The relapsing nature and varying geographical prevalence of ulcerative colitis (UC) implicates environmental factors such as diet in its aetiology.In order to determine which foods might be related to disease activity in UC a new method of dietary analysis was developed and applied. Eighty-one UC patients were recruited at all stages of the disease process. Following completion of a 7 d diet diary, clinical assessment including a sigmoidoscopic examination  to 6 (very active disease)) was conducted. Food weights for each person were adjusted (divided) by the person's calorific intake for the week. Each food consumed was given a food sigmoidoscopy score (FSS) calculated by summing the products of the (adjusted) weight of food consumed and sigmoidoscopy score for each patient and occurrence of food and dividing by the total (adjusted) weight of the food consumed by all 81 patients. Thus, foods eaten in large quantities by patients with very active disease have high FSSs and vice versa. Foods consumed by <10 people or weighing <1 kg for the whole group were excluded, leaving 75 foods.High FSS foods were characterized by high levels of the anti-thiamin additive sulfite , i.e. bitter, white wine, burgers, soft drinks from concentrates, sausages, lager and red wine. Caffeine also has anti-thiamin properties and decaffeinated coffee was associated with a better clinical state than the caffeine containing version. Beneficial foods (average intake per week) included pork (210 g), breakfast cereals (200 g), lettuce (110 g), apples and pears (390 g), milk (1250 ml), melon (350 g), bananas (350 g), bacon (120 g), beef and beef products (500 g), tomatoes (240 g), soup (700 g), citrus fruits (300 g), fish (290 g), yogurt (410 g), cheese (110 g), potatoes (710 g) and legumes (120 g).The dietary analysis method described provides a new tool for establishing relationships between diet and disease and indicates a potentially therapeutic diet for UC. Ulcerative colitis (UC) is a chronic, relapsing mucosal disorder that extends in continuous fashion proximally from the rectum and is limited to the colon. The aetiology of UC includes a genetic component possibly involving an abnormal cell-mediated immune response to commensal enteric bacteria in the large intestine. The relapse/remission pattern of the disorder and substrate driven nature of microbial metabolism in the large bowel implicate environmental factors such as diet.Apart from nutritional repletion, dietary measures do not play a role in the management of UC. Nonetheless, attempts to link the cause of UC with specific foods date back at least 50 years. Many foIt has been proposed that sulfide, produced in the large bowel from either amino acid fermentation or sulfate reduction, may be a triggering factor in the inflammatory process of UC -16. ReceThe main source of inorganic sulfur, predominantly sulfate, in the diet are the S (IV) family of additives; the sulfiting agents. Sulfites have been used as food preservatives since the 17th century and are amongst the most widely accepted and versatile of additives. Sulfiting agents, denoted by E220–228 in Europe and generally recognized as safe (GRAS) substances in the USA, include sulfur dioxide, hydrogen sulfites, sulfites and metabisulfites. Sulfiting agents are cheap, easy to use and extremely effective at preventing microbial growth and reducing spoilage. They seHowever, there are some problems with sulfite use,20. In tA study of diet and disease activity in UC using a 7 d dietary diary, a full assessment of disease activity and a method of dietary data analysis that allows trends in food consumption not apparent using customary dietary software was therefore undertaken.Eighty-one UC patients were recruited and informed consent obtained. Ethical permission was granted by Tayside Committee on Medical Ethics, Dundee, UK (ref 007/00). As it was important to have a range of disease activities present, recruitment included patients at all stages of the disease. Patients were excluded if clinical examination or histology indicated Crohn's disease or indeterminate colitis, if there was a positive stool culture for pathogens or if the patient had antibiotic treatment within 3 months preceding the start of the study.All the UC patients were asked to complete a 7 d diet diary. The die7d diet diaries were coded and analyzed using Tinuviel, WISP v3.0 nutritional analysis software . Due to the variation in the sulfiting protocols and widespread use of sulfiting agents, current tables of food composition do not contain inorganic sulfur values and cannot be used to quantify intake. Instead of quantitating the intake of particular dietary components, foods and food groups were assessed in their entirety using the method described in the dietary data analysis section (below).Clinical assessment included history, physical examination and global clinical grading, plus full blood count, liver function tests and inflammatory markers. Patients were examined by rigid sigmoidoscopy or flexisigmoidoscopy and graded on a scale 0–6  according to the macroscopic appearances of the rectal mucosa at a distance 5–10 cm from the anal verge. The cliPatterns of dietary intake associated with disease activity became apparent through the study of the dietary diaries, e.g. high intakes of sulfite containing foods coupled with a modern processed, convenience diet was associated with a high sigmoidoscopy score. Traditional dietary coding (WISP) did not show any such clear associations between micro or macro nutrient intake and sigmoidoscopy score. Traditional dietary analysis was therefore thought to be missing important patterns in dietary data and a new method of dietary assessment was subsequently developed.This new method used the following procedure. To calculate the association of a particular food with clinical score, each food or food group consumed was given a food sigmoidoscopy score (FSS) calculated by summing the products of food weight and sigmoidoscopy score for each occurrence of the food or food group and dividing by the total weight of the food or food group contained in all diaries. In order for each diary to make equal contributions to the FSSs, the weight of each food was adjusted using the calorific intake for each person. This procedure was carried out separately for every food item recorded in the 7 d diet diaries but is explained below using the example of red wine.Red wine score = (Σv(i)s(i))/Σv(i) for i = 1 to 81     equation 1.Where: -i is the 7 d dietary diary number (n = 81).v(i) is the volume  of red wine recorded in 7 d dietary diary i.s(i) is the sigmoidoscopy score associated with 7 d dietary diary (i).Thus foods eaten in large quantities by patients with high levels of disease activity will have high scores and vice versa. The denominator in the above equation is the total volume of the food in question from all diaries  so the food scores can be equated with the effect of a typical portion of the food in question on the sigmoidoscopy scores of the patients. This procedure is repeated for every food item. Foods or food groups were excluded from the analysis if 10 or fewer people consumed them or if they made up less than 1 kg of the total intake of the entire population. The decision as to where food group boundaries lay was made depending on the size of the group and whether the differences between the foods were considered important for this study.Dietary data was exported from WISP to Microsoft EXCEL 98 . A worksheet containing the core headings; Patient ID, food description, weight and patient sigmoidoscopy score was completed. The data was then sorted by food description and each food copied to a separate EXCEL file. Equation 1 was then used to calculate food sigmoidoscopy scores for each food in a manner similar to the example in table 2 see .2)) combined with t tables provided corresponding significance levels.Correlation values for scatter plots were obtained using the linear regression function in EXCEL. The equation t = r √((n-2)/(1-r2 = 0.25 (n = 81).Of the 81 patients recruited 43 were male and 38 female. The average age (range) of the males and females were respectively 53 (26–78) y and 47 (19–74). The distribution of sigmoidoscopy scores is shown in fig Table 3 see  shows thThe dietary diaries were assessed for completeness by comparing calorific intakes with expected values for the sexes. Expected  versus a2 = 0.07, p < 0.02).Foods for which regulations exist in the EU permitting sulfite addition are shown in table 4 see  29]. Ty. Ty29]. Decaffeinated coffee appeared better for the UC patient than the caffeine-containing counterpart. Decaffeinated tea is not shown on table 3 because it was only drunk by 9 people but had a FSS of 1.71 versus 2.01 for the caffeine-containing product. Whole fruit consumption appeared better than the corresponding juice (e.g. fruit juice scored 2.43 compared to citrus fruits at 1.96 and apples at 1.67).An average thiamin concentration (mg / 100 g) for each food or food group is also shown in table 3. There is a significant correlation (p < 0.005) between this thiamin value and the food's sigmoidoscopy score.Ulcerative colitis is considered to have a genetic component. Twin studies have shoOf all the dietary components studied in relation to UC risk and disease severity, milk has probably received the most attention. Andreson was the In order to ascertain whether dietary antigens may sustain the mucosal inflammatory response, two prospective controlled trials have investigated the effectiveness of bowel rest and total parenteral nutrition as primary therapy in the management of acute UC,36. NeitThe dietary analysis procedure proposed here has the potential to highlight trends in dietary data that would not be apparent using traditional dietary analysis software and could be useful in the study of other diseases with dietary associations. This system would highlight any possible dietary factors both positive and negative, not just sulfite. The proposed method is less reductionist than traditional coding as it assesses the risk of each food item or group rather than the risk from the foods' (quantitated) constituents. Part of the power of this study derives from the availability of a sigmoidoscopic grading (0–6) of the severity and extent of the disease. This grading provides the statistical variable that is normally obtained from a non-UC control group. Other alternative systems for analysis of disease risk for dietary components are; the use of disease occurrence odds ratios between the top and bottom quartiles of intakes, and assessing the correlation coefficients between disease activity and intakes. The odds ratio method loses data and data accuracy by characterizing intakes as high, high middle, low middle and low and then discarding the middle two quartiles. The correlation method is dependent on spread. The proposed system has neither of these disadvantages. The food sigmoidoscopy score calculation does rely on the assumption that the sigmoidoscopy score is an approximately linear scale, i.e. a sigmoidoscopy score of 6 is caused by the consumption of a double portion of a harmful food item of sigmoidoscopy score 3. This could be argued to be reasonable. Both the sigmoidoscopy grading and dietary analysis method are validated methodologies. The food sigmoidoscopy score is simply a mathematical function of these two variables. As all data is transformed according to the same simple rules any statistical treatment of the results is as valid as statistical treatment of the raw data.Whilst clinical activity indices were used to generate analogous scores to the food sigmoidoscopy scores, the results from these measurements are not included in this paper. Clinical activity index involves subjective measurements such as a feeling of well being. Thus, the food orders generated by these measurements were not thought to be as accurate as those generated by the sigmoidoscopy scores.The consensus of previous studies on diet and UC pointed to the modern, processed, highly refined, Western diets as being damaging. The results presented here linking diet with disease activity are broadly in agreement with this. Additionally they propose a new risk factor for UC, namely intake of sulfited foods.The involvement of diet in UC is controversial. Differences in dietary intake between patients and controls could be a result of changes in diet brought on by the symptoms of the disease process. While tet al. demonstrated that the thiamin status of adult humans depends on carbohydrate intake[Alternatively, the relevance of sulfite to UC may be because of its ability to degrade thiamin (particularly at colonic pH). Thiamin deficiency manifests itself in the nervous and cardiovascular systems. It is unlikely that it is the status of the patient that is important, but rather the amount of thiamin available to the gut microflora. An example of the importance of thiamin to the gut microflora is the requirement of the probiotic bacteria, lactobacilli, for thiamin. Thiamin status is influenced by a number of factors. Firstly, thiamin intake; in foods such as pork, fortified cereals and legumes which are good sources of thiamin, intakes were associated with improved clinical state. Traditional dietary analysis did not reveal a significant correlation between thiamin intake and sigmoidoscopy scores though no allowance is made in dietary coding software for the reduction in thiamin content caused by sulfite usage. Secondly, carbohydrate intake; Elmadfa te intake. CarbohyHowever, there was a sub group (n = 8) of this population who recorded an intake of either vitamin B complex or multivitamins. This sub group did not have a mean sigmoidoscopy score significantly lower that the general UC population. It is likely that vitamin B1 is a factor in the disease process but not the only nutritional one.An additional possible interpretation for the experimentally determined food order is the carbohydrate nature and content of the foods. Carbohydrates, such as the α-amylase resistant starch (RS) and prebiotics, escape digestion in the small intestine and provide an energy substrate for the colonic microflora. Both prebiotics  and resistant starch  have been hypothesised to improve the colonic health of the host. For RS, resistance to digestion is a function of the morphology of the starch granules and their crystalline organisation, which is determined by the botanical source of the starch and the processing it has undergone before being eaten. PrebiotAny dietary advice provided to ulcerative colitis patients should be based on the FSS table. The table is of course imperfect because of experimental error, natural variation and the associations between foods. For example, milk and cereal are coded separately but are often consumed together. Thus the magnitude of the difference in the FSSs for these two foods is less than if they'd been independent variables. Suggestions have been made in this discussion as to the factors responsible for the FSS order and to distill these factors into the advice given in Table 5 see . This taThe list of dietary risk factors for colon cancer bears a A dietary analysis method is described that provides a new tool for establishing relationships between diet and disease. This method has been applied to the study of ulcerative colitis and points to sulfite and caffeine as being harmful, with thiamin and resistant starch being potentially therapeutic. For the first time, dietary guidelines for ulcerative colitis patients, including food portion sizes have been developed.Ulcerative colitis (UC); Food sigmoidoscopy score (FSS); European Union (EU); Odds ratio (OD) and Confidence interval (CI).The author(s) declare that they have no competing interests.JHC, EAM and LME contributed to the study design and the writing of the manuscript. EAM was the co-ordinator of the study and along with ST had responsibility for managing the patients and coding the diaries. LME developed the dietary data analysis protocol assisted by RC. CK and JHC performed the sigmoidoscopic examinations of the patients. All authors read and approved the final manuscript.Review of studies of diet and ulcerative colitis (UC).Click here for fileFood sigmoidoscopy score (FSS) calculation example for red wine (NB incomplete data set used).Click here for fileFoods consumed in order of food sigmoidoscopy scores (FSS).Click here for filePermitted levels of sulfite in the UK.Click here for fileProposed dietary advice for ulcerative colitis patients.Click here for file


By contrast, CD45+:Sca1+ isolated from injured Pax7 −/− muscle were incapable of forming myoblasts. Infection of CD45+:Sca1+ cells from uninjured muscle with retrovirus expressing Pax7 efficiently activated the myogenic program. The resulting myoblasts expressed Myf5 and MyoD and differentiated into myotubes that expressed myogenin and myosin heavy chain. Infection of CD45−:Sca1− cells from Pax7 −/− muscle similarly gave rise to myoblasts. Notably, infection of Pax7-deficient muscle with adenoviral Pax7 resulted in the de novo formation of regenerated myofibers. Taken together, these results indicate that Pax7 is necessary and sufficient to induce the myogenic specification of CD45+ stem cells resident in adult skeletal muscle. Moreover, these experiments suggest that viral transduction of Pax7 is a potential therapeutic approach for the treatment of neuromuscular degenerative diseases.CD45 The gene Pax7 is shown to be a key regulator of muscle cell differentiation in specific populations of adult stem cells during muscle tissue regeneration Skeletal muscle regeneration has long been considered to be mediated solely by monopotential skeletal muscle stem cells known as satellite cells . However+ cells  injury. Importantly, a large proportion of CD45+:Sca1+ cells isolated from regenerating muscle acquire myogenic potential and appear to represent a significant source of myogenic progenitors during regenerative myogenesis . Four days following ctx injury, a significantly higher proportion of CD45+:Sca1+ cells  and a reduced proportion of CD45−:Sca1+ cells were observed in Pax7 −/− muscle   A.+:Sca1+ cells from wild-type muscle 4 d after ctx injury (+:Sca1+ cells purified from uninjured muscles (+:Sca1+ cells purified from regenerating  +/Pax7− muscle (4 d post-ctx). By contrast, Pax7 −/− CD45+:Sca1+ cells from regenerating muscle did not give rise to any MyoD-expressing (n = 3 independent isolations with three mice per experiment). Taken together, these results support a central role for Pax7 in the myogenic specification of CD45+:Sca1+ cells in response to acute muscle damage.By immunohistochemical analysis, Pax7 protein was upregulated in 6%–10% of CD45x injury B and 1C. muscles . Further muscles D and 1E  muscles F and 1G pressing H and 1I pressing J and 1K Adenoviral and retroviral expression systems were developed to ectopically introduce the Pax7 gene into putative adult stem cell populations. Pax7 was efficiently expressed from retrovirus (HAN-Pax7) in C3H10T1/2 fibroblasts and other cell cultures A. Stable+ progenitors, cells were fractionated from uninjured muscle and infected with Pax7-expressing retrovirus. Strikingly, CD45+:Sca1+ cells expressed Myf5 (data not shown) and MyoD  did not activate Myf5nLacZ expression . CDSC-Pax7 cells displayed a stellate, fibroblastic morphology that was distinct from the round, refractile appearance of primary satellite-cell-derived myoblasts. Proliferating CDSC-Pax7 cells expressed the myogenic determination bHLH factors, Myf5 . These experiments indicated that Myf5 was expressed at high levels in proliferating CDSC-Pax7 cells  using primers that amplify a sequence within the Pax7 3′ UTR that is not present in the viral-Pax7 vector  expression profile in CDSC-Pax7 cells contrasted with the pattern observed in satellite-cell-derived primary myoblasts ; Wt-Mb. 7 vector B. This r+:Sca1+ cells (data not shown).CDSC-Pax7 cells were originally derived from cells expressing cell surface CD45 and Sca1 proteins. Flow cytometry was employed to determine whether expression of these markers was maintained in vitro. Notably, CDSC-Pax7 cells continued to express high levels of Sca1 (approximately 90% of cells showed intense staining), but CD45 expression was extinguished C. Interedystrophin-deficient) muscle. Specifically, 1 × 105 CDSC-Pax7 cells were injected into the tibialis anterior (TA) muscle of 4- to 6-week-old mdx:nude mice. Mdx mice carry a point mutation in the dystophin gene and are a mouse model of Duchenne muscular dystrophy . Interestingly, ectopic expression of Pax7 in CD45−:Sca1− cells isolated from Pax7 −/− muscle resulted in the expression of Myf5 protein in more than 50% of infected cells (n = 3)  A–7C. Ana (n = 3) D–7F. Imp (n = 3) G–7I and  (n = 3) J–7L in PIn these experiments we cannot formally exclude the possibility that Pax7 promoted the survival and proliferation of committed myoblasts. However, given the extremely low number of myogenic cells recovered in culture (less than 0.7%), the low efficiency of primary myoblast infection (approximately 5%–10%), and the absence of any Myf5- or MyoD-expressing cells in control HAN-puro cultures, our results strongly suggest that Pax7 induces myogenic specification in a nonmyoblast, CD45- and Sca1-negative cell.Pax7 −/− muscle. Adenoviral particles (1 × 108) expressing either Pax7 (Ad-Pax7) or the bacterial β-galactosidase gene (LacZ) (Ad-LacZ) were injected directly into injured TA muscles of 4- to 6-week-old Pax7 −/− animals 2 d after administration of ctx (n = 3). Immunohistochemistry for Pax7 in adenovirus-infected muscles demonstrated widespread Pax7 expression primarily in mononuclear cells within the damaged tissue (data not shown).To investigate whether Pax7 was sufficient to stimulate myogenesis in vivo, adenovirus was used to ectopically express Pax7 in damaged Pax7 −/− muscle as evidenced by the increased number of Desmin-positive . In three independent experiments, ctx-damaged TA muscle from neration E. By conyofibers E. Therefyofibers F. Togeth+ muscle-derived adult stem cells. First, CD45+:Sca1+ cells isolated from regenerating Pax7 −/− muscle were incapable of undergoing myogenic specification  also differentiated in vivo, readily contributing to the regeneration of dystrophic muscle . The identification of additional markers expressed by adult muscle-derived stem cells is required to more thoroughly explore these issues.A confounding result from our study was the inability of Pax7 to induce myogenesis in CD45scle see . Severalic cells . We alsoic cells A. The puPax7 −/− muscles is likely to invoke nonspecific and indirect changes to the muscle microenvironment . Taken together, these results imply the presence of Pax7 −/− muscle progenitors that require the activity of Pax7 to generate sufficient numbers of myoblasts for effective regeneration. Further studies will be required to molecularly characterize the responsive cells and their developmental relationship to other muscle stem cell populations.Although CD45ells see . Moreovescle see . Skeleta+:Sca1+ cells (CDSC-Pax7) (see The dominant expression of Myf5 in Pax7-infected CD45ax7) see A suggestax7) see . Taken tMyf5nLacZ,  embryos display significantly reduced numbers of LacZ-expressing myogenic progenitors  were generously provided by Drs. A. Mansouri and P. Gruss . Mdx:nu mice were provided by Dr. T.A. Partridge , CD45.2-FITC (104), Sca1-PE, or FITC (D7), all from BD Biosciences Pharmingen  and CD45-TC (30-F11) from Caltag Laboratories . Primary antibodies were diluted in cell suspensions at 1:200. After two washes with cold PBS supplemented with 2% FBS, cells were separated on a MoFlo cytometer . Sort gates were strictly defined based on isotype control stained cells and single antibody staining. Dead cells and debris were excluded by gating on forward and side scatter profiles. Sorting was performed using single cell mode to achieve the highest possible purity. The purity of sorted populations was routinely greater than 98%.Mononuclear cells were recovered from uninjured hindlimb muscles or from ctx-damaged TA muscles of eviously . Cells w6 and 107 cfu per ml. pHAN-puro was used to produce puromycin-resistant virus for controls.Retrovirus was produced according to the 3-plasmid HIT system with plasmids pHIT60, pHIT456, and pHAN-puro as described elsewhere . pHIT60 +:Sca1+ or CD45−:Sca1− cells were spun down, counted, and 20,00–50,000 cells were then cultured overnight on collagen-coated 4-well chamber slides in HAM's F10 medium (Invitrogen) supplemented with 20% FBS, antibiotics, and 10 ng/ml Stem Cell Factor . The following day, cells were incubated for 6 h with retrovirus at a 1:1 ratio (complete medium: retrovirus supernatant) with 8 μg/ml polybrene . After infection, cells were rinsed twice with PBS, and all cells were replated in myoblast growth medium. After 48 h, infected pools were selected in 1 μg/ml puromycin (Sigma) to establish stable CDSC-Pax7 lines. C3H10T1/2 cells were incubated overnight with MyoD, Pax7, or puro virus and 8 μg/ml polybrene.Purified CD4510 pfu/ml.Adenovirus (type V) was prepared using the Ad-Max adenovirus creation kit . Ad-Pax7d (cDNA as described above) and Ad-LacZ were expressed from the murine CMV promoter. Adenovirus was purified in CsCl gradients by centrifugation, dialyzed against sterile PBS, and frozen down in 15% glycerol at −80 °C. Titres of purified adenovirus were determined by plaque assays on 293 cells and were always above 10Cell cultures were lysed in RIPA extraction buffer  supplemented with protease inhibitors . The extracts were normalized for protein content using Bio-Rad dye . Forty micrograms of lysate was separated by sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis and transferred to PVDF filters . Filters were probed with antibodies to Pax7 ; Myf5, 1:1000 ; MyoD, 1:1000 ; myogenin ; and α-tubulin, 1:2000 . Secondary detection was performed with horseradish peroxidase-conjugated antibodies (Bio-Rad). Protein expression was visualized using the ECL Plus kit .Pax7 −/− and wild-type littermates were anesthetized with Halothane gas. Twenty-five microliters of 10 μM ctx  was injected into the midbelly of the TA muscle, using a 29½ G insulin syringe. Mice were sacrificed at 4 d or 2 wk after ctx injection. For adenovirus infections, 25 μl of sterile PBS containing 108 particles of purified Ad-Pax7 or Ad-LacZ was injected 2 d after ctx injection into damaged TA muscles with a 29½ G insulin syringe.Four- to six-week-old 5 cells/25-μl in sterile PBS for cell transplantation. Cells were injected directly into the TA midbelly of 4- to 6-wk-old mdx:nude mice. Mice were sacrificed 2 mo after cell injections to analyze the myogenic contribution of transplanted cells.Primary CDSC-Pax7 cells cultured in myoblast conditions were trypsinized, washed twice with PBS, and suspended at 5 × 10−:Sca1− fractions of hindlimb muscle of 4- to 6-wk-old  +/+Pax7 or  +/−Pax7 mice. Myoblasts and CDSC-Pax7 cells were maintained in HAM's F-10 medium (Invitrogen) supplemented with 20% FBS and 2.5 ng/ml bFGF (Invitrogen) on collagen-coated dishes. CDSC-Pax7 cells and primary satellite-cell-derived myoblasts were differentiated for 1–3 d in DMEM supplemented with 5% horse serum. C3H10T1/2 and HEK 293 cells were obtained from the ATCC  and maintained in DMEM supplemented with 10% FBS.Primary satellite-cell-derived myoblasts were established from purified CD45For analysis of regeneration and enumeration of regenerated myofibers, TA muscles were isolated, embedded in OCT /20% sucrose and immediately frozen in liquid nitrogen. Ten-micrometer cryosections (cross sections) from the TA midbelly at the site of ctx injection were stained with Hematoxylin and Eosin. Central myonuclei in regenerating muscles were counted on at least two independent cross sections of the entire TA muscle per mouse analyzed. Fibers were further identified by immunostaining with antibodies specific to Desmin, 1:200 ; dystrophin, 1:500 (Sigma); Pax7 (DSHB); or embryonic fast MyHC  followed by secondary detection with anti-mouse FITC conjugated antibody, 1:200 . Sections were analyzed on a Zeiss  Axioplan 2 microscope.Myf5nLacZ expression was detected by X-Gal reaction as described previously ; MyoD, 1:200 ; all MyHC ; Myf5, 1:1000 ; Pax7 (DSHB); and myogenin . Secondary detection was performed using fluorescein- or rhodamine-conjugated antibodies, 1:200 (Chemicon). eviously .Pax7 mRNA was performed using the GeneAmp PCR Core kit . RT-PCR using 1 μg of total RNA was conducted as per manufacturer's instructions with the following modifications. cDNA synthesis was extended for 1 h at 42 °C, and 5 μl of the first-strand RT product was used for PCR amplification. PCR conditions for endogenous Pax7 were 94 °C for 5 min; 35 cycles of 94 °C for 45 s, 56 °C for 45 s, 72 °C for 45 s; and finally 72 °C for 7 min. The PCR primers span intron 8 of the Pax7 gene (Pax7-exon8-fwd 5′ gct acc agt aca gcc agt atg 3′ and Pax7-exon9-rev 5′ gtc act aag cat ggg tag atg 3′) and amplify sequence in the 3′-UTR of the gene that is not contained in the viral Pax7 expression cassette. RT-PCR products were analyzed by electrophoresis through a TAE-ethidium-agarose gel.Total RNA was extracted using RNeasy kits , according to manufacturer's instructions. RT-PCR analysis for endogenous 32P-dCTP radiolabeled cDNA fragments as probes (Northern blot studies were performed according to standard techniques using random-primed sciences). FifteenThe accession numbers for the proteins discussed in this paper are Desmin (LocusLink ID 13346), mouse MyoD (GenBank NM_010866), MyoD (LocusLink ID 17927), Myogenin (LocusLink ID 17928), Pax7 (LocusLink ID 18509), and Pax7d isoform (GenBank AF_254422).


It is widely accepted that the development of atherosclerosis starts at an early age. However, there are very few studies evaluating the prevalence of the common clinical and behavioral cardiovascular disease (CVD) risk factors among children, especially in developing countries. The aim of the present cross-sectional survey was to evaluate the distribution of blood lipid profile and various behavioral  factors related to CVD risk and its relationships to paternal (PEL) and maternal educational level (MEL) among primary schoolchildren in Turkey.In three major metropolises in Turkey , a random sample of 1044 children aged 12 and 13 years old was examined. ANOVA was applied to evaluate the tested hypothesis, after correcting for multiple comparisons (Tukey correction).After controlling for energy and fat intake, physical activity status and Body Mass Index (BMI), it was found that mostly PEL had a significant positive effect for most of the subgroups examined (Lower vs. Higher and Medium vs. Higher) on TC and HDL-cholesterol and a negative effect on TC/HDL ratio for both genders. Furthermore, both boys and girls with higher PEL and MEL were found to have higher energy intake derived from fat and protein than their counterparts with Medium and Lower PEL and MEL, while the opposite was observed for the percentage of energy derived from carbohydrates.Our study provides indications for a possible association between an adverse lipid profile, certain dietary patterns and Higher PEL and MEL among schoolchildren in Turkey. These findings underline the possible role of social status, indicated by the degree of education of both parents, in developing certain health behaviors and health indices among Turkish children and provide some guidance for Public Health Policy. Cardiovascular disease (CVD) is the primary cause of mortality in developed countries and generates a major burden of morbidity throughout life ,2. AdditThe continuing modernization and technological advancement of the developing world has brought about rapid lifestyle changes , which are known to have a major impact on the development of CVD and other chronic diseases ,9. Many As far as Turkey is concerned, the available data on the possible effect of parental educational level on the clustering of CVD risk factors among children are limited. Turkey, which has experienced rapid urbanization and industrialization in the past few decades, is a middle-income country, located between Europe and Asia and bordering the Mediterranean, Aegean and Black Seas. The purpose of the present study was to examine the influence of PEL and MEL on CVD risk factors, namely the lipid profile and behavioral indices, among children of Turkey. Expanding our knowledge and understanding on the interrelationship of all these parameters could effectively guide Public Health Policy in early prevention of CVD risk factors in childhood.th grade primary school children registered in the selected schools, 1044 pupils  were finally examined. Inclusion of subjects was on a voluntary basis; prior to acceptance, children's parents or guardians were fully informed about the objectives and methods of the study and were asked to sign a consent form.The first phase of this cross-sectional study took place during March-May 2001 and the second phase during January-May 2002. The study population consisted of primary schoolchildren aged 12 and 13 years old, living in three urban areas of Turkey, namely Istanbul, Ankara and Izmir. Out of totally 1320 6The study population was selected from nine primary schools (three from each city) using the multi-stage sampling method. Schools were selected taking into consideration the available records of the Ministry of National Education and the National Statistical Center of Turkey, in an attempt to obtain a representative sample from the overall population. In the case of Ankara and Izmir data were collected from three public schools respectively, while in the case of Istanbul sample came from one private and two public schools. All children in the same class were invited to participate in the study to avoid ethical problems.Approval to conduct this survey was granted by the Ethical Committee of Marmara University and the Turkish Ministry of Education.Baseline data was collected during face-to-face interviews with children by a team of trained personnel. The data collected from the children consisted of physiological indices, namely biochemical and behavioral indices, such as dietary habits, estimation of energy and nutrient intake and physical activity assessment. These data are presented in more detail below.A coded questionnaire was developed and administered to the study participants, in order to obtain information on socioeconomic conditions and their personal characteristics, such as family size, parental academic qualifications, occupation and car ownership. Both Paternal (PEL) and Maternal Educational Level (MEL) were classified into three categories. "Lower" PEL and MEL corresponded to illiterate, literate with no formal education and to primary school graduates. "Medium" PEL and MEL was attributed to those parents, who had completed junior high school or high school, whereas the category of "Higher" PEL and MEL included parents who possessed a college or a university diploma.Early morning venous blood samples were obtained from each child for biochemical screening tests, following a 12-h overnight fast. Professional staff performed venipuncture, using vacutainers to obtain 10 ml of whole blood. Blood was centrifuged for plasma separation at the local University Hospital and 1.5 ml aliquots were pipetted into plastic Eppendorf tubes and stored at -80°C. When blood collection from each city was completed, all samples were sent to Marmara University, Faculty of Health Education, where the actual biochemical analysis took place.Total cholesterol (TC) was determined using Allain's method , while FLDL-C = TC - (HDL-C + TG/5) .2).Body weight was measured using a digital scale (Seca) with an accuracy of ± 100 g. Subjects were weighed without shoes, in the minimum clothing possible, i.e. underwear. Standing height was measured without shoes to the nearest 0.5 cm with the use of a commercial stadiometer, with the shoulders in relaxed position and arms hanging freely . Body MaFood consumption of children was assessed by the 24-hour recall technique on three consecutive days. Dietary data was collected from children during a face-to-face interview with a trained dietician. Dieticians were trained as a group to minimize inter-observer variation. During the interview, food models and photos of common Turkish dishes of various portions, as well as household cups and measures were used to define amounts, in order to obtain as accurate information as possible, regarding the type and amount of food and beverages consumed during the previous day. Macronutrient and micronutrient intake were calculated using the food database available in Marmara University, Faculty of Health Education. This database contains Turkish food composition tables for all food, including cooked Turkish dishes. Information on processed foods was obtained from food companies and national as well as international fast food chains.Physical activity during school hours and leisure time was assessed using a standardized physical activity diary completed by the children for two consecutive weekdays and one weekend day. A member of the research team crosschecked diary information during daily interviews. The diary was constructed for searching various physical activities ranging from mild to vigorous , while tActivities were classified into two groups, namely Sedentary and Light Activities (< 4 METs) and Moderate to Vigorous Physical Activities (MVPA) (> 4 METs). Typical activities in the Sedentary and Light category were watching TV, board and computer games, studying and extra curricular classes , etc. The MVPA category included activities such as walking, bicycling, rhythmic-gymnastics, dancing, basketball, soccer, athletics, tennis, swimming, running up and down, jumping rope and general participation in active outdoors games. Given the age group, MVPA was defined as continuous vigorous activity causing sweating and heavy breathing for periods longer than 15 minutes, but with occasional breaks in intensity, rather than the strict aerobic definition of 20 continuous minutes appropriate for adults.th and 75th percentile for not normally distributed variables. The 2- sample Z test was used to compare proportions regarding values above normal range among the different categories of parental education for both genders. One- way analysis of variance (ANOVA) with a Tukey post- hoc analysis and multivariate analysis of variance (MANOVA) controlling for certain confounding factors, was conducted to determine whether differences derived from the comparison of continuous variables among the educational categories were statistically significant. For not normally distributed variables the non- parametric univariate Mann-Whitney test was used to verify the statistical significance of the existing differences among the three educational categories. All analyses were conducted using the SPSS 10.0 statistical software package for Windows. In all analyses a 5% significance level was used.Descriptive statistics of continuous variables were expressed as the Mean ± standard deviation (s.d.), as well as median and 25Table Table Dietary and physical activity characteristics of the under study population are reported in Tables Despite the traditional notions of CVD as a Western disease of "affluence", more than three quarters of global CVD mortality now occurs in middle- and lower- income nations  . FurtherAccording to the findings of the current study there was a higher prevalence of "borderline or high risk", with respect to TC levels, for children with Medium and Higher parental educational level Table . SeveralThe effect of diet on serum lipids and lipoproteins has been extensively studied in various clinical trials and epidemiological studies, and although dietary content is clearly an important determinant, several environmental and metabolic factors intervene to modulate the dietary effect 36). Previously conducted surveys in urban regions of Turkey have provided strong evidence that children coming from more affluent and/or well-educated families consumed diets higher in animal protein and fat, while the typical diet of less privileged children was rich in carbohydrates, including cereal grains and sugar (37). These findings, regarding the macronutrient content of the consumed diet, are in consistency with the current study, since children from Medium and Higher PEL and MEL were found to consume higher percentage of energy derived from fat and protein, while the opposite was observed for carbohydrate intake (Table 6. PrevioRegarding physical activity, it is well established that higher levels of physical activity are associated with higher HDL-C and lower TC to HDL-C ratio values in both children and adolescents -42. HoweThe findings of the current study are in agreement with previous surveys conducted in Turkey indicating that parental education, either as a single estimate of social status or combined with other social and/or economic indices, is a "reliable index" of socioeconomic status ,46. BothThe underlying influence of socio-economic status on health status is far more complex than diet and physical activity alone, since many other factors interact in producing the differences observed for serum lipids or other health indices. Based on the data provided by the current study it could be concluded that the differences regarding the lipidemic profile of children with different parental educational level could possibly be attributed to the differences observed on their dietary and macronutrient intake. Any attempt for the development and implementation of a health and education programme should consider these findings and set priorities accordingly. However further research is needed in order to develop an effective index for assessing socioeconomic level, as well as in better understanding its multidimensional role on public health. All these will help the public health authorities to develop effective strategies, which will efficiently tackle these health issues early in life.The pre-publication history for this paper can be accessed here:


Fucus vesiculosus (bladderwrack), as well as other brown kelp species, lower plasma cholesterol levels. Since cholesterol is a precursor to sex hormone biosynthesis, kelp consumption may alter circulating sex hormone levels and menstrual cycling patterns. In particular, dietary kelp may be beneficial to women with or at high risk for estrogen-dependent diseases. To test this, bladderwrack was administered to three pre-menopausal women with abnormal menstrual cycling patterns and/or menstrual-related disease histories.Rates of estrogen-dependent cancers are among the highest in Western countries and lower in the East. These variations may be attributable to differences in dietary exposures such as higher seaweed consumption among Asian populations. The edible brown kelp, P = 0.04) following 700 mg/d, which decreased further to 92.5.0 ± 3.5pg/ml (P = 0.03) with the1.4 g/d dose. Mean baseline progesterone levels rose from 0.58 ± 0.14 to 8.4 ± 2.6 ng/ml with the 700 mg/d dose (P = 0.1), which increased further to 16.8 ± 0.7 ng/ml with the 1.4 g/d dose (P = 0.002).Intake of bladderwrack was associated with significant increases in menstrual cycle lengths, ranging from an increase of 5.5 to 14 days. In addition, hormone measurements ascertained for one woman revealed significant anti-estrogenic and progestagenic effects following kelp administration. Mean baseline 17β-estradiol levels were reduced from 626 ± 91 to 164 ± 30 pg/ml (These pilot data suggest that dietary bladderwrack may prolong the length of the menstrual cycle and exert anti-estrogenic effects in pre-menopausal women. Further, these studies also suggest that seaweed may be another important dietary component apart from soy that is responsible for the reduced risk of estrogen-related cancers observed in Japanese populations. However, these studies will need to be performed in well-controlled clinical trials to confirm these preliminary findings. Epidemiological studies show that incidence rates of estrogen-dependent diseases such as cancers of the breast, endometrium and ovary are among the highest in Western, industrialized countries, while rates are much lower in China and Japan ,2. TheseUndaria pinnatifida) and kombu (Laminaria japonica). These species and the Atlantic brown kelp, bladderwrack (Fucus vesiculosus), have been shown to exert powerful anti-hypertensive activity related to angiotensin-I-converting enzyme inhibition [Studies show that Japanese women have longer menstrual cycle lengths (greater than the 28 day average) and lower circulating estrogen levels compared to Western populations -17, whichibition , to posshibition , and to hibition . Other chibition ,28, immuhibition , anti-prhibition ,31, and hibition , have beIntake of bladderwrack, as well as other brown kelp species, also has been shown to alter cholesterol metabolism and to significantly lower plasma cholesterol levels ,34. A poThree pre-menopausal women with abnormal menstrual cycling histories volunteered for the present study. An abnormal menstrual cycle was defined as one or more of the following: menstrual cycles of <26 or >32 days in length; menstrual cycles consisting of >8 menstruating days; or anovulatory menstrual cycling. Study subject characteristics are outlined in Table The protocol was approved by the Committee for the Protection of Human Subjects of the University of California at Berkeley. The nature of the study was explained, and written informed consent was obtained from all study subjects.Dried, powdered bladderwrack was obtained from Maine Coast Sea Vegetables  and encapsulated in 350 mg capsules. Two capsules were administered daily for the low dose treatment (700 mg) and four capsules were administered daily for the high dose treatment (1.4 g). Bladderwrack dosage levels were chosen to fall within the range of reported dietary seaweed intakes (10–25%) of the total diet reported for Japanese populations ,22. ThisDetails of the study protocol are outlined in Table All hormone assays were performed by Quest Laboratories , an outside-certified clinical laboratory. Serum 17β-estradiol and progesterone levels were measured in duplicate by radioimmunoassays. Blank and control sera were run with each assay. The coefficient of variation (a measure of laboratory error) was consistently low (<15%) for 17β-estradiol and progesterone.Statistical analyses were performed by unpaired t-tests (2-sided) with a commercially available statistical software package . All statistical tests were considered significant for p ≤ 0.05. Results are referred to as borderline significant for 0.05 < p ≤ 0.10.There were no adverse side effects reported and bladderwrack was well tolerated by all three women.P < 0.002), which increased by approximately 5 additional days to 31.2 ± 1.1 days following administration of the higher dose (P < 0.001). In Subject 2, the average cycle length increased 5.5 days, from 23.0 ± 1.7 to 28.5 ± 0.7 days (P = 0.03). Subject 3 exhibited a 4-day increase in menstrual cycle length from 27.3 ± 0.6 to 31.5 ± 0.7 days with the 700 mg dose (P = 0.005) that increased by approximately 6 more days to 36.0 ± 2.8 days with the 1.4 g dose (P = 0.01).Following treatment, all women exhibited a significant increase in menstrual cycle lengths Figure . SpecifiP = 0.06) with the low dose and to 4.5 ± 0.7 average days with the high dose (P < 0.003). Subject 2, who only took the low dose, also experienced a marked reduction in number of days of menstruation, from 8.0 ± 1.0 to 5.3 ± 2.5 days (P = 0.06). Subject 3 exhibited a decrease in total menstruating days averaging from 6.3 ± 1.5 to 5.8 ± 0.4 days (P = 0.65) with the low dose, and to 3.5 ± 0.7 days (P = 0.10) with the 1.4 g/d dose. Subjects 1 and 3, who both suffered from endometriosis, reported substantial alleviation from pain during menstruation and throughout the menstrual cycle following bladderwrack treatment.Along with increased menstrual cycle lengths, all women reported marked reductions in blood flow and average number of days of menstruation following bladderwrack treatment Figure . SubjectSubject 1 also reported an ovulatory cycle following 2 months on the 700 mg/d kelp intervention, and continued to have ovulatory cycles while on the 1.4 g/d dose.P = 0.04) with the low dose (700 mg/d), which decreased further to 92.5 ± 3.5 pg/ml (P = 0.03) with the higher dose (1.4 g/d). Furthermore, mean baseline progesterone level rose from 0.58 ± 0.14 to 8.4 ± 2.6 ng/ml with the lower 700 mg/d dose (P = 0.1), which increased further to 16.8 ± 0.7 ng/ml with the 1.4 g/d dose (P = 0.002).A significant anti-estrogenic and progestagenic dose response was observed in plasma estradiol and progesterone levels in Subject 1 Table . SpecifiThe results of this preliminary pilot study suggest that bladderwrack consumption can effectively increase the length of the menstrual cycle and reduce the total number of days of menstruation in pre-menopausal women. These effects were most marked in the two women that had shorter than average cycles (16 and 23 days) versus the normal range of 26 to 32 days seen in women in Western populations. Menstrual cycles were further lengthened with increasing dose, which may suggest a linear dose-response effect. However, since there was not a sufficient washout period between the 700 and 1400 mg/d doses, an effect of length of time of dosing rather than a dose response effect cannot be ruled out. Nonetheless, these marked increases in menstrual cycle length may have beneficial health effects in lowering risk of estrogen-dependent diseases such as endometriosis and ovarian, endometrial, and breast cancers as reported in a number of studies -38. MensBladderwrack consumption also led to a marked reduction in circulating 17β-estradiol levels and an increase in progesterone levels in a subject who exhibited high serum estrogen levels and progesterone deficiency prior to the intervention. While estrogen's proliferative effects on mammary gland development and endometrial and breast tumorigenesis are well documented, progesterone's role in these processes is not as well defined. Studies show that progesterone deficiency is associated with increased endometrial cancer incidence , and thaDue to the small number of subjects and the lack of a control group, this study will need to be repeated in a larger, randomized population of women with placebo controls. Other weaknesses of the present study are the lack of data on luteinizing hormone and follicular stimulating hormone levels which would provide pertinent information regarding the effects of dietary bladderwrack on ovulation and the luteal and follicular phases of the menstrual cycle. The potential beneficial impact that dietary bladderwrack may have on abrogating symptoms of endometriosis warrants a closer look at a larger population of women suffering from this disease. However, studies should also be performed in women with normal menstrual cycles who have sex hormone levels within clinically normal ranges to determine the impact of dietary kelp on menstrual cycling patterns and hormone levels in the general population.The observed responses to bladderwrack consumption in this study suggest that dietary modification may lead to significant changes in the regulation of the menstrual cycle by increasing the length of the cycle, stimulating ovulation, and lowering the estrogen/progesterone ratio in pre-menopausal women. Such changes may be beneficial particularly with regard to women at high risk of estrogen-dependent diseases or who are experiencing fertility problems. Results from these preliminary experiments also suggest that bladderwrack administration may alleviate hypermenorrhea and dysmenorrhea, which may provide some relief in the treatment of endometriosis. Although these reported effects are generally in a beneficial direction, their clinical significance is yet to be determined in a well-controlled study.in vitro studies are currently underway in our laboratory to elucidate the potential mechanisms and clinical relevance of bladderwrack bioactivity, and to identify and isolate the active components involved.The critical role of hormones in breast, endometrial, and ovarian cancers in women and prostate cancer in men has long been recognized. Given the vast rise of these cancers in the U.S. and our limited success with prevention and treatment, there is clearly a need for the identification of novel, non-cytotoxic chemopreventive agents. Future investigations should clarify the role of bladderwrack and other seaweed species on estrogen and progesterone metabolism, to evaluate its potential binding affinity to estrogen and progesterone receptors, and to determine its effects on proliferation in hormone-sensitive tissues. These investigations should also be expanded to include effects of bladderwrack on other sex hormones including the androgens and gonadotropins. In this regard, animal and None declared.The pre-publication history for this paper can be accessed here:


Magnetic nanoparticles have been significantly used for coupling with biomolecules, due to their unique properties.Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chloride using two different base solutions. Glucose oxidase was bound to the particles by direct attachment via carbodiimide activation or by thiophene acetylation of magnetic nanoparticles. Transmission electron microscopy was used to characterize the size and structure of the particles while the binding of glucose oxidase to the particles was confirmed using Fourier transform infrared spectroscopy.The direct binding of glucose oxidase via carbodiimide activity was found to be more effective, resulting in bound enzyme efficiencies between 94–100% while thiophene acetylation was 66–72% efficient. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when subjected to thermal and various pH conditions. The overall activity of glucose oxidase was improved when bound to magnetic nanoparticlesBinding of enzyme onto magnetic nanoparticles via carbodiimide activation is a very efficient method for developing bioconjugates for biological applications The immobilization of biomolecules onto insoluble supports is an important tool for the fabrication of a diverse range of functional materials or devices . Enzyme In the last decade, nanosize materials have been widely used as support for this purpose. Among these materials, magnetic nanoparticles are very popular when used in conjunction with biological materials including proteins, peptides, enzymes -9 antibo3O4) are biocompatible superparamagnetic materials that have low toxicity and strong magnetic properties [in vivo examination including magnetic resonance imaging, contrast enhancement, tissue specific release of therapeutic agents, hyperthermia [in vitro binding of proteins and enzymes [Magnetite  is a homodimer flavoprotein containing two active sites per molecule [Magnetite nanoparticles have been used as support material for binding of enzymes including yeast alcohol dehydrogenase  and lipamolecule ,13. It cmolecule ,14,15. Amolecule  examined3O4 magnetic nanoparticles using two binding methods, the direct binding via carbodiimide activation of amino functionalized particles and binding to thiophene-functionalized acetylated nanoparticles. A comparison of the stability and activity of glucose oxidase immobilized to magnetite using different protocols will lay the foundation for magnetic immunoassays. The size and structure of the nanoparticles were characterized using Transmission electron microscopy (TEM) and Fourier Transform Infrared (FTIR) spectroscopy, respectively. The stability, activity, and kinetic behavior of bound glucose oxidase were also examined.Here, we report the stability and enzymatic activity of glucose oxidase immobilized onto FeAspergillus niger was purchased from VWR international . Carbodiimide-HCl (1-ethyl-3-(3-dimethyl-aminopropyl), ammonium hydroxide, sodium hydroxide, acetic anhydride, glucose, bovine serum albumin (BSA), iron (II) chloride tetrahydrate 97 % and iron (III) chloride hexahydrate 99%, were obtained from Sigma-Aldrich St Louis (USA). 11-bromoundecanoic acid was obtained from TCI America Portland, USA. The Biorad Protein Assay Reagent Concentrate was purchased from Biorad Laboratories . Thiophene-2-thiolate was obtained from Alfa Aesar MA, USA. Iodine was obtained from Mallinckrodt Kentucky, USA, and acetonitrile was obtained from EMD Chemicals . Sodium phosphate monohydrate and potassium phosphate dihydrate were acquired from EM Science . Sodium carbonate was obtained from Orion Research Inc. . Magnetic nanoparticles (Fe3O4) were prepared by chemical co-precipitation of Fe2+ and Fe3+ ions in a solution of ammonium hydroxide (magnetic nanoparticles I or Fe3O4 I), or sodium hydroxide (magnetic nanoparticles II or Fe3O4 II) followed by a treatment under hydrothermal conditions [4OH solution (30%) or NaOH 3 M at a pH 10. The precipitates were heated at 80°C for 35 min under continuous mixing and washed 4 times in water and several times in ethanol. During washing, the magnetic nanoparticles were separated from the supernatant using a magnetic separator of strength greater than 20 megaoersted (MOe). The particles were finally dried in a vacuum oven at 70°C. The dried particles exhibited a strong magnetic attraction.Glucose oxidase (specific activity 200 units/mg protein) from cles Fe3O were prenditions . Iron (I3O4 nanoparticles I and Fe3O4 bound glucose oxidase (GOX-Fe3O4 I) were washed with phosphate buffer pH 7.4 and 0.1 M Tris, pH 8.0, and then used for activity and stability measurements. NaCl was added to enhance the separation of the magnetic nanoparticles [Magnetic nanoparticles I (50 mg) produced, using a solution of ammonium hydroxide were added to 1 mL of phosphate buffer (0.05 M. pH 7.4). After adding 1 mL of carbodiimide solution (0.02 g/mL) in phosphate buffer (0.05 M. pH 7.4), the mixture was sonicated for 15 min. Following the carbodiimide activation, 2 mL of glucose oxidase (1000 units /mL) was added and the reaction mixture was sonicated for 30 min at 4°C in a sonication bath. The magnetic nanoparticles were separated from the mixture using a magnetic separator. The precipitates containing FeCandida rugosa lipase on the γ-Fe2O3[3O4 was added to 5 g of 11-bromoundecanoic acid dissolved in 15 mL of ethanol. 11-bromoundecanoic acid was covalently linked to the nanoparticles surfaces by heating the mixture with microwave irradiation for 10 min. Functionalization of the particle was achieved through nucleophilic substitution with the 2-thiophene thiolate. In practice, 2-thiophene thiolate (7 mL) was added to the mixture containing the particles and heated in microwave for 5 minutes. The mixture was washed with ethanol and transferred in a round bottom flask. Acetic anhydride (4 mL) and 34.6 mL of iodine (0.01N) were successively added to the particles and agitated. The mixture was heated for 1 h under reflux condition [A second functionalization protocol using a modification of the strategy adopted to immobilize e γ-Fe2O3 was implondition . The parFor the attachment of glucose to nanoparticles, 2 mL of the GOX solution (1000 units/mL) was added to 50 mg of functionalized magnetic nanoparticles in a test tube and sonicated at 15°C for 3 h. The supernatant containing unbound enzymes was separated from the magnetic nanoparticles using the magnetic separator, and the enzymes bound to magnetic nanoparticles were then used for activity determination. A schematic of the procedures used for both attachments are presented in Figure A = (Ci - Cs)* V     (1)A is the amount of bound enzyme, Ci and Cs are the concentration of the enzyme initially added for attachment and in the supernatant, respectively (mg/mL) and V is the volume of the reaction medium (mL). The size of Fe3O4 magnetic nanoparticles, GOX-Fe3O4 I and GOX-Fe3O4 II were characterized by transmission electron microscopy  and structure by FTIR spectroscopy . The samples for TEM analysis were prepared as follows: a drop of magnetic nanoparticles was dispersed in nanopure water. The resulting solution was sonicated for 4 min to obtain better particle dispersion. A drop of the dispersed solution was then deposited onto a copper grid and dried overnight at room temperature. The binding of GOX onto the magnetic nanoparticles was investigated using FTIR spectroscopy. Samples for FTIR analysis were prepared in phosphate buffer pH 7.4. The activity of bound GOX was determined by measuring the initial rate of formation of hydrogen peroxide at a given temperature following the formation of a red quinoneimine dye. The principle of enzymatic determination of the activity of glucose oxidase is described as follows: Glucose is oxidized by glucose oxidase to gluconate and hydrogen peroxide. Phenol + 4-AAP, in the presence of peroxidase (POX), produces a quinoneimine dye that is measured at 500 nm using a Beckman Du Spectrometer to provide an absorbance that is proportional to the concentration of glucose in the sample. The reaction is described as follows:Where 3O4 and mixed by vortex. A solution of free GOX of the same molar concentration was used to evaluate the activity of the free enzyme for comparison. The solution was incubated at various temperatures (37–80°C) at specific intervals of time (30 min) and the supernatant was separated from GOX-Fe3O4 using a magnetic separator. 10 μL aliquots of the supernatant were then taken for determining the concentration of hydrogen peroxide following the procedure by Trinder [The activity of glucose oxidase was measured as follows. An assay mixture was prepared by mixing 500 U of horseradish peroxidase, 0.015 mmol of 4-aminoantipyrine (4-AAP), 0.025 mmol of phenol and 5 mmol of glucose in 50 mL of phosphate buffer solution (0.05 M. pH 7.4) to result in a glucose concentration of 0.1 M. To start the enzymatic reaction, 2 mL of the assay solution was added to 15 mL centrifuge test tubes containing GOX-Fe Trinder . The actABS sample denotes the absorbance of the sample, ABS Std is the absorbance of the standard solution, and C the concentration of glucose in the sample.Where 3O4 I and GOX-Fe3O4 II was estimated by determining the concentration of glucose in the sample at various temperatures. A solution of the assay mixture was added to the various centrifuge test tubes containing bound or free enzymes. The samples were stored in a water bath at specific temperatures  and the absorbance was monitored at fixed time intervals to determine the glucose content. The effect of pH on GOX was monitored by measuring the initial rate of glucose oxidation by glucose oxidase in different phosphate and carbonate buffer solutions of pH (5–10) at 25°C.The effect of temperature on the free GOX, GOX-Fe3O4 I, and GOX-Fe3O4 II were determined by measuring the residual activity of the enzyme at 25°C, after being exposed to different temperatures (37–80°C) in phosphate buffer  for 30 min. Aliquots of the reacting solutions were taken at periodic intervals (every 30 min for 6 h) and assayed for enzymatic activity as described above. The first-order inactivation rate constant, k was calculated from the equation:The thermal stability of free GOX, GOX-FeA = ln A0 - kt     (5)ln A0 is the initial activity, A is the activity after time t (min) and k is the reaction constant.where 3O4 I, GOX-Fe3O4 II, or free GOX solutions were stored at 25°C in phosphate buffer (0.05 M. pH 7.4) for 33 days. Thereafter, 3 mL of the assay solution was added, and the residual activity of GOX was assayed.The storage stability was examined by measuring the change in the concentration of glucose at room temperature at different time intervals (4 days). Test tubes with samples of GOX-Fe3O4 I and GOX-Fe3O4 II, Km and Vmax were determined by measuring the initial rates of glucose oxidation (0.2–1 mM) by glucose oxidase (0.25 mg/mL) in phosphate buffer (pH 7.4) at 25°C.The kinetic parameters of free GOX, GOX-Fe3O4 I and Fe3O4 II, and their GOX bound counterparts shown in the TEM micrographs in Figures  were estimated from the double reciprocal plots of the initial rates of glucose oxidation by GOX. The double reciprocal plots are presented in Figure Vmax and Km for GOX are shown in Table Vmax of free GOX, GOX- Fe3O4 I and GOX-Fe3O4 II were 0.731, 0.803, and 0766 μmol/min mL and, the corresponding Km values were, 0.383, 0.208, and 0.237 mM, respectively. The Vmax value of GOX immobilized on magnetic nanoparticles (I and II) was higher than that of the free enzyme. The highest Vmax was obtained with GOX-nanoparticles I. Since a low Km indicates a high degree of affinity of the enzyme to substrate [3O4 II and GOD-Fe3O4 I, respectively. The high affinity of the enzyme to the substrate may be explained by a favorable change in the structural organization of the enzyme due to the immobilization procedure [The most important aspect of this study is related to the retention of biocatalytical activity of GOX after binding to magnetic nanoparticles. The amount of bound GOX was estimated using the UV-vis spectrophotometer and the catalytic activity of free and bound GOX was compared. Kinetic parameters . It can be observed that at 37°C, the enzyme retained its activity for about 80 minutes before showing a slight decrease. At 50, 60, 70 and 80°C the activity decreased as the temperature increased in both systems. In GOX- Fe3O4, the remaining activity was 23% at 50°C and 15% at 60°C after 270 min. For this time period and duration, the remaining activities were 9% and 0%, respectively for GOX-Fe3O4 II. A similar trend was observed at 70 and 80°C with a more drastic loss of activity from the GOX-Fe3O4 II protocol. Loss of activity occurred more rapidly in GOX-Fe3O4 II indicating that direct binding through carbodiimide activation provides a greater thermal stability to GOX. The effect of temperature on the activities of free, GOX-Fe3O4 I, and GOX-Fe3O4 II at pH 7.4 are presented in the Arrhenius plots  at 50, 60 70, and 80°C. The rate constants increased with increasing temperature in the order GOX-Fe3O4 I, GOX-Fe3O4 II and free GOX. Here again, binding GOX to magnetic nanoparticles minimized structural denaturation due to heat treatment. Covalent binding was expected to provide the enzyme with the protection against structural denaturation due to the unfavorable solvent-protein interactions, and thus result in activation effect [3O4 II had a lower stability at higher temperatures compared to GOX-Fe3O4 I. The reason for this difference could reside in the stability of the binding, since the binding methods are so far the major difference between these systems. Indeed with GOX-Fe3O4 I, the binding of the enzyme occurred through the amino groups on the surfaces of the particles and the carboxylic groups of proteins in the enzymes [3O4 II the N atom to which the enzyme is attached shared a double bond with the carbon atom which is less stable than the amide bond.The effect of temperature on the activity of free GOX was examined by measuring its relative activity when stored at various temperatures. Figure n effect , a possi3O4 I, and GOX-Fe3O4 II at 25°C at pH 7.4. Each data point was the average of duplicate measurements (coefficient of variation of the measurements was between 1 and 5%). The activity decreased with time in all the systems. Total loss of activity was observed after 20 days for the free GOX and 28 days for GOX-Fe3O4 II while GOX-Fe3O4 I retained 26% activity after 33 days of storage under identical conditions. The stability of the enzyme was found to improve upon binding to the magnetic nanoparticles but the most significant improvement in stability was observed for the GOX-Fe3O4 I nanoparticle complex. The fixation on the surface of the magnetic nanoparticles has been a tangible argument supporting the prevention of auto-digestion of the enzyme and lysis, and the subsequent conservation of its activity [Loss of storage stability is a major concern in enzyme preservation. The storage stability of the enzyme was examined for 33 days. Figure activity . This maactivity  and a su3O4 II complex. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when the complex was subjected to thermal and pH variations. This study shows that binding onto magnetic nanoparticles can allow the enzyme to acquire the conformational and structural arrangement for a better activity and stability, and suggests that binding of enzyme onto magnetic nanoparticles via carbodiimide activation was efficient for creating bioconjugates for a variety of applications in health and food safety.Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chlorides using two different base solutions. GOX was bound to the particles by direct attachment via carbodiimide activation and chemically via covalent attachment onto thiophene acetylated magnetic nanoparticles. Confirmation of the binding was demonstrated by FTIR spectroscopy and the sizes of the particles were characterized by TEM. The direct binding of GOX via carbodiimide activation was more effective and resulted in binding efficiency in the range between 94–100% while the binding efficiency was only between 66–72% for the GOX-FeDrs. Gilles Kouassi and Joseph Irudayaraj were the primary authors. They were responsible for the concept and experimental plan of the article. Dr Gregory MacCarty was the secondary author and contributed to the overall effort.


Sentinel lymph node (SLN) biopsy is an effective tool for axillary staging in patients with invasive breast cancer. This procedure has been recently proposed as part of the treatment for patients with ductal carcinoma in situ (DCIS), because cases of undetected invasive foci and nodal metastases occasionally occur. However, the indications for SLN biopsy in DCIS patients are controversial.The aim of the present study was therefore to assess the incidence of SLN metastases in a series of patients with a diagnosis of pure DCIS.A retrospective evaluation was made of a series of 102 patients who underwent SLN biopsy, and had a final histologic diagnosis of pure DCIS. Patients with microinvasion were excluded from the analysis. The patients were operated on in five Institutions between 1999 and 2004.Subdermal or subareolar injection of 30–50 MBq of 99 m-Tc colloidal albumin was used for SLN identification. All sentinel nodes were evaluated with serial sectioning, haematoxylin and eosin staining, and immunohistochemical analysis for cytocheratin.Only one patient (0.98%) was SLN positive. The primary tumour was a small micropapillary intermediate-grade DCIS and the SLN harboured a micrometastasis. At pathologic revision of the specimen, no detectable focus of microinvasion was found.Our findings indicate that SLN metastases in pure DCIS are a very rare occurrence. SLN biopsy should not therefore be routinely performed in patients who undergo resection for DCIS. SLN mapping can be performed, as a second operation, in cases in which an invasive component is identified in the specimen. Only DCIS patients who require a mastectomy should have SLN biopsy performed at the time of breast operation, since in these cases subsequent node mapping is not feasible. Ductal carcinoma in situ (DCIS) is defined as the proliferation of malignant epithelial cells in the mammary ductal system, with no evidence of invasion of the basement membrane. By definition, the disease is localized to the breast, with no spread to regional nodes or distant sites.In the pre-mammography era, DCIS was rarely diagnosed, accounting for only 1 to 2% of all breast cancers. The increasing use of screening mammography in recent years has resulted in a dramatic increase in the diagnosis of DCIS, which now accounts for roughly 20% of all mammographically detected cancers [Axillary treatment in patients with a diagnosis of pure DCIS is controversial. By definition, pure DCIS is not invasive and cannot spread to the regional nodes, so axillary staging should not be indicated in DCIS patients. In practice, however, nodal metastases are found in 1 to 2% of patients with a diagnosis of pure DCIS, and this paradoxical condition usually depends on a focus of invasion being missed by the pathologist because of a sampling bias . The intThe aim of the present study was to determine the prevalence of SLN metastasis in a multi-institutional series of patients with a diagnosis of pure DCIS in order to determine the usefulness of routine SLN biopsy in such cases.Our series consisted of 102 patients with a final histopathological diagnosis of pure DCIS who underwent SLN biopsy in five Institutions between January 1999 and January 2004. Patients with DCIS with microinvasion were excluded from the study.All patients underwent SLN biopsy after they had given their written informed consent, and had decided whether they would undergo a completion ALND if SLN were found to be positive.On the day before surgery, all the patients received an injection of 30–50 MBq of 99 m-Tc-nanocolloidal albumin  in 0.2 cc of saline.In patients with palpable tumours, the radiocolloid was injected subdermally into the cutaneous projection of the tumour. In those with non-palpable tumours, the lesion was localized by stereotactic or ultrasonographic placement of a self-retaining wire and the cutaneous projection was marked by an ink spot on the skin, which served as a guide for subdermal radiocolloid injection. Patients with extensive microcalcifications or multifocal tumours were given subareolar radiotracer injection. Finally, patients who underwent delayed SLN biopsy after excision of the primary lesion were given subdermal radiotracer injection in two fractions, at the sides of the surgical scar.Twenty minutes and 2 hours after injection, scintigraphic images were obtained using a large-field-of-view gamma camera  equipped with a parallel hole, low energy and a high resolution collimator.Primary tumour resection with simultaneous SLN biopsy was performed in patients with a preoperative diagnosis of DCIS made by stereotactically-guided directional vacuum-assisted biopsy and in those with mammographical finding pathognomonic of DCIS, with or without a preoperative cytologic diagnosis of malignancy. A diagnostic excisional biopsy was performed in patients with a doubtful pre-operative diagnosis, and SLN biopsy was performed as a second operation in those with a final histologic diagnosis of DCIS.As a rule, in patients undergoing conservative surgery for non-palpable lesions, an intraoperative x-ray examination of the resected specimen was performed, in order to confirm that complete tumour excision had been achieved.SLN biopsy was performed 16 to 24 hours after radiocolloid injection. When conservative surgery was performed, the SLN was excised through the incision used for resecting the primary tumour, if located in the upper outer quadrant, whereas a separate axillary incision was performed if the tumour was located in another breast site. A gamma-ray-detecting probe was used for intraoperative SLN identification. All lymph nodes with a probe-detected radioactive count >10% of that of the hottest node were excised.The excised breast lesions were sampled with serial cuts and the margins were identified by ink.The histopathologic diagnosis and classification of DCIS was made according to the Holland grading system . The VanFor definitive histological examination, two consecutive 5 μm thick tissue sections were cut from a paraffin block at two levels, 40 μm apart from each other. The sections were then stained with haematoxylin-eosin and immunostained with monoclonal antibody to cytokeratin.The 102 patients with pure DCIS had a median age of 59.4 years (range 37 to 85). The histologic characteristics of the tumours are reported in Table Of the 102 patients, 20 (19.6%) had palpable, and 82 (80.4%) non-palpable breast tumours.Seventy-four patients (72.5%) were treated with conservative surgery (quadrantectomy or lumpectomy) and 28 (27.5%) with mastectomy.Ninety-one patients underwent primary tumour resection with simultaneous SLN biopsy: 16 on the basis of a preoperative histologic diagnosis of DCIS obtained by a vacuum-assisted large core biopsy, 63 with a preoperative cytology suggestive of malignancy and 12 only on the basis of the mammographical findings alone.Patients with a doubtful preoperative diagnosis underwent excisional biopsy of the breast lesion, and the 11 patients with pure DCIS at definitive histology, underwent SLN biopsy and, if required, definitive treatment of the primary tumour as a second operation.A total of 147 SLNs were identified and excised from the 102 patients: a single SLN was found in 61 cases (59.8%), two SLNs in 37 (36.3%), and three SLNs in 4 (3.9%).A positive SLN was found in one (0.98%) patient, who had one micrometastases (0.6 mm) detected by haematoxylin-eosin staining. The pathologic review of the surgical specimen confirmed that the tumour had been completely excised, and no microinfiltration was detected. In this SLN positive case, the primary tumour was a micropapillary DCIS, G2, with a diameter of 16 mm. The patient did not undergone previous microbiopsy or FNAC, and SLN biopsy was performed at the time of the primary tumour excision. Completion ALND was not performed.Indications for routine SLN biopsy in patients with a diagnosis of pure DCIS are still controversial. The first study proposing SLN biopsy for DCIS patients came from the H. Lee Moffitt Cancer Center in the year 2000 : in a stThe Memorial Sloan-Kettering Cancer Center group reviewed patients with "high risk" DCIS and found that 9/76 (12%) patients had SLNs positive for metastases. The authors suggested that SLN biopsy should be performed in all DCIS patients with one or more of the following characteristics: palpable or mammographic mass, suspicion of microinvasion, multicentric disease, high nuclear grade or necrosis [On the other hand, in subsequent studies negligible rates of nodal involvement were found in patients with pure DCIS: Kelly et al.  found noThe variability in the reported rates of nodal metastases probably reflects differences in the accuracy of pathological evaluation of the primary breast tumour: extensive sampling and a thorough histological examination of the DCIS are of crucial importance in ruling out microinvasive foci. Microinvasive DCIS is a different pathological entity, with a well-defined metastatic potential, and should be excluded from studies evaluating the role of axillary staging in non-invasive cancer.Our results support the view that the presence of axillary nodal metastases in patients with a final histopathological diagnosis of pure DCIS is a very unusual phenomenon, if the primary breast tumour has been completely excised and thoroughly examined by the pathologist. We, therefore, believe that routine SLN biopsy in all DCIS patients represents an over-treatment and should be avoided.Enthusiasm for SLN biopsy in DCIS patients is partly due to the low morbidity of the procedure. However, complications such as lymphedema, seroma, infection and sensory neuropathy have been reported after SLN biopsy -15. MoreFinally, the policy of performing primary tumour excision with simultaneous SLN biopsy in all patients with a preoperative diagnosis of DCIS may incur a risk of performing axillary biopsy in patients with benign breast lesions, since intraoperative frozen section histology is usually unreliable in patients with small areas of microcalcifications.We therefore believe that this procedure should be used cautiously, being reserved for cases in which a real advantage can be expected.Another factor that has prompted interest in SLN biopsy for DCIS patients is the widespread use of image-guided core needle biopsy for the diagnosis of mammographically-detected abnormalities, a diagnostic technique that often fails to identify invasive foci: 14 to 29% of patients with a preoperative core needle biopsy of DCIS are found to have invasive cancer at surgical excision ,17, and It has been claimed that some features of DCIS  are associated with a higher risk of invasive disease and nodal metastases. Therefore, some authors suggest that SLN biopsy should be reserved for all patients with these "high- risk" DCIS ,21,22. HThe majority of patients in our series presented with low risk DCIS: most of the tumours were small (<1 cm) and only 20/102 patients had a palpable breast mass. Therefore, we cannot rule out that a higher incidence of microinvasion and nodal metastases might be found in a patient population with more aggressive forms of DCIS. However, in our series, the one DCIS patient with SLN metastasis had a small micropapillary, G2, non-palpable tumour.In our opinion, clinical judgement should be used in patients with large solid tumours or diffuse comedo-type DCIS, bearing in mind that most of these patients require a mastectomy and are therefore candidates for SLN biopsy.Our results confirm that the finding of SLN metastasis in pure breast DCIS is a very rare occurence, if the primary tumour has been completely excised and microinvasion has been ruled out by a thorough histologic examination. Therefore, our current policy is to avoid routine SLN biopsy at the time of primary tumour resection in the presence of a preoperative diagnosis of DCIS. We reserve this procedure, as a second operation, for cases in which an invasive component is identified at the histologic examination of the surgical specimen. Only patients with a diagnosis of DCIS requiring a mastectomy or a wide resection close to the axilla should undergo concomitant SLN biopsy.However, particularly if a large high-grade tumour is found, patients should be informed of the risk that invasive disease may be found, and that a second procedure for SLN biopsy may be required. In these cases, the patient can decide whether to undergo a potentially unnecessary SLN biopsy at the time of the first operation or whether to run the risk of requiring a second operation.The author(s) declare that they have no competing interestsGZ planned the study and drafted the manuscriptPC, RM, ZF, GS, PB, PP, AB contributed with their own cases to the final multi-institutional seriesRM and GC reviewed the pathologic specimens of selected cases and drafted the histopatology section of the manuscriptMEP and GM were in charge of data collectionML and DN co-ordinated the studyThe pre-publication history for this paper can be accessed here:


Mathematical measures of complexity shed light on why some concepts are inherently more difficult to learn than others I suspect, nevertheless, that he was not very capable of thought. To think is to forget a difference, to generalize, to abstract. In the overly replete world of Funes there were nothing but details, almost contiguous details.” —Jorge Luis Borges, “Funes the Memorius”We are told scientists are divided into experimentalists and theoreticians. The dialectic description of the dynamics of science, with one tribe gathering data and collecting evidence and another tribe providing form to these observations, has striking examples that argue for the importance of synthesis. The 16th century revolution, which settled the way in which we see the sky today, is probably one of the best examples of how comparatively ineffective each of these tribes can be in isolation. Tycho Brahe, the exquisite observer, who built, calibrated, and refined instruments to see in the sky what no one else could, collected the evidence to prove a theory that Copernicus had already stated years before (in a book he dedicated to the Pope). It was only many years later that Galileo established the bridge between theory and observation; he understood the data in terms of the theory and thereby cemented the revolution. Copernicus's statements, showed Galileo, were not only figments of his imagination; they were an adequate description of the universe as he and Brahe had observed it.Since my first steps in biology, after a prompt departure from physics and mathematics, I have looked for such encounters between theory and experiment. I began studying the visual system and the series of fundamental works by In the middle of the last century, working on the problem of communications, languages, codes, and channels, Claude Shannon proposed a very elegant theory that formalized intuitive ideas on the essence (and the limits) of communications . One of About four years ago, Jacob Feldman published a paper, similar in spirit, proposing a simple explanation for a long-standing problem in cognitive science about why some concepts are inherently more difficult to learn than others . An artiTo visualize the intuitive nature of his theory it helps to do a thought experiment. Imagine a set of objects defined by, say, three features: form, color, and size. And imagine, for simplicity, that each feature is binary, that is, there are only two possible cases. Size is big or small, color is yellow or red, and shape can be either a triangle or a circle. This defines, of course, a universe of eight objects. We can now define categories within this universe: for example, all the circles (a category of four exemplars), or all the big and yellow objects (two exemplars), or all the triangles that are not red (again two) . We can also define a category by enumeration, for example, the small red triangle, the big yellow circle, and the small yellow circle (three exemplars). Some rules (and thus the groups defined by these rules) are intuitively easier to define than others. “All the circles” is an easier statement to make (and probably to remember) than “small circles and yellow big objects.” This notion of difficulty is what Kolmogorov's theory formalized, stating that complexity was the length of the shortest definition  of a given set. From this thought experiment, we can understand the logic of Feldman's paper, which showed that. Kolmogrov complexity is very closely related to our intuitive notion of conceptual difficulty. Feldman presented subjects with all possible categories (of a fixed number of exemplars) in different universes and showed that the critical parameter to rank the difficulty of a given subset was its Kolmogrov complexity. Moreover by explicitly presenting all the members and the nonmembers of a category to naïve subjects, he showed that we can spontaneously reduce a category to its minimal form and remember it without any explicit instruction. Thus, what Feldman found, following the original ideas of Shepard, was that our psychological measure of complexity—our difficulty in defining and remembering a category or concept—is also determined by the Kolmogorov complexity that describes it.This essay is, in a way, about how we avoid becoming Borges's character Funes, who could not understand repeated observations as exemplars of a common rule and thus could not synthesize and categorize. Simply, he could not think. Probably the most disappointing moment of Feldman's paper comes at the very end, where it deals with its (somehow unavoidable) recursive quest. Understanding why some concepts are difficult to learn may itself be difficult to learn. Modern mathematics, together with Kolmogorov complexity and information theory, has taught us another fundamental concept that may be relevant when trying to understand the logic of the mind. In a long series of paradoxes enumerated by Bertrand Russell, Kurt Goedel, and others, we learn that a formal system that looks at itself is bound to fail. At the very end of his paper, Feldman writes, “In a sense, this final conclusion [that psychological complexity is Boolean complexity] may seem negative: human conceptual difficulty reflects intrinsic mathematical complexity after all, rather than some idiosyncratic and uniquely human bias.” Who invented mathematics? The Martians? On the contrary, I believe this result supports a more naturalistic and less Platonic conception of mathematics. Formal ideas in mathematics are not arbitrary constructions of an arbitrary architecture; rather, they reflect the workings of the brain like a massive collective cognitive experiment. Mathematics does not only serve to help us understand biology; mathematics is biology. We are not less original if our thoughts resemble our mental constructions, we are just consistent. It is within this loop, this unavoidable recursion—mathematics understanding the logic of the brain—that we will have an opportunity to test, as some conspire, whether among all the wonders evolution has come out with, the ultimate might be a brain good enough to avoid the risk of understanding itself.


To quickly control infectious disease outbreaks, extensive information is required to identify the source and transmission routes, and to evaluate the effect of containment policies. Traditionally, scientists have used travel- and contact-tracing methods, but the recent SARS epidemic showed that sequence-based techniques for pathogen detection can also be important tools to help understand outbreaks. Jianjun Liu and colleagues adapted mass spectrometry (MS)–based genotyping, already used as a high-throughput way of detecting single nucleotide polymorphisms in human DNA, to the analysis of the SARS virus from clinical samples.The major breakthroughs against SARS were the discovery of the SARS coronavirus (SARS-CoV) as the etiological agent and the sequencing of the SARS genome. Liu's colleagues at the Genome Institute of Singapore had previously shown that common genetic variants in the SARS-CoV genome could be used as molecular fingerprints to help trace the route of infection. However, as “sequence analysis of large numbers of clinical samples is challenging, cumbersome, and expensive,” they felt that “what is needed is a rapid, sensitive, high throughput, and cost-effective screening method.” Towards this goal, Liu and colleagues now demonstrate that an MS-based technique can quickly yield accurate information on clinical isolates (in this case from the 2003 SARS outbreak in Singapore).The scientists demonstrate the sensitivity of the assay in detecting SARS-CoV variations and test it further in cultured viral isolates and uncultured lung tissue samples of SARS-CoV. They analyzed isolates taken from 13 patients with SARS at different stages of the Singapore outbreak, identified nine sequence variations, and discovered a new primary route of introduction of the virus into the Singapore population. They also found a Singaporean origin for a German case of SARS, a result that could not be derived from standard sequencing methods. The analysis of the uncultured lung tissue also found different sequences in a single patient, which suggested the presence of multiple viral sequence variants in one host.The study suggests that MS-based genotyping can be used for large-scale genetic characterization of viral DNA from clinical samples. The researchers found that the method was accurate and sensitive, with a 95% success rat e for detecting sequence variations at low virus concentrations. The MS-based assay allows high-throughput analysis and complements the “gold standard” direct sequence analysis method, which is used to identify new sequence variations. As such, it is particularly useful for investigating agents for which extensive sequence information exists.Liu and colleagues propose that the most efficient method for a large-scale population investigation would be initial characterization of a genome sequence by direct sequence analysis in a subset of samples, followed by MS-based analysis of informative genetic variations. Altogether, their results suggest that MS-based genetic analysis can help real-time investigations in disease outbreaks.