comparaison of YASS and Gapped BLAST, parameters should be set so that both algorithms have the same selectivity level.Note, however, that for the chosen parameters, the YASS criterion is slightly less selective than that of Gapped BLAST as it includes any two non-overlapping seeds but also includes pairs of seeds overlapped by at most 5 bp. The selectivity can be estimated by the probability of a hit at a given position in a random uniform Bernoulli sequence (see [-6). This is achieved with seed weight 8 for Gapped BLAST and group size 11 for YASS (while keeping seed weight 9). In this configuration, and for sequences of score 25, YASS turns out to be considerably more sensitive on sequences up to 80 bp and is practically as sensitive as Gapped BLAST on longer sequences (data not shown). At the same time, YASS is more time efficient in this case, as Gapped BLAST tends to compute more spurious individual seeds that are not followed by a second hit, which takes a considerable part of the execution time. This is because the YASS seed is larger by one nucleotide, and the number of spurious individual seeds computed at the first step is then divided by 4 on large sequences.To compare the sensitivity of YASS and Gapped BLAST for an equal selectivity level, we chose a parameter configuration such that both algorithms have the same estimated selectivity  and more sensitive Finally, we point out another experiment we made to bring more evidence that the group criterion captures a good sensitivity/selectivity trade-off. We monitored the partition of the execution time between the formation of groups and their extension by dynamic programming (data not shown). It appeared that YASS spends approximately equal time on each of the two stages, which gives a good indication that it provides an optimal distribution between the two complementary parts of the work.k-words, i.e. short strings of contiguous nucleotides. Recently, it has been understood that using spaced seeds allows to considerably improve the sensitivity. A spaced seed is formed by two words, one from each input sequence, that match at positions specified by a fixed pattern – a word over symbols # and _ interpreted as a match and a don't care symbol respectively. For example, pattern ##_# specifies that the first, second and fourth positions must match and the third one may contain a mismatch.So far, we defined seeds as lossless filtration for approximate pattern matching, namely for the problem of detecting all matches of a string of length m with q possible substitution errors (an (m, q)-problem). The use of some specific spaced seeds for this problem was proposed earlier in [PatternHunter  was the rlier in . Yet earrlier in  to coverrlier in .m, q)-problem was proposed in [The advent of spaced seeds raised new questions: How to choose a good seed for a local alignment algorithm? How to make a precise estimation of the seed goodness, or more generally, of a seed-based local alignment method? In , a dynamposed in . The algposed in  has beenposed in  for hiddposed in . A complposed in . Papers posed in ,20 preseOther extensions of the contiguous seed model have been proposed. BLAT  uses contransition-constrained seed model. Its idea is based on the well-known feature of genomic sequences that transition mutations (nucleotide substitutions between purins or between pyrimidins) occur relatively more often than transversions (other substitutions). While in the uniform Bernoulli sequence transitions are twice less frequent than transversions, in real genomic sequences this ratio is often inverted. For example, matrices computed in [ti/tv) is greater than one on average. Transitions are much more frequent than transversions in coding sequences, as most of silent mutations are transitions. ti/tv ratio is usually greater for related species, as well as for specific DNA .Here we propose a new puted in  on mouseweight of a transition-constrained seed is defined as the sum of the number of #'s plus half the number of @'s. This is because a transition carries one bit of information while a match carries two bits.Transition-constrained seeds are defined on the ternary alphabet {#, @, _}, where @ stands for a match or a transition mismatch (A ↔ G, C ↔ T), and # and _ have the same meaning as for spaced seeds. The Group criterion. The only non-trivial algorithmic issue raised by this combination is how to efficiently compute the group size during the formation of groups out of found seeds. In YASS, this is done via a special finite automaton resulting from the preprocessing of the input seed.Note that using transition-constrained seeds is perfectly compatible with the group criterion described in Section ti/tv ratio. The sequence length is set to 64, a typical length of a gapless fragment in DNA alignments. We are interested in seed weights between 9 and 11, as they represent a good sensitivity/selectivity compromise.To estimate the detection capacity of transition-constrained seeds, we first use the Bernoulli alignment model, as done in ,19,20. WTable ti/tv ratio. This is shown in Figure ti/tv ratio is over 1, which is typically the case for real genomic sequences.A natural question is the efficiency of transition-constrained seeds depending on the Neisseria Meningitidis, S. Cerevisiae, Human X chromosome, Drosophila). The alignments were computed with different seeds of small weight, to avoid a possible bias caused by a specific alignment method. We then designed optimal spaced and transition-constrained seeds of weight 9–11 with respect to this Markov model. Table Homologous coding sequences, when aligned, usually show a regular distribution of errors due to protein coding constraints. In particular, transitions often occur at the third codon position, as these mutations are almost always silent for the resulting protein. Markov models provide a standard modeling tool to capture such local dependencies. In the context of seed design, papers -18 propoS. Cerevisiae (chromosomes IV, V, IX, XVI) and Neisseria meningitidis (strains MC58 and Z2491). The experiments were made with our YASS software [Group criterion. The experiments used seeds of weight 9 and 11, obtained on Bernoulli and Markov models  than two match-or-transition pairs.A series of comparative tests has been carried out to compare the sensitivity with traditional approaches. Several complete bacterial genomes ranging from 3 to 5 Mb have been processed against each other using both YASS and the b12seq programs (NCBI BLAST package 2.2.6.). The tests used the scoring system +1/-1 for match/mismatch and -5/-1 for gap opening/extension. The threshold E-value for the output was set to 10 , and the sequence filtering was disabled. YASS was run with its default seed pattern #@#__##__#_##@# of weight 9 which provides a good compromise in detecting similarities of both coding and non-coding sequences.-6 found by each program, and the number of exclusive alignments were reported. By "exclusive alignment", we mean every alignment of E-value less than 10-6 that does not share a common part (do not overlap on both sequences) with any alignment found by the other program. To take into account a possible bias caused by splitting alignments into smaller ones (X-drop effect), we also computed the total length of exclusive alignments, found by each program.For each test, the number of alignments with E-value less than 10Experiments are summarized in Table group criterion, based on statistical DNA sequence models, combines the advantages of both single-seed and double-seed criteria. Transition-constrained seeds account for specificities of real DNA sequences and allow to further increase the search sensitivity with respect to spaced seeds. Both options have been implemented in YASS software available at .In this paper, we introduced two independent improvements of hit criteria for DNA local alignment. The Transition-constrained seeds could be further extended using the idea of vector seeds , i.e. byAnother new direction for further improving the efficiency is a simultaneous use of several seed patterns -24, compS1 and S2 be the input sequences of length m and n respectively. Each of them can be considered as a succession of m - k + 1 (respectively n - k + 1) substrings of length k, called k-words. If a k-word of S1 matches another k-word of S2, i.e. S1[i..i + k - 1] = S2[j..j + k - 1] for some i ≤ m and j ≤ n, then these two k-words form a seed denoted <i, j>. Two functions on seeds are considered: For a seed <i, j>, the seed diagonal d(<i, j>) is m + j - i. It can be seen as the distance between the k-words S1[i..i + k - 1] and S2[j..j + k - 1] if S2 is concatenated to S1, For two seeds <i1, j1> and <i2, j2>, where i1 <i2 and j1 <j2, the inter-seed distance D(<i1, j1>, <i2, j2>) is the maximum between |i2 - i1| and |j2 - j1|. The problem considered in this Section is to derive conditions under which two seeds are likely to be a part of the same alignment, and therefore should be grouped together. More precisely, we want to be able to compute parameters ρ and δ such that two seeds <i1, j1> and <i2, j2> have a probability (1 - ε) to belong to the same similarity iffWe first introduce some notations used in this section. Let D(<i1, j1>, <i2, j2>) ≤ ρ,     (1)d(<i1, j1>) - d(<i2, j2>)| ≤ δ.     (2)|k-words occur at close diagonals.The first inter-seed condition insures that the seeds are close enough to each other. The second seed diagonal condition requires that in both seeds, the two ρ and δ.We now describe statistical models used to compute parameters p for a match and (1 - p) for a mismatch. To estimate the inter-seed shift Dk, we have to estimate the distance between the starts of two successive runs of at least k matches in the Bernoulli sequence. It obeys the geometric distribution of order k called the Waiting time distribution [Consider two homologous DNA sequences that stem from a duplication of a common ancestor sequence, followed by independent individual substitution events. Under this assumption, the two sequences have an equal length and their alignment is a sequence of matched and mismatched pairs of nucleotides. We model this alignment by a Bernoulli sequence with the probability ribution ,26:ρ such that the probability  is (1 - ε) for some small ε.Using this formula, we compute k inside a Bernoulli sequence of length x. In a Bernoulli sequence of length x, the probability of the event Ip,x,r of having exactly r non-overlapping runs of matches of length at least k is given by the following recursive formula:Note that the Waiting time distribution allows us to estimate another useful parameter: the number of runs of matches of length at least r non-overlapping seeds of length at least k inside a repeat of size x. The recurrence starts with r = 0, in which case  and is computed through the Waiting time distribution.This gives the probability of having exactly  allows us to infer a lower bound on the number of non-overlapping seeds expected to be found inside a similarity region. In particular, we will use this bound as a first estimate of the group criterion introduced later.The distribution Indels (nucleotide insertions/deletions) are responsible for a diagonal shift of seeds viewed on a dotplot matrix. In other words, they introduce a possible difference between d(<i1, j1>) and d(<i2, j2>). To estimate a typical shift size, we use a method similar to the one proposed in [posed in  for the q at each of l nucleotides separating two consecutive seeds. Under this assumption, estimating the diagonal shift produced by indels is done through a discrete one-dimensional random walk model, where the probability of moving left or right is equal to q, and the probability of staying in place is 1 - 2q. Our goal is to bound, with a given probability, the deviation from the starting point.Assume that an indel of an individual nucleotide occurs with an equal probability i after l steps is given by the following sum:The probability of ending the random walk at position  and consider the power Pl(x) = al.xl +…+ al-.xl-. Then the coefficient ai computes precisely the above formula, and therefore gives the probability of ending the random walk at position i after l steps. We then have to sum up coefficients ai for i = 0,1, -1, 2, -2,..., l, -l until we reach a given threshold probability (1 - ε). The obtained value l is then taken as the parameter δ used to bound the maximal diagonal shift between two seeds.A direct computation of multi-monomial coefficients quickly leads to a memory overflow, and to circumvent this, we use a technique based on generating functions. Consider the function