Many researchers are interested to know if there are any improvements in recent treatment results for metastatic breast cancer in the community, especially for 10- or 15-year survival.Between 1981 and 1985, 782 and 580 female patients with metastatic breast cancer were extracted respectively from the Connecticut and San Francisco-Oakland registries of the Surveillance, Epidemiology, and End Results (SEER) database. The lognormal statistical method to estimate survival was retrospectively validated since the 15-year cause-specific survival rates could be calculated using the standard life-table actuarial method. Estimated rates were compared to the actuarial data available in 2000. Between 1991 and 1995, further 752 and 632 female patients with metastatic breast cancer were extracted respectively from the Connecticut and San Francisco-Oakland registries. The data were analyzed to estimate the 15-year cause-specific survival rates before the year 2005.The 5-year period (1981–1985) was chosen, and patients were followed as a cohort for an additional 3 years. The estimated 15-year cause-specific survival rates were 7.1%  and 9.1% (95% CI, 3.8–14.4) by the lognormal model for the two registries of Connecticut and San Francisco-Oakland respectively. Since the SEER database provides follow-up information to the end of the year 2000, actuarial calculation can be performed to confirm  the estimation. The Kaplan-Meier calculation for the 15-year cause-specific survival rates were 8.3% (95% CI, 5.8–10.8) and 7.0% (95% CI, 4.3–9.7) respectively. Using the 1991–1995 5-year period cohort and followed for an additional 3 years, the 15-year cause-specific survival rates were estimated to be 9.1% (95% CI, 3.8–14.4) and 14.7% (95% CI, 9.8–19.6) for the two registries of Connecticut and San Francisco-Oakland respectively.For the period 1981–1985, the 15-year cause-specific survival for the Connecticut and the San Francisco-Oakland registries were comparable. For the period 1991–1995, there was not much change in survival for the Connecticut registry patients, but there was an improvement in survival for the San Francisco-Oakland registry patients. Prospective trials have the disadvantages of requiring a long time to complete, and using highly selected patient subgroups in tertiary centers. While one waits for the results to mature, this delays additional research to improve treatment. If there were a method that allowed earlier prediction of the results of prospective trials, advances in cancer treatment could be attained within a shorter time period.There is a parametric lognormal model, proposed by Boag -3 that hThe prognosis for metastatic breast cancer is generally poor and therefore it is believed that statistical prediction models for long-term survival rates are not needed. Nevertheless, specific subgroups of metastatic breast cancer patients exist, for which depending on the treatment given, the prognosis is improved so that some patients can survive for some time, particularly for those with limited organs involvement such as involvement with bone and/or skin only. In this situation, for which the present study was relevant, a prediction model, even for metastatic breast cancer, can be useful.Breast cancer, among other cancers, has the highest incidence in women, and many studies are currently in progress to assess treatment regimens. If, even for a subgroup of patients, the 10- and 15-year survival rates can be predicted from follow-up data available only 3 years after a 5-year diagnosis period, this would be a useful means of obtaining study results earlier than would otherwise have been possible. For example, a 15-year survival rate calculated by the Kaplan-Meier method requires at least some patients to have been followed for 15 years. In addition prediction model such as the lognormal model can also be used to review the progress of treatment results for a specific period from a treatment center, and to compare that with another specific period of the same treatment center to evaluate the potential impact for any possible change in treatment policy or guideline.Boag's lognormal model for long-term cancer survival rates has been available for use for some 50 years. When the lognormal model was first proposed in the 1940s, it was difficult to implement because of a lack of computing power, and lack of good quality long-term follow-up data from cancer registries. Since 1970s the model has been used by some authors in breast cancer, cervix uteri cancer, head and neck cancer, intraocular melanoma, choroidal-ciliary body melanoma, and small cell lung cancer . CurrentBetween 1981 and 1985, 782 and 580 female patients of metastatic breast cancer were extracted respectively from the Connecticut and San Francisco-Oakland registries from the SEER database using SEER*Stat 5.0 software. The two registries were chosen because they are two of the earliest registries, with a large population. The data used were survival time, vital status, cause of death, age at diagnosis, and race.The cause-specific survival was defined as the interval from the date of diagnosis to the date of death from breast cancer or the last follow-up date for censoring purposes, if the patient was alive and still being followed at the time of analysis. The survival time of the uncured group of patients who died of breast cancer had been verified to follow a lognormal distribution previously .Next, between 1991 and 1995, 752 and 632 female patients of metastatic breast cancer were extracted respectively from the two registries. The data were used to estimate the 15-year cause-specific survival rates before the year 2005. To be comparable, for both the 1981–1985 and 1991–1995 cohorts, the staging system used was the SEER historical system . The choice of 1981–1985 and 1991–1995 has the advantage that the two time periods are not too far apart otherwise there would be too much changes of medical practice. These time periods have a minimum of 5 years follow-up.The overall survival rates (OSR) of the two time periods were calculated using the Kaplan-Meier method. The actual relative survival rates (RSR) were calculated using SEER*Stat 5.0 software. The modified version of period analysis  was applThe validation of the lognormal model has two phases. Phase 1 tests the goodness of fit to a lognormal distribution of the survival time of those cancer patients who died with their disease present, termed an uncured group with a fraction of 1-C, where C is the cured proportion of patients. The lognormal distribution is similar to the normal distribution in that if the variable in the normal is time t, the variable in the lognormal is the logarithm of t. In other words, the investigators attempt to show that the logarithm of the survival time follows a normal distribution. Phase 2 attempts to show that the lognormal model, using short-term follow-up data, can predict long-term survival rates comparable to those calculated by the Kaplan-Meier life-table method with long-term available. This model can be used to estimate long-term cause-specific survival rates (CSSR) by a maximum likelihood method  from only short-term follow-up data. The maximum likelihood method is used to estimate the CSSR at time τ, and is calculated as [C+(1-C)·Q]·100%, where Q is the integral of the lognormal distribution between the limits time τ and infinity.The lognormal statistical model had been validated in stages III and IV breast cancer in a previous publication that survival rates could be estimated several years earlier than is possible using the standard life-table actuarial method . The surA 5-year period of diagnosis could be selected and patients followed as a cohort for an additional 3 years. The current study was for metastatic breast cancer patients treated between 1981 and 1985, with follow-up to the end of year 2000, making the series ideal for validating purposes. For example, for cases diagnosed during the 5-year period, prediction of the 15-year survival rate was made using data at the follow-up cutoff date of December 31, 1988 (i.e., 3 years after 1985). The 15-year survival rate prediction was then validated by Kaplan-Meier life-table calculations using the follow-up data available in 2000.For metastatic breast cancer patients treated between 1991 and 1995, and follow-up to the end of year 2000, prediction of the 15-year survival rate was made using data at the follow-up cutoff date of December 31, 1998 (i.e., 3 years after 1995) before the year 2005.From the cohort of 1981–1985 inclusively, 782 patients from the Connecticut registry were followed to the end of 1988. The lognormal model predicted the 15-year CSSR to be 7.1% (95% CI, 1.8–12.4). The 15-year CSSR was 8.3% (95% CI, 5.8–10.8) validated by the Kaplan-Meier calculation using actuarial follow-up data up to the end of year 2000.From the cohort of 1981–1985 inclusively, 580 patients from the San Francisco-Oakland registry were followed to the end of 1988. The lognormal model predicted the 15-year CSSR to be 9.2% (95% CI, 3.9–14.5). The 15-year CSSR was 7.0% (95% CI, 4.3–9.7) validated by the Kaplan-Meier calculation using actuarial follow-up data up to the end of year 2000.Using the same method, the cohort of 1991–1995 inclusively, 752 patients from the Connecticut registry were followed to the end of 1998. The lognormal model predicted the 10-year CSSR to be 12.6% (95% CI, 7.3–17.9). The 10-year CSSR was 11.3% (95% CI, 7.8–14.8) validated by the Kaplan-Meier calculation using actuarial follow-up data up to the end of year 2000. The lognormal model predicted the 15-year CSSR to be 9.1% (95% CI, 3.8–14.4), which cannot be validated before 2005.For the cohort of 1991–1995 inclusively, 632 patients from the San Francisco-Oakland registry were followed to the end of 1998. The lognormal model predicted the 10-year CSSR to be 17.0% (95% CI, 12.1–21.9). The 10-year CSSR was 15.9% (95% CI, 11.4–20.4) validated by the Kaplan-Meier calculation using actuarial follow-up data up to the end of year 2000. The lognormal model predicted the 15-year CSSR to be 14.7% (95% CI, 9.8–19.6), which cannot be validated before 2005.For the period 1991–1995, there was not much change of only about 2% absolute percentage point in the predicted 15-year CSSR for the Connecticut registry, but there was an improvement of about 6% absolute percentage points for the San Francisco-Oakland registry when compared with the period 1981–1985 15-year CSSR, which was validated by the Kaplan-Meier calculation. . Therefore, overall survival cannot be predicted. The maximum likelihood method is the most accurate method for fitting the lognormal model with the smallest mean squared error. However, there are some requirements for its use. The maximum likelihood method fails to converge to a stable solution using the initial estimates if there is extensive censoring within the data. This occurs if patients are lost to follow-up or die from coincidental non-cancerous causes. The frequency of failure to yield a successful fit for lognormality was greater when one-fourth of cases were designated as lost to follow-up. Gamel vergence .et al. [et al. [et al. [lognormal with a median diameter equal to 3.5 cm. The patients were subdivided into 3 groups according to the histological grade. In each subgroup there was a significant correlation between tumor size and the probability of distant spread. The distributions were lognormal and the median size was markedly larger for grade 1 tumors.Prognostic factors in patients with distant metastases at the time of diagnosis were investigated by Rudan et al. , and Cha [et al. , primary [et al.  found th [et al. . Tubiana [et al.  have fouet al. were found to approximate the lognormal distribution reasonably well.A number of quantitative postmortem observations regarding the size distribution of metastases have been published -23. ThesA hypothesis was proposed by Kendal  that theet al. [Why is the lognormal model applicable to so many organ sites ,12,25-36et al.  found thet al. [et al. [In order to determine whether current programs for the management of metastatic breast cancer have led to improved patient survival, Debonis et al.  determin [et al.  studied et al. [The tumor registry at Yale-New Haven Hospital, which began recording data in 1920, was utilized by Todd et al.  to examiet al. [Farrow et al.  documentet al. [et al. [Maggard et al.  also fou [et al.  examined [et al. . For womThe above-mentioned results are consistent with that from the present study: the Connecticut registry has lower CSSR than the San Francisco-Oakland registry for the period 1991–1995. The Connecticut cohort has median age at diagnosis of 66 (range 25–103), while the San Francisco-Oakland cohort has lower median age of 63 (range 26–96). It could be argued that new treatments evolved in the recent decade have improved the survival of the breast cancer patients, and younger patients benefit more than the older patients. Apart from treatment offered, changes of survival rates over time or geographical areas can be due to co-morbidities or other characteristics such as race, age, and differences in staging procedures.For the period 1981–1985, the 15-year cause-specific survival for the Connecticut and the San Francisco-Oakland registries were comparable. For the period 1991–1995, there was not much change in survival for the Connecticut registry patients, but there was an improvement in survival for the San Francisco-Oakland registry patients.CSSR: Cause-specific survival. SEER: Surveillance, Epidemiology, and End Results. OSR: Overall survival rate. ASR: Absolute survival rate. RSR: Relative survival rate.None declared.PT: Data analysis and writing of manuscript. EY, VVH, GC, GV: Critical appraisal of manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Heterogeneity of cartilage tumours may confound accurate diagnosis and grading resulting in under and over treatment. Improved preoperative assessment of malignancy and grade would be invaluable for developing a rational plan for treatment. We examined correlations between nuclear tracer avidity and malignancy grade in cartilage tumours.Between 1996 and 2000, 92 consecutive patients with cartilaginous tumours  underwent nuclear scanning. Thallium-201 (TL-201) and pentavalent dimercaptosuccinic acid (DMSAV) were used as nuclear isotopes. Scanning with these agents was performed on separate days 48 hours apart. Static and SPECT images were obtained at 30 m and 4 h after injection of nuclear tracer. Pathology review was undertaken blinded to the results of the nuclear scans and correlations between histologic results and trace uptake at 4 hours examined.25 patients with negative DMSAV had benign tumours. 15/17 tumours with positive TL-201 had malignant tumours. 11/13 patients with both positive DMSAV and TL-201 scans had intermediate or high grade tumours and 4 of these developed metastases. We have developed an algorithm for the management of patients with tumours that aims to avoid over treatment of low grade tumours and under treatment of high grade tumours.Functional nuclear scanning with TL-201 and DMSAV complements other imaging modalities in the management of cartilaginous tumours. Traditionally, the determination of malignancy and its grade in cartilage tumours has been from the combination of history, radiographic features with or without computed tomography and histologic examination -3. More Functional nuclear scans employ isotopes that become substrates for various cellular metabolic cycles and therefore, are useful for determining the metabolic activity in these tissues . Given tWe now report our experiences with 2 radio-siotopes, Thallium-201 (Tl-201) and pentavalent dimercaptosuccinic acid (DMSAV) for determining the metabolic activity of cartilage tumours, and their value in differentiating between malignant and benign cartilage tumours in 92 consecutive cartilage tumours. We describe their role in the development of our current regimen for treating cartilage tumours.Between January 1997 and December 2000, 92 consecutive patients were referred to our institution for investigation and management of suspected chondral tumours. There were 50 females and 42 males with a median age of 45 years (range 16–87 years).Tumours were located in the humerus (17), chest wall (3), femur (27), hand (3), knee (3), vertebra (1), scapula (7), tibia (16), pelvis (9), foot (4), radius (2). All patients had previously unoperated tumours and no patient presented with metastases.All patients were examined with plain radiographs, computed tomography and magnetic resonance imaging. From 1997 onward, TL-201 and/or DMSAV scans were also performed on patients with suspected chondral tumours.Tl-201 and DMSAV scintiigraphy were conducted at two centers (SVH, PMCI) with similar imaging protocols. All studies were conducted using a gamma camera. A pre-determined dose of radioisotope was administered intravenously and scintigraphic images obtained at 30 minutes (early phase) and 3–4 hours (late phase) after injection in all cases. Early phase static images were acquired over the area of interest and late phase images consisted of whole body imaging and SPECT over the area under investigation. A low energy high resolution, parallel-hole collimator was used and image acquired in a 128 × 128 matrix for 5 minutes. A simple grading system was devised for late phase isotope uptake with NO UPTAKE indicating no tumour uptake greater than background activity and INCREASED UPTAKE indicating definite activity greater than the background level. The background level referred to is that of the tissue within which the tumour arose, that is, bone. We selected the late phase results for correlation with the results of histological examination of the surgical specimens because early uptake at 30 minutes may also represent peritumoural inflammation or vascularity, which may confound the interpretation of results. In contrast, late phase uptake represented true tumour uptake of isotope.The pathologist (JS) is the designated lead pathologist on the Victorian Bone Tumour Registry. For the purpose of this study, assessments of surgical tissue and histologic re-evaluation were conducted without prior knowledge of the results of the functional nuclear scans. There were 50 benign tumours and these consisted of enchondromata (29), osteochondromata (17), chondroblastoma (3), and chondromyxoid fibroma (1). There were 42 malignant tumours and these consisted of 38 central chondrosarcoma and 4 dedifferentiated chondrosarcoma. Of these malignant tumours, 22 were graded as grade I, 12 as grade II and 8 as grade III according to.All tumours in this study were treated with excision. In those tumours that were considered to be benign or grade 1 malignancy as based on history, examination, plain radiography, magnetic resonance imaging and functional scanning, were treated by careful intralesional curettage, burring with a high speed dental burr, pulsatile lavage, chemical cautery with phenol and then the defect filled with polymethylmethacrylate cement. In those tumours that were considered to be clearly malignant and interpreted as grade 2 and higher or if the functional nuclear scans showed significant uptake, wide resection was employed.Because of the histologic heterogeneity of cartilage tumours, the majority of tumours were not biopsied prior to definitive surgery. Biopsy was considered if there was doubt about the diagnosis, or if the anticipated treatment was potentially far greater than may have been required. If biopsy was preferred but could not be safely performed preoperatively, for example, a periacetabular tumour, frozen section drill biopsy would be conducted as part of the initial surgical approach to the tumour.No patients were lost to follow-up. Patients with malignant tumours were reviewed every 3 months for the first 2 years and 6 monthly after that for a further 2 years with a plan for yearly review for the following 4 years. Computed tomography of the chest was performed every 6 months, and plain radiographs obtained of the operated area at each visit.The median follow-up was 3.7(0–6) years. At last review, 85 patients were alive without disease, 2 were alive with pulmonary metastases. 3 patients had died of metastatic disease, one patient died from intraoperative complications and 1 patient died from a pulmonary embolus 1 month after surgery. There were no local recurrences.Eighty seven of ninety two patients underwent thallium scanning. Of these, 17 patients had increased uptake on the delayed scans, and 70 patients had no uptake on the delayed scans.Eighty three of ninety two patients underwent DMSA(V) scanning. Of these, fifty eight patients had increased uptake on the delayed scans and twenty five patients had no uptake on the delayed scans.Seventy eight of ninety two patients had both thallium and DMSA(V) scanning. There was no uptake with Thallium and DMSA(V) scanning in twenty patients. Thallium scanning was negative and DMSA(V) was positive in forty five patients. There was uptake on both Thallium and DMSA(V) scanning in 13 patients. There was no case where there was thallium uptake without DMSA(V) uptake.a. Thallium scans Of the 50 benign tumours, 45 underwent thallium scanning. Forty-three tumours had negative scans and 2 had positive scans. Of the 42 malignant tumours, 27 had negative thallium scans and 15 had positive scans.b. DMSA(V) scans Of the 50 benign tumours, 47 had DMSA(V) scans. Twenty five of these tumours had negative scans and 22 had positive scans. Of the 42 malignant tumours, 36 had DMSA(V) scans. All 36 of these were positive. No malignant tumour was DMSA(V) negative.c. Combined thallium and DMSA(V) scans There 78 tumours that were scanned with both thallium and DMSA(V). Of the 42 benign tumours, 2 had positivity forboth thallium and DMSA(V), 20 had positivity only for DMSA(V) and 20 had no uptake on either scan.Of the 36 malignant tumours that were scanned with both isotopes, 11 showed positivity for both thallium and DMSA(V), while 25 tumours showed positivity for DMSA(V) only. No malignant tumour was negative to thallium scanning.a. Thallium scans Of the 42 chondrosarcomas, 7 grade II and 8 grade III tumours had positive thallium uptake. No grade I tumour had thallium uptake. Twenty two grade I tumours, and 5 grade II tumours showed no uptake.b. DMSA(V) scans Thirty six of the 42 chondrosarcomas had DMSA(V) scans. All were positive (20 grade I, 11 grade II, 5 grade III). No chondrosarcoma was DMSA(V) negative.No patient with a negative thallium scan developed metastasis. Six of seventeen patients with positive thallium scans developed metastases. In contrast, four of fifty eight patients with positive DMSA(V) scans developed metastases. In patients where both scans were performed, four of thirteen patients developed metastases.Anatomic imaging such as computed tomography  and magnConventional technetium monodiphosphonate skeletal scans are employed to identify uni-or multifocal disease when bone tumours are suspected . For posReliance on preoperative histologic diagnosis for determining the nature of a cartilage lesion can also be difficult. Biopsy of a suspicious cartilage tumour is most helpful when clear malignancy is demonstrated because a benign finding does not necessarily exclude malignancy. The histologic heterogeneity of cartilage tumours, however, is well recognized and unless the biopsy accurately targets the most malignant part of the tumour, there is a risk that the histologic diagnosis may under-report the state of malignancy if this should exist.Functional nuclear scans reflect the metabolic activity of tumours and may provide important information regarding their biologic behaviour. In this regard, hypermetabolic tumour tissue is likely to be more active than surrounding normal tissue and this difference may be valuable for distinguishing between benign and malignant tumours. Similarly, when dealing with grades of malignancy, a higher level of metabolic activity may be expected from high grade tumours in comparison to lower grade counterparts. Since the late 1970s, thallium Tl-201) scanning has been used extensively as a safe method to assess ventricular function in patients with myocardial ischaemia and the results have been interpreted as reflecting relative levels of myocardial metabolic activity. As Tl-201 is a potassium analogue, its uptake into cells depends on the sodium potassium ATPase dependent pump. Tl-201 is a readily available, cyclotron produced radionuclide that decays by electron capture with a half-life of approximately 73 hours. The liver, spleen, kidneys, myocardium, thyroid, choroids plexus of the lateral ventricles and testis normally demonstrate avidity for Tl-201, with very minimal uptake in healing surgical wounds. Tl-201 is thought to accumulate less well within connective tissue, which contains inflammatory cells and almost undetectable in necrotic tissue. Localization of Tl-201 within tumours appears to be influenced by tumour vascularity, tumour cellularity, the metabolic rate of the tumour and the histological type of tumour. Thallium uptake studies 1 scannin in patieOur study has demonstrated that the combined use of Tl-201 and DMSA(V) scanning may be correlated with the benignity, malignancy and grade of cartilaginous tumours. For a tumour that is hetereogeneous not only on radiologic but also pathologic appearance, the use of functional nuclear scanning may help to differentiate particularly between the low grade and benign lesions. Of note, no malignant lesion had a negative DMSA(V) scan, and only 2 out of 45 benign tumours showed thallium uptake in our study.While this modality of investigation does not replace the value of combining elements from the clinical presentation, plain radiographic changes and pathology, functional nuclear scanning may increase the confidence of diagnosing a low grade or benign tumour, and the availability of information preoperatively may also be helpful for tumours that are deeply situated and difficult to biopsy. With regard to biopsy, localized thallium or DMSA(V) positivity may be useful for guiding tissue sampling from the most metabolically active site, thus enhancing the procurement of potentially the most aggressive/malignant part of the tumour .Although, the median duration of follow up in this study was limited to 3.7 years, the high proportion of metastases in patients whose sarcomas were thallium positive may suggest a role for thallium scanning as a prognostic indicator for metastasis in chondrosarcoma. Interestingly, the combination of DMSA(V) with thallium scanning did not improve the prognostic value for metastases. This may be attributed to the fact that the spread of DMSA(V) positivity amongst the tumours was greater than thallium positivity, whereas the latter was positive only in patients with grade II and III chondrosarcomas, which are the ones that mainly metastasise.We cannot explain why 2 out of 45 benign tumours were thallium positive. The specimens were reviewed by a panel of pathologists expert in bone pathology and none conferred a diagnosis of sarcoma. In both of these cases, DMSA(V) positivity was also observed. It is important to note that these tumours occurred in the hand and foot, although not part of the syndrome of enchondromatosis. It is appreciated that small bone enchondromata may behave locally in a very aggressive manner without any cytologic evidence of malignancy . HoweverAs a result of this study, we now undertake the following sequence of scanning and treatment Figure . All patSurgical treatment for cartilage lesions vary widely . IntraleThe management of grade 1 tumours remains controversial. Some authors have recommended intralesional curretage, adjuvant treatment and cementation for these lesions ,46. In aThere has been rising interest in studying non-invasive techniques of imaging cartilage tumours to try and determine their biologic aggressiveness prior to definitive surgery. The advantages of functional nuclear scanning with DMSA(V) and thallium are that they are easy to perform in the majority of nuclear medicine departments, the isotopes are easily available and the costs are not prohibitive. We have found that the information derived from our study complements other imaging modalities and apart from improving our understanding of cartilaginous tumours, has also assisted us in developing a strategy for their treatment.The author(s) declare that they have no competing interests.TL-201 – Thallium -201DMSA(V) – Pentavalent dimercaptosuccinic acidProf. Peter Choong Surgeon, carried out surgery, participated in clinical and diagnostic practice. Wrote and prepared manuscriptDr. Toshikunisada Orthopaedic oncology fellow, collected diagnostic dataDr. Stephen Schlicht Nuclear physician, Participated in manuscript preparation, and conduct of diagnostic testsDr. Rodney Hicks Nuclear physician, Participated in manuscript preparation and conduct of diagnostic testsDr. John Slavin Pathologists, participated in blinded review of patients histology


E. coli and B. subtilis both regulate the same task, namely, excitation and adaptation to environmental signals. Both pathways employ many orthologous genes. Yet how these orthologs contribute to network function in each organism is different. To investigate this problem, we propose what is to our knowledge the first computational model for B. subtilis chemotaxis and compare it to previously published models for chemotaxis in E. coli. The models reveal that the core control strategy for signal processing is the same in both organisms, though in B. subtilis there are two additional feedback loops that provide an additional layer of regulation and robustness. Furthermore, the network structures are different despite the similarity of the proteins in each organism. These results demonstrate the limitations of pathway inferences based solely on homology and suggest that the control strategy is an evolutionarily conserved property.Comparable processes in different species often involve homologous genes. One question is whether the network structure, in particular the feedback control structure, is also conserved. The bacterial chemotaxis pathways in  E. coli and B. subtilis, differences that are hard to predict on the basis of sequence homology aloneComputational modeling reveals some important differences in the networks that regulate chemotaxis in  Escherichia coli and Bacillus subtilis, swimming alternates between smooth runs and reorientating tumbles. Smooth runs require that the flagellar motors spin counterclockwise, whereas tumbles result from clockwise spins. Bacteria follow a random walk that is biased in the presence of gradients of attractants and repellents by alternating the frequency of runs and tumble. Owing to their small size, most bacteria are unable to sense chemical gradients across the length of their body. Rather, they respond only to temporal changes. In particular, their stimulated response always returns to prestimulus levels despite the sustained presence of attractants or repellents. Sensory adaptation involves a rudimentary form of memory that allows bacteria to compare their current and past environments. Bacteria regulate chemotaxis using a network of interacting proteins. The basic mechanism in flagellated bacteria involves receptor-mediated phosphorylation of a cytoplasmic protein (CheY) that binds to the flagellar motor and changes the spin direction , E. coli does not adapt  indicate that CheY interacts with the receptors. This model provides one possible feedback mechanism for methylation-independent chemotaxis. The other possibility is CheV. While either CheY or CheV is sufficient for methylation-independent chemotaxis, the model predicts that both feedback loops are necessary to generate the oscillations that are observed in the cheBCDR strains . However in the mutant, there are no complementary changes at residue E637, as it cannot be methylated.When thylated . Cast in removed . These r of CheY  or when  of CheY , we propB. subtilis. While in E. coli CheB phopshorylation is not necessary for adaptation . Likewise, when the majority of CheY is unphosphorylated, CheA is not repressed and residue E630 is preferentially methylated (activating residue). This feedback loop provides a regulatory mechanism for adaptation otherwise absent in aptation , it formaptation . This feaptation . HowevercheC and cheD, chemotaxis genes present in B. subtilis and missing in E. coli, are not treated explicitly in the model. Mutations to either gene are modeled implicitly by perturbing the kinetic parameters governing CheA activation and selective methylation. CheC is homologous to the P2 domain of CheA and the N-terminal domain of FliM .receptor . Adaptatnhibited . The cueB. subtilis. On the one hand, we expect that the rates and concentrations are comparable to their E. coli counterparts, given that many B. subtilis chemotaxis proteins complement in E. coli. On the other hand, the additional feedback loops involving CheV and CheY could mask differences in the rates and concentrations between the two species. Unlike E. coli, many properties of the B. subtilis model, such as the steady-state bias and adaptation time, are insensitive to the kinetic parameters, suggesting that perhaps chemotaxis is more robust in B. subtilis than in E. coli. For lack of a better alternative, we used E. coli parameters for the B. subtilis model when available, as they produce results in the B. subtilis model consistent with experimental measurements.Few kinetic measurements have been made for B. subtilis model were inferred from mutants and lack explicit experimental confirmation. There are a number of experiments that could test the predictions made by the model, and we describe just a few. One experiment is to correlate receptor methylation with CheA activity in vitro using purified components (cheV mutants (e.g., cheBCDR versus cheBCDRV).Many regulatory interactions proposed in mponents . This inmponents . The in E. coli  binds only inactive receptors (IT) and that phosphorylated CheB (PB) binds active receptors (AT). In a simplified version of the model  and that CheB demethylates active receptors (AT) at residue 630 and inactive receptors (IT) at residue 637. If we simplify the model, the concentrations of receptors with residues methylated at 630 (m630)and 637 (m637) are described by the following two differential equations:The YK is the Michaelis constant for phosphorylated CheY and the receptor. Subtracting m = m630 – m637. We assume the concentration PY is proportional to the concentration of active receptors. The relative rate of methyation at residue 630 in E. coli model, E. coli forms memory using the absolute level of receptor methylation m, and B. subtilis forms memory using the differential level of receptor methylation Δm. The structure of Equations The difference between the two species is how receptor methylation forms memory. m and Δm—are equivalent.Both mechanisms are robust; adaptation does not depend on the values of the kinetic parameters. Robust adaptation requires feedback with integral memory . The samE. coli chemotaxis; changes in the relative level of CheR expression did not alter the ability of E. coli to adapt to attractants  and, as a result, are subject to different selective pressures, so it is difficult to explain their differences without further investigating the role of their environment. There is also the issue of sensitivity; E. coli is able to sense gradients in concentrations spanning five orders of magnitude. As formulated, both models fail to capture this observed behavior. Other mechanisms, such as receptor clustering . The results are similar when either gene is deleted. A subpopulation (60%) of B. subtilis cheBCDR cells oscillates when stimulated with chemoattractants (cheD mutants weakly respond to chemoattractants, suggesting that the coupling between the receptor and kinase is attenuated. These results suggest that CheD, which deaminates glutamine residues on the receptors (In the absence of CheR and CheB, computer simulations, consistent with experiments , 1993b, ractants . To moderactants A. Wild-teceptors , enhancecheBCDR mutants oscillate (60%). The remaining cells partially adapt to the addition of attractants . Matlab m-files are available from T exist in either an active (AT) or inactive (IT) state. Let iT denote the concentration of receptor complexes with i residues methylated and αi(L) denote the probability that the receptor complex iT is active when the concentration of chemoattractant is L. The concentration of active receptors isThe chemotaxis model combines the two-state model proposed for adaptation by i(L) are given by the expression0 = 0; α1 = 0.1; α2 = 0.5; α3 = 0.75; α4 = 1; αL0 = 0; αL1 = 0; αL2 = 0.1; αL3 = 0.5; αL4 = 1; LK = 10 μM ( = 10 μM .We modeled the phosphorylation cascade using the mechanism and parameters proposed by A and pA denote the concentrations of CheA and phosphorylated CheA, Y and pY denote the concentrations of CheY and phosphorylated CheY, B and pB denote the concentrations of CheB and phosphorylated CheB, M denotes the concentration of FliM, and [pMY] denotes the concentration of phosphorylated CheY bound to FliM.iT, the rate of demethylation is Bαri(L)iT and the rate of methylation is Br(1 – αi(L))iT, whereWe modeled receptor methylation using the mechanism proposed by i(L))iT and the rate of demethylation is proportional to the concentration of active receptors αi(L)iT. A simple mass balance yields the following set of differential equations for the receptors:rk = 0.255 s–1; RK = 0.251 nM; Bk = 0.5 s–1; BK = 5.5 nM; A + pA = 5 nM; B + pB = 2 nM; Y + pY + [pMY] = 17.9 nM; M + [pMY] = 5.8 nM; and T0 + T1 + T2 + T3 + T4 = 5 nM  active receptors is given by the expressionThe te model . Let Tijiji is the probability that the receptor complex ijT adopts an inactive conformation. The concentration of weakly active receptors is given by the expressionL) is the probability that a weakly active receptor adopts an active conformation. The concentration of weakly inactive receptors is given by the expressionE. coli model, there are two boundary conditions: fully methylated receptors and unmethylated receptors. Furthermore, methylated receptors are active (α = 1) and unmethylated receptors are inactive (α = 0). In the B. subtilis model, there are four boundary conditions: T20, T02, T11, and T00. Furthermore, the methylated receptors T11 and unmethylated receptors T00 are partially active. We needed, therefore, to distinguish additional states to construct a robust model involving activity-dependent methylation.E. coli model, we assume that the kinetics for ligand binding are fast and employ the quasi-steady-state assumption for simplicity. The probabilities αij(L) and iji(L) are given by the expressionsIn a similar manner to the 20 = 1; α10 = 0.4, α11 = 0.2; α00 = α01 = α02 = 0; α020 = 1; α010 = 0.99, α011 = 0.8; α000 = α001 = α002 = 0; i02 = 1; i01 = 0.99, i11 = 0.8; i00 = i10 = i20 = 0; i002 = 1; i001 = 0.4, i011 = 0.2; i000 = i010 = i020 = 0; β = 0.2; β0 = 0.8; LK = 10 μM. The parameters were inferred from tethering experiments, where the attractant asparagine is added and then removed in a flowcell containing wild-type cells and the rotation of the flagellar motor is observed (observed pY) binds receptors. We model receptor binding with the following two differential equations:The model assumes that CheY negatively regulates CheA activity. The model assumes that only phosphorylated CheY  and Ik = 0.5 [pTY](2 + 10IT + 0.1WIT). The term C denotes the concentration of inactive receptor complexes. Evident from the expressions for A kand Ik, weakly active and inactive receptors contribute less to the state of the receptor complex.B. subtilis is an extension of the model proposed for E. coli. The key differences are the addition of CheV and the loss of CheZ. We used a Michaelis–Menten-type expression to model inhibition of the CheA kinase by unphosphorylated CheV (V). There is no dedicated phosphatase for CheY in B. subtilis. However, the motor switch appears to enhance the CheY dephosphorylation when phosphorylated CheY is bound to the motor (The model for the phosphorylation cascade in he motor . We assuB. subtilis, we used the parameters from the E. coli model when available. The parameters for CheV and CheY dephosphorylation were chosen so that the dynamics of the model were similar to those observed in tethering experiments involving wild-type bacteria and cheBCDR mutants ( mutants .ijT, the rate of demethylation for residue 630 is Bαrij(L)ijT and the rate of demethylation for residue 637 is ijB ir(L)ijT, whereFor simplicity, we used Michaelis–Menten kinetics to model the methylation reactions. Similar results were obtained using mass action kinetics. For the receptor 1ij R Trand the rate for residue 637 is 2ijR Tr, wherepY and vice versa. A simple mass balance yields the following differential equation for the receptors:E. coli model: rk = 0.255 s–1; RK = 0.251 nM; Bk = 0.5 s–1; BK = 5.5 nM; A + pA = 5 nM; B + pB = 2 nM; Y + pY + [pMY] + [pTY] = 17.9 nM; M + [pMY] = 5.8 nM; T20 + T10 + T00 + T01 + T02 + T11 = 5 nM; [T] + [pTY] = 5 nM. The model assumes that the concentration of CheV is 8 nM: V + pV = 8 nM.cheBCDR strain described in ACTo model oscillations for the Ak = 0.001T (1 + 0.1WAT) and Ik = 0.001[pTY] (2 + 0.1WIT) with the initial condition T00 = 5 nM. The concentrations of CheB and CheR were set to 0 to account for their deletion. The subpopulation that partially adapts was modeled by setting the concentration of CheV = 4 nM. In this formulation, receptors adopt either a weakly active or weakly inactive conformation. We also induced a timescale separation necessary for a relaxation oscillator by decreasing the transition rate between active and inactive receptor complexes by a factor of 500. This change produced oscillations with a period of 100 s.AC:To model precise adaptation with simple negative feedback by CheY as described in Ak = 0.1[T]WAT and Ik = 0.1[pTY]WIT with the initial condition T00 = 5 nM. The concentrations of CheB and CheR were set to 0. We also needed to change the model for receptor binding:Ak = 0.01/(10 + L) + 0.036L/(10 + L).http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the genes and gene products discussed in this paper are E. coli CheA (AAC74958) and B. subtilis CheA (CAB13516), E. coli CheB (AAC74953) and B. subtilis CheB (CAB13506), B. subtilis CheC (CAB13518), B. subtilis CheD (CAB13519), E. coli CheR (AAC74954) and B. subtilis CheR (CAB14188), B. subtilis CheV (CAB13274), E. coli CheW (AAC74957) and B. subtilis CheW (CAB13517), E. coli CheY (AAC74952) and B. subtilis CheY (CAB13506), E. coli CheZ (AAC74951), and B. subtilis FliY (CAB13505).The GenBank (


Merkel cell (neuroendocrine) carcinoma is a small round blue cell malignant neoplasm that primarily presents in the skin. The diagnosis of Merkel cell carcinoma in a pleural fluid is challenging because of the morphological similarity to many other malignant neoplasms. Immunohistochemical stains can be essential to establish the diagnosis of Merkel cell carcinoma.A 77 year-old woman presented with a mass in her right buttock thought clinically to be a boil or sebaceous cyst. Upon histopathologic review including immunohistochemical analysis, a diagnosis of Merkel cell carcinoma was rendered. Wide-excision and sentinel lymph node biopsy revealed negative margins and no evidence of metastasis. Ten months later she complained of bone pain and a bone scan revealed multiple lesions. An abdominal CT scan revealed a T4 vertebral mass and local radiotherapy was administered. Two months later the patient presented with shortness of breath. A chest radiograph showed an effusion and thoracentesis was performed. The fluid was confirmed to contain metastatic Merkel cell carcinoma by cytology and immunohistochemical analysis.Merkel cell carcinoma is an aggressive neoplasm that can, despite careful surgical management, occasionally present as a malignant pleural effusion in a relatively short time period. Immunohistochemical analysis can aid in confirming this rare outcome. The Merkel cell is the namesake of an F. Merkel who in 1875 described distinct round cells in the basal layer of the epidermis . Toker fA 77 year old female presented to her primary care physician with a complaint of a right buttock mass. Her past medical history was significant for colonic adenocarcinoma treated surgically, twelve years prior. The skin overlying the 2 × 3 cm firm, non-fluctuant, non-tender mass was erythematous. Initially the mass was thought to be a boil or carbuncle. Six weeks later during a visit with a surgical oncologist for routine follow-up of her colonic adenocarcinoma resected 13 years prior, the patient noted that the mass was becoming quite painful and enlarging rapidly. The patient was then taken to the OR for local excision. Pathology revealed a small round blue cell tumor forming large nests and infiltrating as single cells between thick strands of fibrous tissue in the dermis and extending to the subcutaneous fat. There were abundant mitoses, a lack of necrosis or obvious vascular invasion. The epidermis was uninvolved. The histologic and immunochemical findings supported the diagnosis of Merkel cell carcinoma. A wide local excision and sentinel lymph node biopsy were performed. One sentinel lymph node was negative for malignancy and there was no residual tumor identified in the excision. No adjuvant therapy was given.Ten months after the initial presentation the patient had increasing shoulder pain that radiated into the chest and flank. A CT of the thoracic spine revealed a 3.0 cm mass in the area of the fourth thoracic (T4) vertebra. A CT guided needle biopsy of the area revealed metastatic Merkel cell carcinoma. A bone scan revealed additional areas of concern in the right humerus and bilateral distal femurs. The patient received radiation therapy to the thoracic spine and right distal femur for palliation of bone pain.A year after first presenting for a right buttock mass, the patient was admitted to the hospital for increasing dyspnea. A chest radiograph revealed a large right pleural effusion, which was drained for both therapeutic and diagnostic purposes. The fluid was grossly bloody and cytology revealed abundant, round, basophilic single cells, many in mitosis, with characteristic granular, "salt and pepper" nuclear chromatin Figure . A cellbThe diagnosis of Merkel cell carcinoma in pleural fluid is challenging partially because of its rarity. To our knowledge this is the first case report of Merkel cell carcinoma in a pleural fluid characterized with immunohistochemical stains. Wason and Friedman described the only other reported case of pleural effusion due to metastatic Merkel cell carcinoma in 1985 . In thatSince Merkel cell carcinoma rarely appears in pleural fluid and small cell lung carcinoma frequently does the pathologist must have the salient cytomorphologic features of Merkel cell carcinoma in mind to make the diagnosis in the absence of clinical history. The most characteristic features of Merkel cell carcinoma in pleural fluid include convolution of nuclei and lack of prominent nuclear molding. Once Merkel cell carcinoma has entered the differential diagnosis the case can be pursued.With immunohistochemistry, it is now possible to definitively diagnose Merkel cell carcinoma in pleural fluid without electron microscopy. The immunohistochemical profile of Merkel cell carcinoma is distinct and has been well established on paraffin embedded tissue . Tumor cMetastatic Merkel cell carcinoma as the cause of a malignant pleural effusion is a rare occurrence. However, such an aggressive neoplasm as Merkel cell, which is often widely metastatic, may be a more common cause of malignant pleural effusion than recognized. Morphologically, the tumor cells in a body fluid could easily be mistaken for a malignancy of another origin. In the work-up of the pleural effusion caused by a small round blue cell process, inclusion of immunohistochemical markers specific to Merkel cell carcinoma may be prudent.The authors declare that they have no competing interests.


Understanding the influences of educational and economic variables on food consumption may be useful to explain food behaviour and nutrition policymaking. The aim of this study was to evaluate the importance of educational and economic factors in determining food pattern in Portuguese adults.A cross-sectional study in a representative sample of Portuguese adults (20977 women and 18663 men). Participants were distributed in four categories according to years of education (≤4, 5–9, 10–12, and >12) and income (≤314 euros, 315–547 euros, 548–815 euros, and >815 euros). Logistic regression models were fitted to estimate the magnitude of the association between food groups and education/income, adjusting for confounders.In both genders, the odds favouring milk, vegetable soup, vegetables, fruit, and fish consumption, increased significantly with education, for those having >12 years of education compared to those with ≤4 years; the odds favouring wine, and spirits consumption decreased significantly with education, for those having >12 years of education compared to those with ≤4 years. In males, the odds favouring starchy foods and meat consumption decreased significantly with income, while for milk, the odds increased with higher income (those having >815 euros compared to those with ≤314 euros).The low and high income groups are or tend to be similar in regard to several food groups consumption, and access to education/information appears to be the key element to a better food pattern as indicated by higher frequency of milk, vegetable soup, vegetables, fruit, and fish consumption. There is a large published literature on associations between socio-economic position and chronic disease, with socioeconomically disadvantaged groups experiencing higher mortality and morbidity rates for coronary heart disease, noninsulin dependent diabetes mellitus and some cancers -4. ChronThe relationship between socio-economic factors and diet has been examined on the basis of food and nutrient intake and the results are mixed, sometimes contradictory, and often the observed differences are small ,10. WhenIn the context of European countries, economic development and increased purchasing power have recently changed the food availability situation . On the The aim of this study was to evaluate the importance of educational and economic factors in determining food choice in a representative sample of the Portuguese general adult population.Data for this study were drawn from the Portuguese third National Health Survey  carried out in 1998–1999. The study sample included all subjects (20977 women and 18663 men) older than 18 years, who reported their education level, income, physical activity, smoking habits, weight, height, and food intake when participating in the National Health Survey. Subjects were selected from 21808 households distributed according to the five regions of Portugal , using a multi-stage random probability design. This probabilistic sample is representative of the Portuguese population from the Continental area (Azores and Madeira islands were not included). The survey response rate was 82%.Trained interviewers conducted face-to-face interviews with the person in each household and inquired participants on social and demographic characteristics, smoking status (non-smokers, ex-smokers, smoking less than one cigarette per day, and smoking one or more cigarettes per day), weight, height , food and beverages intake, and daily physical activity .Occupational physical activity was measured using the respondent's own occupation at the time of the survey. Respondents were asked about what best characterized their daily occupational activity, namely: usually seated and walking during short periods of time; standing activities or walking during long periods of time without carrying loads to often; carrying light objects or walking upstairs/downstairs several times; heavy physical work or carrying heavy objects; or don't know.Respondents were asked to describe their leisure-activity using the following classification: heavy training and competitive sports more than once a week; running or practicing recreational sports or gardening activities ≥4 hours per week; walking for pleasure, bicycling (light effort) or doing other light activities ≥4 hours per week; reading, watching television or other sedentary activities; and don't know. Respondents were also asked to provide information about whether they had regular activities (once or more per week) such as running or bicycling (enough to make them feel tired).Respondents were asked twelve questions related to their intake of central food groups and beverages, namely milk, vegetable soup, meat, fish, vegetables, fruit, bread, starchy foods (pasta/rice/potatoes), beer, spirits, Port Wine, and wine, and the consumption was recorded as a yes (when the respondent indicated the consumption of the food) or no answer. Because the data were collected by interviewers within the framework of an epidemiological study that was not specifically designed to assess quantitative aspects of nutritional and food intake, the dietary assessment method employed generic classifications of food groups, rather than specific varieties or species , or quantitative measures. Consumption of these food items was determined by asking "For each of the listed food items please indicate those consumed": "during the day before the interview" (vegetable soup, meat, fish, vegetables, fruit, bread, and starchy foods – pasta, rice and potatoes); "during the week before interview" (beer, spirits, and Port Wine); and "daily consumed" (milk and wine).Respondents were asked to provide information about whether they had attained further education since leaving school and if so, the highest qualification completed. Respondent's education was subsequently classified in four levels of education: less than 4 years, 5–9 years, 10–12 years, and more than 12 years.Respondent's were asked to estimate the total income  received by all household members in the last month and to indicate this using a single measure comprising ten narrow-ranged income categories. This measure was subsequently re-coded into four categories according the number of salaries: less than 315 euros, 315–547 euros, 548–815 euros, and >815 euros.Separate logistic regression models were fitted for male and female to estimate the magnitude of the association between food groups consumption and education or income categories, adjusting for age, BMI, smoking habits, physical activity and income/education. An exploratory approach was chosen in the selection of explanatory variables in order to control for as many potentially significant variables as possible in the regression model. The choice of variables  was based on findings reported in the literature, our own experience with specifically Portuguese factors associated with food consumption and their associations with the variables of interest; education was also adjusted for income and vice versa.p-value of less than 0.05 was considered statistically significant. Statistics were performed using SPSS 12.0.Student's t-tests, ANOVA, Spearman rank correlation analyses and chi-squared tests were used to compare BMI, age, frequency of smoking, physical activity categories between genders to determine the degree to which those variables correlated with education and income. A 2 versus 25.6 ± 3.83, p < 0.001). There was a lower proportion of smokers among women compared to men (8.2% versus 30.5%, p < 0.001). General characteristics  by education and income categories are presented in Tables The study sample comprised 20977 women (52,9%) and 18663 men, with mean ages of 50.3 (±18.88) and 47.7 (±18.51) years, respectively; BMI was significantly lower in women than in men , being the odds ratios, respectively, 2.60 2.24–3.01), 1.20 (1.05–1.38), 1.75 (1.44–2.13), 1.92 (1.49–2.49), and 1.40 (1.23–1.60) for those having >12 years of education compared to those with ≤4 years, after adjusting for age, BMI, smoking habits, physical activity and income (Tables .01, 1.20In men, similar odds ratios were observed for milk, vegetable soup, vegetables, fruit, fish, bread, wine, and spirits Tables  and 4. HNo such significant trends were observed for these food groups and income with the exceptions of meat and starchy foods (other than bread) consumption, in men, which decreased with increasing income (p trend ≤ 0.022), and milk consumption which increased with increasing income Tables  and 4.The main finding of the present study is that educational attainment was more frequently associated with food choices than income. There is general agreement among researchers -23 that Over the last years, several studies have attempted to identify the influence of socioeconomic factors on individual's dietary intake -28. Our Several studies have concluded that a strong relationship exists between countries' per capita national incomes and nutrition -32. The Data from the Portuguese Household Budget Surveys (using the DAta Food Networking – DAFNE – classification system), shows similar results to ours in relation to the positive association between education attainment and the availability of fruits, fish, milk and alcoholic beverages but some different data in regard to other foods  . CuriousIn our study, there was also a significant trend in the consumption of milk in men, being more frequently ingested with increasing income. As suggested by Axelson,  positiveThe association between milk consumption and socio-economic position is sometimes contradictory. Cristofar and Basiotis , for exaConsumption of alcoholic beverages, such as wine and spirits, in both genders, and beer in men, exhibited significant decreases in their frequency of intake, with increasing education levels. In Portugal, alcoholic beverages consumption is a major public health problem . In DAFNInterestingly, in our study, the consumption of bread (in both genders) and other starchy foods (in women), decreased with increasing number of years of education; men seem to abandon starchy foods (other than bread) consumption under condition of higher incomes. It is possible that higher educated individuals tend to avoid foods that are considered as being more fattening or rich in energy, such as bread and other starchy foods ,44. ReseOne of the most interesting findings in terms of economic constraints and food consumption relationship in our study, is the few significant associations between income and food choices, even though the well established links between economic and material resources, food availability and dietary quality . By contIn our study, it remains to be explained the different pattern of associations between income and important food groups (milk versus fruit and vegetables, for example) and the different pattern of associations between food choices and income in each gender (e.g., milk and starchy foods). Several reasons may explain specific differences in the findings of our study compared with those of previous mentioned studies, including differences in populations sampled , differences in assessment of education or income, differences in dietary assessment  and differences in analytic methods . Nonetheless, results from our study indicate that the associations between food choice were stronger in relation to educational attainment than income categories. Differences in food choices according the level of education reflect that more knowledge may influence the perceived relationship between diet and health as well as the perceived outcomes of following a healthy diet .Despite differences in food consumption according education and income, in our study we could not assess if these differences were also evident on the energy and nutrient level, which was a limitation. British data point to micronutrient and antioxidant intakes as the most likely nutritional influences on health inequalities . NeverthIf a country like Portugal wants to change the adult food choice behaviour, or in other words, wants to reach certain dietary goals, the support of applied research like ours is needed in order to plan the right strategies for promoting healthy diets. Confidence in a significant positive causal link between per capita national income and individual nutrition reinforces the importance of economic growth  but alsoWhereas income-related dietary differences suggest ameliorative responses through the potential of the economic system, differences based on education point to initiatives such as nutrition education programmes ,54. AccoRegardless of the reasons explaining the complex and diversified patterns of economic and educational associations of food consumption found in Portugal, the findings of this study suggest that education and income have distinct associations with food choice. The low and high income groups are or tend to be similar in regard to the majority of food choices, and access to education appears to be the key element to a better food pattern as indicated by higher frequency of milk, vegetable soup, vegetables, fruit, and fish consumption.The author(s) declare that they have no competing interests.PM and PP designed the study. PM and PP did the statistical analysis, and PM wrote the paper. PM and PP reviewed the final version of the paper.The pre-publication history for this paper can be accessed here:


Highlights of Bela Julesz's scientific career in visual neuroscience Throughout his career, Bela Julesz created new scientific disciplines by remarkable combinations of seemingly disparate approaches. The selection of his major discipline, which would eventually be called visual neuroscience, may have been serendipity or choice.When the unexpected Soviet invasion of Hungary in 1956 spurred his emigration to the United States, Bela Julesz, with his Hungarian doctorate in engineering, joined the numerous mathematical luminaries working at AT&T Bell Laboratories, such as John Tukey, Harry Nyquist, Claude Shannon, and John Kelly. One of the projects underway at the time was the creation of long random-number binary sequences that did not repeat. Bela told the story that he was assigned the problem of testing these number generators; he decided to use the best pattern recognizer that he knew of—the human visual system. The random bits of zeros and ones drawn from the random number sequences were plotted as sequential rows in an image. Any repeats, any correlations across space, would be instantly seen by the human visual system as patterns in the random dots. What caused Bela to choose this unusual approach to looking for patterns, combining computers and vision? His doctoral thesis research in network theory and television signals clearly influenced him, but it was quintessential Bela to give himself a hand up into a new field by building on his base of knowledge, moving in a new and unexpected direction using mathematical and psychological insight. He termed this talent “scientific bilingualism” .This success in exploiting the visual system, and the intellectual freedom intrinsic to the design of Bell Labs, provided Bela with the opportunity to use these new random dot patterns to explore the visual system. Most of us know well that we can use the small differences in the images in each eye to see depth. Sir Charles Wheatstone showed in 1838 that if two different perspective images were observed through a stereoscope so that each eye observed only one view, a startlingly realistic three-dimensional image occurred. Oliver Wendell Holmes, stereoscope enthusiast, wrote of the experience that “the shutting out of surrounding objects, and the concentration of the whole attention, which is a consequence of this, produce a dreamlike exaltation…in which we seem to leave the body behind us and sail away into one strange scene after another, like disembodied spirits” .Journal of the Optical Society of America, where it was rejected; the Bell Labs Technical Journal holds the now classic paper [Journal of the Optical Society of America published Bela's second paper [The basis of this three-dimensional perception was hotly debated between Wheatstone and fellow physicist Sir David Brewster.  Brewster opined that perspective was the source of the apprehension of an object's shape. Wheatstone insisted that the images in the each eye had identifiable landmarks that were combined to assign depth to the landmarks. Bela read much of the literature of that time, and he must have seen two greats as wrestling without either finding the overwhelming hold to pin down the other. More than one hundred twenty years after Brewster and Wheatstone, Bela realized that his random dot patterns could be used to probe this question. What Bela did was create a pair of identical random dot patterns. When viewed binocularly through a stereoscope (i.e., fused), they would be seen as a single surface. Then Bela took a central region from the right random dot pattern and displaced it minutely to the right. Now when the two patterns were fused, the central square was not seen double, but after a moment or two, eerily moved into depth, behind the surrounding region. In 1960, Bela's experiment with what eventually became known as Julesz random dot stereograms unambiguously demonstrated that stereoscopic depth could be computed in the absence of any identifiable objects, in the absence of any perspective, in the absence of any cues available to either eye alone. It was a perfect combination of psychological and mathematical insight and technology that solved this puzzle. (It is an interesting aside that Bela sent his first report to the ic paper . The Jound paper .) The stFoundations of Cyclopean Perception (1971) that early in the vision process the two images from the two eyes were combined to form a single view, imbued with inherent depth information. The perceptual “cyclops within us” was proposed to analyze the visual world first, before the motion, color, and contrast systems began their perceptual operations. Bela's book is full of powerful visual experiments that make this point irrefragably; from his psychophysical analysis, binocular vision forces unexpected constraints on the rest of vision, Q.E.D. Foundations of Cyclopean Perception is still considered one of the classics of modern psychophysics and continues to have profound relevance to both those entering the field and established investigators—over thirty years after its publication. At the time of his death, Bela had begun working on a second edition.Bela proposed in his book His success in determining the sequence of visual processing using random dot stereograms led Bela to propose that the anatomical hierarchy of the visual system could be understood in part through visual psychophysics—he termed this approach “psychoanatomy.” His ingenious use of the stereogram established a new approach in the field of vision research and presaged the now common use of carefully controlled computational techniques in brain science. By this time Bela's reputation was established, and in 1983, he received a prestigious MacArthur Fellowship—the “genius award.” He used the funds for travel, including an annual peregrination to the California Institute of Technology, where I first met Bela in 1985.His seminars and lecture courses were enthusiastically received and endorsed by countless students, post-doctoral trainees, and faculty, as evidenced both by his formidable reputation and through the numerous citations of his work. His approach to presenting his research was modest and gently self-deprecating. He always encouraged young scientists; his joy and passion in their science were transmitted both through his warm persona and his suggestions of directions for future study. His insights guided my development of random dot kinematograms (i.e., movies) to examine how motion could be used to construct three-dimensional form . He collBela was a fount of ideas, each building on the prior's advance. His later passions were explorations of texture and attention, notably with Jonathan Victor and Dov Sagi. Bela's appealing hypothesis that textons (putative elements of textures) are represented at a cellular level is now questionable . Bela waDialogues on Perception (1995), a wide-ranging intellectual effort, in which he uses classic dialectics to question both his own successes and those of his chosen field. In the book one reads of two competing intellects, a Bela who believes in his contributions to science and another Bela who is constantly belittling and judging his contributions.In 1989, Bela retired from Bell Labs (by then he was a department head) and joined the Department of Psychology at Rutgers University to establish the Laboratory of Vision Research. Bela continued investigating mechanisms of form, texture, and stereopsis; his presence led to numerous studies into the implications of his original findings as well as new investigations into computational vision. His collaborations greatly aided the establishment of neuroscience at Rutgers. Bela wrote Throughout his career Bela Julesz was able to add language after language to his research imperative, becoming a true scientific polyglot. Although his arrival in the United States was propelled by political events beyond his control, his intellectual directions followed a chosen path “less traveled by, and that has made all the difference.” In 1956, an engineer set out from Hungary. By 2003, his unique combination of mathematical precision combined with deep biological insight had carried him to elegant solutions for seemingly intractable problems in visual neuroscience. Bela was always in dialogue, often with others, and often with himself. In the process, he would gently drive each of us, and himself, forward to our final destination of understanding the brain. Bela Julesz died on December 31, 2003, forty-seven years to the day after starting at Bell Laboratories.


Since the mid eighties, responsiveness is considered to be a separate property of health status questionnaires distinct from reliability and validity. The aim of the study was to assess the strength of the relationship between internal consistency reliability, referring to an instrument's sensitivity to differences in health status among subjects at one point in time, and responsiveness referring to sensitivity to health status changes over time.We used three different datasets comprising the scores of patients on the Barthel, the SIP and the GO-QoL instruments at two points in time. The internal consistency was reduced stepwise by removing the item that contributed most to a scale's reliability. We calculated the responsiveness expressed by the Standardized Response Mean (SRM) on each set of remaining items. The strength of the relationship between the thus obtained internal consistency coefficients and SRMs was quantified by Spearman rank correlation coefficients.Strong to perfect correlations (0.90 – 1.00) was found between internal consistency coefficients and SRMs for all instruments indicating, that the two can be used interchangeably.The results contradict the conviction that responsiveness is a separate psychometric property. The internal consistency coefficient adequately reflects an instrument's potential sensitivity to changes over time. Responsiveness, a concept introduced in the mid-eighties by bio-medical researchers, is considered to be an essential measurement property of health status questionnaires, distinct from reliability and validity . HoweverWe used three datasets comprising the scores of patients on three widely used health status instruments on two moments in time in order to assess health changes. The first dataset was from a randomized clinical trial investigating the effects of arm and leg rehabilitation training on the functional recovery of stroke survivors using the 10-item Barthel (basic) activities of daily living scale . PatientOnly subjects with no missing values at baseline or follow-up were included in the analysis. The Barthel dataset had no missing values, the SIP datasets had 13 % (16/120), 28% (64/227) and 0.7% (1/141) missing values respectively and the GO-QOL had 0.6% (1/164) missing values. For the SIP datasets, there was a mean deterioration in health (1 point), for the Barthel and GO-QOL scales patients were improved at follow-up  reflecting sensitivity to differences in health status among patients , and theFigures Our results contradict the conviction that responsiveness is a separate psychometric property of health scales. Internal consistency reliability, reflecting a scale's sensitivity to cross-sectional differences in health, closely coincided with the instruments' sensitivity to change as measured with the standardized response mean. Our results also reflect what is already known within the framework of classical test theory. A test score cannot correlate more highly with any other variable than its own true score . This imWe used the SRM effect size that uses the standard deviation the change scores and therefore includes all information about the changes on the selected instruments. The results can not generalized to alternative effect sizes such as Cohen's effect size or Guyatt's responsiveness statistic  because In a frequently cited paper, Guyatt et al.  made theDuring the past 20 years, clinimetric research has resulted in about 25 definitions and 30 measures of instrument responsiveness, sometimes referred to as sensitivity to change or longitudinal validity . MoreoveRobert Lindeboom conceived the idea for the manuscript, Mirjam Sprangers and Robert Lindeboom wrote the manuscript, Koos Zwinderman provided the mathematics and rewrote parts of the manuscript in earlier draftsAppendix 1: Is it possible to have one high reliable scale with low 'responsiveness', and a low reliable scale with high 'responsiveness' measuring the same construct?Click here for file


A fresh look at classic work of Thomas Gold on how the inner ear processes sound Sitting in the enveloping quietness of an anechoic chamber, or other quiet spot, you soon become aware that the ear makes its own distinctive sounds. Whistling, buzzing, hissing, perhaps a chiming chorus of many tones—such continuous sounds seem remarkably nonbiological to my perception, more in the realm of the electronic.Even more remarkable, put a sensitive microphone in the ear canal and you will usually pick up an objective counterpart of that subjective experience. Now known in auditory science as spontaneous otoacoustic emission, the sound registered by the microphone is a clear message that the cochlea uses active processes to detect the phenomenally faint sounds—measured in micropascals—that our ears routinely hear. If the ear were more sensitive, we would need to contend with the sound of air molecules raining upon our eardrums.What is that process—the mechanical or electrical scheme that Hallowell Davis in 1983 called the ‘cochlear amplifier’ —which enThat question has engaged my curiosity since the late 1970s, when English auditory physicist David Kemp first put a microphone to an ear and discovered the telltale sounds of the cochlea at work . Siren-lKemp's discoveries are rightly viewed as opening a fresh path to auditory science, and to the tools and techniques for diagnosing the functional status of the cochlea. But in terms of fundamental understanding, a key paper remains that of Thomas Gold more than half a century ago . Still cThe first option, sympathetic resonance, has the advantage of allowing vanishingly small energies to build up, cycle by cycle, into an appreciable motion—like boosting a child on a swing. The second, travelling wave, has the weight of von Békésy's extensive experiments behind it. At the same time, one of the drawbacks of the travelling wave theory is the difficulty of accounting for the ear's exquisite fine tuning: trained musicians can easily detect tuning differences of less than 0.2%. Even von Békésy himself notes, on page 404 of his classic book, that ‘the resonance theory of hearing is probably the most elegant of all theories of hearing’.Gold's work, done in collaboration with RJ Pumphrey , was theGold's pioneering work is now acknowledged to be a harbinger of Kemp's discoveries. But there is one aspect of Gold's paper that is not so widely considered: Gold's experiments led him to favour a resonance theory of hearing. In fact, the abstract of his 1948 paper declares that ‘previous theories of hearing are considered, and it is shown that only the resonance theory of Helmholtz… is consistent with observation’.Gold and Pumphrey did psychophysical experiments in which hearing thresholds were determined for listeners first for continuous pure tones and then for increasingly briefer stimuli of the same frequency. Gold and Pumphrey showed that their results could only be accounted for by considering the cochlea as a set of resonators, each of which responds to a narrow frequency range.(Q), or narrowness in frequency range, of the presumed underlying resonance.In a second neat experiment, listeners had to detect differences between the sound of repetitive tone pips (series one) and those same stimuli but with the phase of every second pip inverted (series two, in which compressions replaced rarefactions and vice versa). Out-of-phase pips should counteract the action of in-phase pips and, following the child-on-swing analogy, rapidly bring swinging to a halt. Therefore, the argument goes, the two series should sound different. By increasing the silent interval between pips until the difference disappeared, the experimenters could infer how long the vibrations (or swinging) appeared to persist and could then put a measure on the quality factor Q of 32 to 300, meaning that the range of response was as little as 1/300-th of the imposed frequency—based on the picture of a broad travelling wave. The second experiment gave comparable results. However, their resonance interpretation has been dismissed because of a methodological flaw in the second experiment: the spectral signatures of the two series are not the same and provide additional cues. Nevertheless, it is not widely appreciated that the first experiment seems methodologically sound, and its results remain persuasive.From the first experiment, Gold and Pumphrey derived values of I think the resonance theory deserves reconsideration. The evidence of my ears tells me that the cochlea is very highly tuned, and an active resonance theory of some sort seems to provide the most satisfying explanation. Furthermore, as well as Gold's neglected experiment, we now know from studies of acoustic emissions that the relative bandwidth of spontaneously emitted sound from the cochlea can be 1/1000 of the emission's frequency, or less. My research, guided by Professors M. V. Srinivasan and N. H. Fletcher, has centred on finding an answer to that most fundamental question: if the cochlea is resonating, what are the resonant elements?A point of inspiration for me is Gold's later discussion of cochlear function —some ninMy research is investigating what Gold's underwater piano strings might be. A suggestion put forward in a recent paper  is that With a prime candidate in place for the resonating elements, this should, I think, prompt us to re-evaluate resonance theories of hearing, which were first put forward by the ancient Greeks and which, irrepressibly, keep resurfacing. The best-known resonance theory was that formulated by Helmholtz, but at that time no satisfactory resonating elements could be identified, and it lapsed until Gold's attempt to revive it. There are other difficulties in reviving a resonance theory of hearing, but I think they can be overcome.If there really are resonant elements in the ear, the outstanding question would be, how are they stimulated? It is conceivable that motion of the conventional travelling wave sets them off, in which case we have an interesting hybrid of travelling wave and resonance. The other possibility, which I favour, is that outer hair cells are stimulated by the fast pressure wave that sweeps through all of the cochlear fluid at the speed of sound in water (1,500 m/s). If that is the case, and outer hair cells are primarily pressure sensors, not displacement detectors, then the ear is a fully resonant, pressure-driven system. New life, perhaps, to that old resonance idea.


Rubella virus (RV) causes severe congenital defects when acquired during the first trimester of pregnancy. RV cytopathic effect has been shown to be due to caspase-dependent apoptosis in a number of susceptible cell lines, and it has been suggested that this apoptotic induction could be a causal factor in the development of such defects. Often the outcome of apoptotic stimuli is dependent on apoptotic, proliferative and survival signaling mechanisms in the cell. Therefore we investigated the role of phosphoinositide 3-kinase (PI3K)-Akt survival signaling and Ras-Raf-MEK-ERK proliferative signaling during RV-induced apoptosis in RK13 cells. Increasing levels of phosphorylated ERK, Akt and GSK3β were detected from 24–96 hours post-infection, concomitant with RV-induced apoptotic signals. Inhibition of PI3K-Akt signaling reduced cell viability, and increased the speed and magnitude of RV-induced apoptosis, suggesting that this pathway contributes to cell survival during RV infection. In contrast, inhibition of the Ras-Raf-MEK-ERK pathway impaired RV replication and growth and reduced RV-induced apoptosis, suggesting that the normal cellular growth is required for efficient virus production. Rubivirus genus of the Togaviridae. It has a positive-sense single stranded RNA genome that is 9762 nucleotides (nt) in length and contains two non-overlapping open-reading frames (ORFs). The 5' proximal ORF encodes the p200 polyprotein precursor for the nonstructural proteins (NSPs) p150 and p90 [Rubella virus (RV) is the sole member of the  and p90 ,4.RV infection usually causes mild disease with few complications. However, infection during the first trimester of pregnancy results in fetal infection, and in more than 75% of cases this leads to the development of congenital abnormalities. These abnormalities include sensorineural deafness, mental retardation, and congenital heart defects, and are collectively termed congenital rubella syndrome (CRS) -12. ApopEukaryotic cells contain a large number of mitogen activated protein kinase (MAPK) signaling cascades that are activated in response to growth factors, cytokines and stress stimuli such as viral infection and UV irradiation. In common with apoptotic proteins, MAPKs are highly conserved and ubiquitously expressed ,15. Thes473 and Thr308. Activated Akt promotes cell survival by phosphorylating and inhibiting a number of pro-apoptotic proteins including BAD, caspase-9, GSK-3β and Forkhead transcription factors [PI3Ks phosphorylate plasma membrane inositol lipids at the 3' position of the inositol ring. These 3'phosphoinsoitides recruit proteins such as Akt and phosphoinositide dependent kinases 1 and 2 (PDK1/2) to the plasma membrane via their pleckstrin homology (PH) domains ,17. At t factors ,19.myc, c-jun, and Elk-1, which regulate cell cycle progression responses [The Ras-Raf-MEK-ERK is a classical MAPK pathway where growth factor-receptor interactions trigger intracellular activation of the small G-protein Ras. Ras recruits and directly activates the MAPK kinase kinase (MAPKK) Raf, which phosphorylates and activates the MAPK kinase (MAPKK) MEK1/2, which in turn activate the MAPK ERK1/2. Activated ERK1/2 translocates to the nucleus where it can activate a number of transcription factors including c-esponses .Activation of PI3K-Akt and Ras-Raf-MEK-ERK signaling cascades during virus infection is thought to play an important role not only in cellular growth and survival, but also in virus replication and growth during both acute and chronic virus infections -25. ThisThe effect of RV infection on PI3K-Akt and Ras-Raf-MEK-ERK pathways was investigated by examining the expression and phosphorylation profiles of Akt, ERK1/2 and their downstream targets. Cell lysates from RV and mock infected RK13 cells were collected 12–96 hours post-infection (p.i.), separated by SDS-PAGE, and analyzed for total and phosphorylated Akt and ERK1/2 by Western blotting. Phosphorylated Akt and ERK1/2 could be detected in RV-infected cells from 48 hours p.i., and band intensity increased from 48–96 hours p.i. compared to total levels Fig. . Phosphomyc and BAD were also examined by Western blotting between 12–96 hours p.i.  at 96 hours p.i. in the mock-infected cells, suggests that cell survival mechanisms may be activated in older uninfected cell cultures. The phosphorylation pattern of downstream target p70S6K did not follow that of Akt and ERK1/2. Apart from being phosphorylated by ERK1/2 and mTOR/FRAP downstream of Akt, p70S6K can be phosphorylated by an array of different proline-directed kinases, including PDK1, PKCζ, JNK and cdc2 which may explain this difference -33.myc, a downstream target of ERK1/2, did not follow the same pattern. Levels of phosphorylated c-myc decreased as infection progressed, which was probably due to its targeted degradation or the action of cellular phosphatases. RV infection has been observed to slow cell cycle progression both in vivo and in vitro [myc is a transcription factor that stimulates cell cycle progression, its de-phosphorylation or degradation as RV infection progresses supports these observations. The expression and activity of c-myc and other downstream transcription factors in relation to the cell cycle during RV-infection requires further investigation. Phosphorylation of BAD, downstream of Akt, could not be detected in RV-infected cells (data not shown). However, BAD is not ubiquitously expressed and therefore may not be produced in the rabbit kidney epithelial cells (RK13) used [The phosphorylation of c-in vitro ,34. As c13) used .Inhibition of PI3K signaling with LY294006 significantly increased the speed and magnitude of RV-induced apoptosis as shown by increased caspase activity, dead floating cells, apoptotic laddering of genomic DNA and decreased cell viability. Thus, RV-induced apoptotic signaling appears to be held in check by host cell survival signals downstream of PI3K. Although inhibition of PI3K did not affect RV replication, virus growth was affected. The speed of apoptotic monolayer death may have prevented production of optimal virus titers.The importance of PI3K survival signaling has been observed with other viruses. Recently phosphorylation of Akt, GKS3β and PKCζ , has been demonstrated in Vero E6 cells early during infection with severe acute respiratory syndrome (SARS)-associated corona virus (CoV) . HoweverInhibition of the MEK1/2 in RK13 cells by U0126 resulted in necrotic monolayer destruction and a significant decrease in cell viability. XTT assay and light microscopy demonstrated that RV infection appeared to delay the effect of U0126. As discussed above, RV infection stimulates ERK activity, downstream of MEK, and may therefore counteract the effect of the inhibitor. Despite this, U0126 impaired RV replication, growth, and induction of apoptosis. Therefore it appears that although RV infection slows the cell cycle progression, cells must be cycling and metabolizing normally for RV replication to occur.in vitro has also been shown to stimulate biphasic activation of MEK1/2 and ERK1/2, and U0126 treatment prevented export of ribonucleoprotein complexes from the nucleus and inhibited virus production [in vitro.ERK1/2 phosphorylation has also been observed during infection with a number of other viruses, and inhibition of ERK1/2 signaling by U0126 has consistently been shown to be detrimental to virus growth. Infection of Jurkat cells with CVB3, for example, leads to up-regulation of ERK1/2 phosphorylation, and elevated levels of phosphorylated ERK1/2 have been observed in the myocardium of mice susceptible to CVB3-induced myocarditis . Treatmeoduction . Inhibitoduction . These fOver-expression of Akt and MEK1/2 increased RV-induced caspase activity in RK13 cells. This response was not due to the transfection procedure, as the increase in caspase activity was not observed in the pUSEamp(+) or lipofectamine controls. Such a response is also seen in malignant cells, which are more readily killed by apoptotic stimuli. Thus, the over-expression of these mitogenic pathways may have resulted in a cell survival response whereby a negative feedback loop occurred that sensitized cells to RV-induced apoptosis. In order to study this further, it would be necessary to construct stable cell lines over-expressing active Akt and ERK1/2 as well as their dominant negative mutants and other signaling proteins.in vitro depends on numerous signaling events. It has been suggested that RV capsid protein, when anchored to the ER can independently induce apoptosis in culture . However this has not been confirmed by other groups and there is conflicting evidence that virus replication and the presence of the RV NSPs (which are necessary for replication) is required [It is clear from the results of this and previous studies that the outcome of RV infection required ,12,41. Irequired ,43. How In vivo studies are difficult, as a reliable animal model does not exist for CRS. However, it may be possible to extrapolate findings from cell culture systems. We used RK13 cells because they are the best cells in which to detect rubella-induced apoptosis; further studies are required to confirm our findings in primary human embryonic cells.In relation to CRS, study of the expression and localization of apoptotic and mitogen activated signaling proteins in RV-infected fetal tissues would be necessary to confirm the theory that the pathogenesis of the disease is related to perturbation of the cell cycle. However as CRS is now rare in the UK and work with fetal tissues is tightly regulated, such a study would be hard to carry out. Stock concentrations of PI3K inhibitor LY294002 [2-(4-Morpholinyl)-8-phenyl-1-4H-1-benzopyran-4-one] and MAPK/MEK inhibitor U0126 [1, 4-Diamino-2, 3-dicyano-1, 4-bis (2-aminophenylthio) butadiene]  were made up in dimethyl sulfoxide (DMSO). In all experiments LY294002 and U0126 were used at concentrations of 30 μM and 15 μM respectively.2 in air, and serum starved overnight. Cells were infected with RV at a MOI of 4 plaque forming units (PFU) per cell and maintained in MEM with 1% FCS until harvested at indicated time points. Where appropriate kinase inhibitors (LY294002 and U0126) were added to the media at the same time as the virus, and were present during subsequent incubation periods. Mock-infected cells were treated and harvested in the same manner as those infected, except that MEM without virus was used during the infection. RV titers, in the presence of inhibitors, were determined by TCID50 assay in RK13 cells as the sample number was too large to perform plaque assays. Inhibitor, virus and serum concentrations were optimized to ensure that the effect of both the virus and the inhibitors could be monitored.Mycoplasma-free rabbit kidney epithelial (RK13) cells were obtained from the European Collection of Cell Cultures and cultured as previously described (3). RV (wild type strain RN) was propagated as previously described (3). For infection, cells were grown to confluence in minimal essential medium (MEM) supplemented with 15 mM L-glutamine and 5% FCS (v/v) (Invitrogen, UK) at 37°C in 5% CO2 tissue culture flasks and transiently transfected with 0.25 μg of control or expression plasmids. Tranfections were carried out in serum-free MEM using Lipofectamine Plus (Invitrogen, UK), according to the manufacturer's instructions. For optimal transfection, cell monolayers were incubated with the DNA-liposome mixture for 5 hours at 37°C. Following transfection, the DNA liposome complexes were removed and replaced with fresh medium. After 24 hours, RV was added to cells, which were maintained on MEM with 1% serum (as above). After an additional 24 hours, cells were analyzed for protein expression by Western blot analysis, and for apoptosis by caspase activity assay.Control and expression plasmids [pUSEamp(+), and constitutively active HA-Akt1 and Myc-MEK1 in pUSEamp(+)] were purchased from Upstate Biotechnology Inc. (UK). RK13 cells were grown to confluence in 25 cmPolyclonal anti-PI3K p85, anti-HA Tag, anti-myc Tag, and monoclonal anti-β-tubulin antibodies were from Upstate Biotechnology inc. (UK). Polyclonal anti-caspase-3 antibody was from Sigma (UK). All other primary antibodies were purchased from Cell Signaling Technology (UK). Cells were treated as described above and at indicated times post-infection (p.i.), washed in PBS and harvested in cell lysis buffer [50 mM Tris, 150 mM NaCl, 1% Triton-X-100, 2 mM EDTA, 2 mM EGTA, 100 μM protease inhibitor cocktail, and 100 μM each of phosphatase inhibitor cocktails 1 and 2 (Sigma, UK)]. Protein concentrations were determined using the BioRad assay (BioRad, Hemel Hemstead, UK), and equal protein loading was determined by Coomassie staining (Invitrogen, Paisley, Scotland). Lysates were electrophoresed on 12% Bis-Tris polyacrylamide gels (Invitrogen, UK) and transferred onto Hybond™ ECL nitrocellulose or PVDF membranes (Amersham Biosciences, UK). Membranes were blocked with 5% non-fat dried milk in PBS containing 0.1% Tween-20, and subsequently incubated with primary antibody (1:1000) overnight at 4°C. Specific antibody binding was detected using horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG (1:2000) (Dako, UK), and immunoreactive bands were visualized using the ECL detection system according the manufacturer's instructions (Amersham Biosciences, UK).2 in air. Cells were treated, in a final volume of 100 μl, with RV and kinase inhibitors as described above. At indicated times p.i., 50 μl of labeling mixture containing XTT (sodium 3'- [1-(phenylaminocarbonyl)-3, 4-tetrazolium]-bis (4-methoxy-6-nitro) and coupling reagent PMS (N-methyl dibenzopyrazine methyl sulphate) (Roche Applied Science, Mannheim, Germany) was added directly to the wells to give final concentrations of 0.3 mg/ml and 2.5 μg/ml respectively. Plates were incubated in a humidified atmosphere (37°C, 5% CO2) for 24 hours. The absorbance of the formazan product was measured at a test wavelength of 450 nm and a reference wavelength of 690 nm.RK13 cells were grown to confluence in 96-well tissue culture plates at 37°C in 5% CODEVD specific caspase activity assay (Promega, UK) was carried out as previously described (3). Briefly, RK13 cells were grown to confluence, and treated with RV, LY294002, and U0126 (as above). Cell lysates were collected at indicated times p.i. and stored at -70°C until required. For the assay, lysates were incubated with colorimetric substrate DEVD-p-NA for 4 hours at 37°C, and absorbance of free pNA cleaved by endogenous caspases-3 and -7 was measured at 405 nm.6 cells according to the manufacturer's instructions . Nucleic acids were precipitated using 3 M sodium acetate, 2-propanol, and ethanol. DNA pellets were dried and re-suspended in 10 mM Tris pH 7.5, 1 mM EDTA. Ladder fragments were electrophoretically separated on 1.5% Tris-Acetate EDTA (TAE) agarose gels. Gels were stained in ethidium bromide solution (5 mg/ml) and fragmented DNA was visualized under UV light.Analysis of apoptotic DNA fragmentation was carried out as previously described (3). Briefly, RK13 cells in 6-well plates were treated with RV, LY294002 and U0126 as above, and harvested 72 hours p.i. Total cellular DNA was extracted from 2 × 10Floating dead cells in the supernatant following infection with RV or drug treatment (as described above) were quantified by trypan blue exclusion staining. The morphological changes to the cells were examined by light microscopy using a Nikon Eclipse TS100 light microscope. Pictures of cells were taken at a magnification of 20X using a Nikon COOLPIX 4500 digital camera and processed with Adobe Photoshop 7.0 software.- reverse transcriptase (Invitrogen, UK). Reverse transcription was performed in 20 μl reaction volumes containing 200 U enzyme, 10 μl sample RNA, 0.5 mM of each dNTP, and 5 pmoles external reverse primer (5'-CCTGTACGTGGGGCCTTTAA-3'). RNA bound to cDNA in RNA-DNA hybrids was removed by incubation of the cDNA with RNase H (Roche Diagnostics, UK) for 20 minutes at 37°C. PCR amplification was carried out using a GC-Rich PCR System (Roche Diagnostics, UK). In the PCR reaction 10 μl cDNA was added to 40 μl of PCR reaction mix to give final concentrations of 1X GC-Rich PCR buffer, 1.5 mM MgCl2, 0.2 mM each dNTP, 0.5 M GC-rich resolution solution™, 0.5 pmole of forward and reverse primers (5'-TAGGAGGTGCCGCCATATCA-3' and 5'-CCTGTACGTGGGGCCTTTAA-3' respectively), and 2U Taq polymerase and a mixture of proof-reading polymerases. The cycling conditions, as recommended by the manufacturer were: 95°C for 3 minutes followed by 10 cycles of 95°C for 30s, 57°C for 30s, 72°C for 1 minute; and 25 cycles of 95°C for 30s, 57°C for 30s, 72°C for 1 minute , and a final extension of 72°C for 7 minutes. Amplified capsid product (1053 b.p.) was electrophoretically separated on 1% Tris-Borate (TBE) agarose gels, stained with ethidium bromide solution (5 mg/ml) and visualized under UV light.Total RNA was extracted from 100 μl tissue culture supernatants, collected at indicated times p.i., using a silica-guanidinium isothiocyanate method [44]. Prior to reverse transcription, RV RNA was heated to 95°C for 1 minute and kept on ice. RNA was transcribed to cDNA using Superscript III RNase HSC conceived of the study, carried out the virological and biochemical assays and drafted the manuscript. JL participated in the design of the study. JMB participated in design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.