Objective: The aim of this study was to characterize using flow cytometry cultured human primary keratinocytes isolated from the epidermis and hair follicles by different methods. Methods: Human keratinocytes derived from discarded fragments of total skin and scalp hair follicles from patients who underwent plastic surgery in the Plastic Surgery Division at UNIFESP were used. The epidermal keratinocytes were isolated by using 3 different methods: the standard method, upon exposure to trypsin for 30 minutes; the second, by treatment with dispase for 18 hours and with trypsin for 10 minutes; and the third, by treatment with dispase for 18 hours and with trypsin for 30 minutes. Follicular keratinocytes were isolated using the standard method. Results: On comparing the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with dispase for 18 hours and with trypsin for 30 minutes, it was observed that the first group presented the largest number of viable cells, the smallest number of cells in late apoptosis and necrosis with statistical significance, and no difference in apoptosis. When we compared the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with trypsin, the first group presented the largest number of viable cells, the smallest number of cells in apoptosis with statistical significance, and no difference in late apoptosis and necrosis. When we compared the results of the group treated with dispase for 18 hours and with trypsin for 10 minutes with the results for follical isolation, there was a statistical difference in apoptosis and viable cells. Conclusion: The isolation method of treatment with dispase for 18 hours and with trypsin for 10 minutes produced the largest number of viable cells and the smallest number of cells in apoptosis/necrosis. The epidermis is formed by multiple layers of keratinocytes and its differentiation process begins in the proliferative basal layer. Keratinocytes are the main cell type of the epidermis, and they are responsible for the formation of the cutaneous barrier.There are 3 populations of keratinocytes present in the epidermis: stem cells, transit amplifying cells, and differentiated cells.2In the stratum corneum, keratin and glycolipids are arranged in successive layers forming the cutaneous barrier, an impermeable functional unit that maintains life. The loss of liquids, proteins, and electrolytes through lesions, as a consequence of the absence of this barrier, leads to important fluid loss, causing hemodynamic instability and also allowing invasion by several pathogenic microorganisms. Depending on how extensive is the cutaneous loss, as in severe burn injuries, this mechanism plays an important role in the morbidity and mortality of these patients.3Diseases that lead to a major skin loss, such as extensive degloving injuries, cutaneous aplasia, epidermolysis bullosa, giant congenital nevi, pyoderma gangrenosum, traumas, and congenital alterations, are ideal for permanent wound coverage with cultured keratinocytes when there is a lack of sufficient skin graft donor sites for wound closure. In burn wounds, large skin surfaces are frequently affected, leaving no donor site available.5The main objective of the treatment of the burn patient is recovery of the lost cutaneous coverage. Major burn patients hardly survive for too long without the cutaneous coverage because of a high infection rate, as the wound contraction and reepithelialization take a long time to be completed. In major burns, donor sites are scarce. Therefore, permanent wound coverage is done with cultured keratinocytes, which allows the patients to survive.The use of a single layer of cultured keratinocytes onto the wound has the disadvantage of presenting with a low graft integration rate and the resulting long immobilization period to allow for wound healing. After the end of this process, new lesions may occur because of the graft fragility.6In the traditional protocol used for isolation of keratinocytes, the efficiency rate of colony formation is very low, about 3% to 4%, producing a very small number of proliferative cells from an initial skin fragment removed from the major burn patient.9These proliferative cells are part of the transit amplifying cell population that belongs to an intermediate stage between the epidermal stem cells and the differentiated cells. This proliferative cell population is found in the epidermis, from which a sample was taken for primary isolation and culture of keratinocytes. A defined population of proliferative cells is also found in the hair follicles, which is the main site for proliferation of keratinocytes for coverage of partial thickness skin injuries.A keratinocyte isolation method that would lead to the formation of a higher number of viable proliferative cells is essential to the success of culture techniques and major burn treatment, since it would produce large amounts of tissue for permanent wound coverage in a short time.The use of enzymes, such as trypsin and dispase among others, associated with the mechanical factor in the isolation of cells from a skin fragment is essential for the culture method. However, it is also necessary that the technique yields viable cells and not dead or apoptotic cells.To better understand the development of the culture of human keratinocytes, it is necessary to characterize the different features of this cell. Since one of the objectives in a cell culture laboratory is improving the culture method, the use of a flow cytometer is of fundamental importance in studying the cellular dynamics of keratinocytes.For many years, biochemical and biophysical measurements of isolated cells have been taken basically by visual analysis with the use of several types of microscope. Many of the methods used for immunohistochemical and cellular analysis became more and more sophisticated after the development of flow cytometers. Flow cytometry is a modern technique used for cellular analysis and it enables simultaneous measurements of multiple parameters, providing a large amount of cellular information from a single sample.Among the examples of measurements that can be taken, physical characteristics, such as size, volume, and refraction and viscosity indexes, as well as chemical characteristics, such as the contents of DNA, RNA, proteins, and enzymes, and antigenic characteristics, such as cell surface molecules that are easily detected by immunofluoroscence using monoclonal antibodies, should be emphasized.10Flow cytometry also allows for a better characterization of cells, making possible the study of the nucleus, its membrane, the cell cycle, cellular apoptosis, and necrosis using specific markers.The aim of this study was to characterize cultured human primary keratinocytes isolated from epidermis and hair follicles by different methods.The primary culture of keratinocytes started with a suspension of isolated cells derived from a full thickness skin of a patient. The suspension was cut into smaller pieces and treated with trypsin, dispase, or a combination of dispase and trypsin to dissociate the cells.−4 mol/L. One millilitre of 10−10 mol/L cholera toxin , 2 mL of penicillin/streptomycin  (Sigma), 2 mL of 0.4 μ g/mL hydrocortisone, 1 mL of 2 × 10−9 mol/L transferrin/triiodo-L-thyronine (Sigma-Aldrich), and 1.3 mL of 5 μ g/mL insulin (porcine) in a final concentration of 5 μ g/mL. The pH was adjusted to approximately 7.2; the medium was sterilized with a 0.22-μ m filter, kept in a refrigerator at 4°C, and produced a red color.The keratinocyte culture medium was a 3:1 mixture of 750 mL Dulbecco's Modified Eagle's Medium (DMEM)  and 250 mL Ham's F-12 medium , totaling a volume of 1 L. This was supplemented with 10% fetal bovine serum (FBS)  and 24 mg of freshly prepared adenine (6-aminopurine hydrochloride) diluted in 20 mL DMEM/Ham's F-12 mixture, resulting in a final concentration of 1.8 × 102 fragments of total skin discarded in the surgical center were obtained in the morning when the cell isolation was performed. The skin fragments were obtained only after the patients were informed about the purpose for which the skin was collected, agreed, and signed an informed consent form. The study was approved by the Ethics Committee of UNIFESP/EPM.Normal human keratinocytes were isolated from the skin of patients who underwent surgical procedures in the Plastic Surgery Division at the Federal University of São Paulo (UNIFESP/EPM). The 1-cmThree different techniques of isolation of epidermal keratinocytes were applied to 3 groups of skin fragments. The traditional technique, applied to the first group, has been described by Green et al13The first step in a laminar flow hood was to wash the skin fragments in tubes containing phosphate buffered saline (PBS) (Sigma) and then dissociate them in 0.05% trypsin (Sigma-Aldrich). The smaller fragments were placed in a preheated shake flask containing 6 mL of trypsin and 6 mL of versene , totaling a volume of 12 mL. After 30 minutes under mechanical agitation and enzyme action at 37°C, the isolation of keratinocytes was accomplished.The supernatant was removed from the solution containing the isolated cells, the solution was centrifuged to remove trypsin and Versene, and the cells were resuspended in 3 mL of keratinocyte culture medium.In the technique of isolation using dispase , applied to the second group of skin fragments, each fragment was subjected to the same washing procedure as was used in the first group, using 30 mL of PBS in eight 50-mL conical tubes to remove contaminants. Following washing, the fragments were placed in a conical tube containing 15 mL of 0.5% dispase for 18 hours at 4°C. Then, 5 mL of Versene was added, the tube content was transferred to a petri dish, and the epidermis was separated from the dermis using 2 delicate clamps. The epidermis was placed in a tube containing 5 mL of 0.25% trypsin for 5 minutes at 37°C. Then, it was blended in a Vortex mixer for 30 seconds, placed again in an incubator for 5 minutes at 37°C, and homogenized by repeated pipetting 30 times. Finally, decantation of the supernatant and centrifugation were performed as in the previous group.In the technique of isolation applied to the third group, dispase was used in a similar way as in the second group. After separating the epidermis from the dermis, the epidermal fragments were placed in a petri dish containing 0.05% trypsin and the isolation was performed for 30 minutes as in the first technique.The protocol for isolation of follicular keratinocytes started with the removal of the follicles from the scalp, discarded from rhytidoplasty, with 2 delicate clamps and movements perpendicular to the surface of the skin fragments to expose the follicle. The follicular material was directly collected in a 60-mm culture dish containing 5 mL of keratinocyte culture medium. The distal region of the follicle was cut with iris scissors and only the bulge and bulb regions of the hair follicles were collected.Following this, all hair follicles were placed in another 60-mm culture dish. For each 50~follicles, 1 mL of 0.05% trypsin was added. The dish was kept in an incubator for 8 minutes at 37°C. Then, the dish was examined under an inverted microscope and the dissociation of keratinocytes from the follicular bulb was observed. Next, the remaining follicles were removed and immediately 1 mL of keratinocyte culture medium was added for each 1 mL of trypsin in order to neutralize the enzyme action.14The set consisting of keratinocytes, trypsin, and culture medium was placed in a 15-mL tube and centrifuged at 800 rpm for 6 minutes. After centrifugation, the cells were resuspended in 1 mL of PBS and analyzed in a flow cytometer.2) (Sigma) added to 10 μL of 50 μg/mL PI buffer solution (BD Biosciences).The flow cytometry technique was used to detect the cell cycle and apoptosis in suspension cells, using propidium iodide (PI)  and Annexin V (BD Biosciences), as in the method described by Nicoletti et al.Cells were resuspended in 100 μL of buffer solution containing PI, incubated for 10 minutes in the dark at room temperature, washed with the buffer solution, and immediately analyzed in the flow cytometer.6 cells/mL resuspended in PBS were centrifuged after the isolation of keratinocytes. The cell pellet was resuspended in 1 mL of Annexin V buffer solution diluted 10-fold in distilled water. Tubes were prepared according to the cell population assayed and cells were resuspended in Annexin V buffer solution. In the first tube was placed 100 μL of cell suspension with 5 μL of Annexin V and 10 μL of PI; in the second tube, 5 μL of Annexin V; in the third tube, 10 μL of PI; and in the fourth tube, only the cell suspension without markers. The solutions were blended in a Vortex mixer and incubated for 20 minutes in the dark at room temperature. The results from tubes 1 and 4 were observed and analyzed. The experiments using flow cytometry were performed in triplicate.To use the Annexin V marker, 2 × 10The statistical analysis of flow cytometry was done in the Cell Quest program, which gives us the results in percentage of cells. One-way analysis of variance (ANOVA) was carried out to test the comparison between age groups, using Bartlett's test for equal variances and Tukey's multiple comparison test. Two-way ANOVA was carried out to study the viability of cells, using Bonferroni posttests.6 cells, were obtained from the age group 0 to 3 years and that the other age groups showed a decrease in the number of isolated cells inversely proportional to the age of the patients, that is, the older the patients in a given group, the lower the number of isolated cells obtained from that group  and side scatter (cell complexity), showing regions of higher cellular frequency corresponding to specific populations studied Fig .After the definition of the flow cytometry protocol for primary keratinocytes, the experiment was performed applying the 3 methods for primary isolation of keratinocytes using different sequences of enzymes and the method for isolation of keratinocytes from follicles. The results of cell viability in each method studied are presented in Figure Since the 1970s, with the publication of the work by Rheinwald and Green,19Different enzymes were used in cell isolation procedures in order to improve the efficiency of the primary culture of keratinocytes.21Recently, new studies have been performed to find out whether there is a link between the isolation of keratinocytes and the age of patients.2 of skin, with a similar average weight. These differences were not important to the results because before the flow cytometry evaluation, we counted the number of cells and a fixed number of cells, 2 × 106cells/mL, were submitted to cytometry.In this age group, 0–15 years, we can also find facilities to have skin fragments from foreskins, which give us almost the same size of fragments, a 1 cmThe 3 age groups used in this study were related to the fact that 50% of the burns occur in the 0–18 years age group, resulting in a high death percentage. In this experiment, it was found that the number of isolated viable cells decreased as the age of the patients increased, with statistical significance, which agrees with the findings in the literature.24Traditionally, the number of live cells in a suspension is estimated by counting, microscopically, the cells that exclude an acidic dye, such as trypan blue. The method can be adapted for the flow cytometer by using PI, which is excluded by viable cells and, when taken up by dead or dying cells, binds to nucleic acids and fluoresces red. The use of flow cytometry has the advantage that a large number of cells can be counted quickly and that the determination of negative/positive cells is objective.Apoptosis induces a variety of changes in the plasma cell membrane, including changes in permeability and alterations in the membrane lipids. During apoptosis, there is a major change in the membrane lipids in that phosphatidyl serine (PS) residues flip from the internal to external membrane. PS binds Annexin V and these changes can be observed by incubating unfixed cells with Annexin V.2+ ions. It is customary to add PI to distinguish cells that have lost integrity of the plasma membrane.Annexin V is a protein that has a high affinity for negatively charged phospholipids, such as PS, in the presence of CaSo, if the cells are negative to PI and Annexin V, they represent viable cells. If they are negative to PI and positive to Annexin V, they are cells in apoptotic state. If they are positive to PI and Annexin V, they are in late apoptotic state, because Annexin V express the PS residues but the DNA is exposed and they also express the PI. And when they are positive to PI and negative to Annexin V, they are in necrotic state, because PS is not present to bind Annexin V.25Different procedures for isolation of keratinocytes were evaluated in this study. The cell group treated with trypsin for 30 minutes followed standard protocol.26In the analysis by flow cytometry, when comparing results of cell treatments with dispase-trypsin (10 minutes) and dispase-trypsin (30 minutes), it was observed that the dispase-trypsin (10 minutes) group presented the largest number of viable cells, the smallest number of cells in late apoptosis and necrosis with statistical significance, and no difference in apoptosis.When comparing isolation of epidermal keratinocytes results obtained with dispase-trypsin (10 minutes) and with the standard method, it was observed that the dispase-trypsin (10 minutes) method presented the largest number of viable cells and the smallest number of cells in apoptosis. The set of analysis for late apoptosis and necrosis showed no statistical difference.Since both the traditional method and the dispase-trypsin (30 minutes) method produced worse results than the dispase-trypsin (10 minutes) method, there was no need to compare them. The method that involved cell treatment with dispase for 18 hours and with trypsin for 10 minutes was pointed as the most efficient in producing viable cells and less damage, such as cells in necrosis and late apoptosis.Therefore, the results obtained with the method that involved treatment with dispase-trypsin (10 minutes) were chosen to be compared with the results obtained from the traditional method applied to follicular keratinocytes. The sets of analysis for apoptosis and viable cells provided statistically significant results.These experiments were used as a first approach in trying to characterize which method of cell isolation would produce the largest number of viable cells and indirectly the largest number of stem cells.27The experiments were done in triplicate, and the remanescent cells, which were not used in the flow cytometry or were cryopreserved, were seeded on flasks to evaluate the behavior after passages. It was observed that the dispase (18 hours) and trypsin (10 minutes) group gave a significantly higher number of colonies in the flasks until fourth passage after rhodamine staining.An extension of this study, with the use of specific markers for stem cells,


Neisseria meningitidis W135 2a: P1.2,5 in Europe. From March 18 to July 31, 2000, some 90 cases of meningococcal infection were reported from nine countries, mostly the United Kingdom (UK) and France; 14 cases were fatal. Although most early cases were in pilgrims, the outbreak spread to their contacts and then to those with no known pilgrim contact. In France and the UK, the outbreak case-fatality rate was compared with the rate reported from national surveillance. The risk of dying during this outbreak was higher in France and the UK, although the difference was not statistically significant. Prophylaxis for all pilgrims and their household contacts was offered in France; in the UK and other European countries, prophylaxis was recommended only for close contacts. No difference in transmission rates following intervention was detected between France and the UK.The 2000 Hajj (March 15–18) was followed by an outbreak of  Neisseria meningitidis serogroup A, occurred in 1987  clonal complex, which is known to cause hyperendemic disease activity In March and April 2000, national reference centers (NRC) for meningococci in several European countries detected a sharp rise in isolates of In most European countries, according to national policies, prophylaxis was recommended only for close contacts of cases. In France, as soon as the outbreak was recognized , the Ministry of Health recommended a 2-day course of rifampicin for all pilgrims and their household contacts, with the objectives of preventing cases in pilgrims’ households and limiting the spread of the outbreak strain in the population.We describe the epidemiology of the outbreak in Europe and evaluate the impact of the French intervention.N. meningitidis of serogroup W135 2a P1.2,5 or belonging to the ET-37 complex. A probable case was defined as illness in a pilgrim or a pilgrim contact, with either invasive disease due to N. meningitidis serogroup W135 of unknown serotype (identified by polymerase chain reaction [PCR] or detection of specific antigens) or with a clinical diagnosis of invasive meningococcal infection without microbiologic confirmation. Cases included were those with dates of hospital admission from March 18, 2000, until July 31, 2000.A confirmed case was defined as invasive disease caused by Nonpilgrim case-patients were classified as 1) living in the same household as a pilgrim during the 7 days before the date of onset, 2) being in contact with a pilgrim but not living in the same household during the 7 days before the date of onset, or 3) having no identified contact with a pilgrim.A questionnaire was sent by e-mail in April 2000 to the National Surveillance Centers in Europe, and interviews of cases were conducted by telephone or in person. For each case, information was anonymously requested on demography, ethnicity, clinical symptoms, medical history, housing situation, meningococcal vaccination history, contact with Hajj 2000 pilgrims, and travel to Saudi Arabia. Results of the microbiologic investigation were obtained from NRC. Additionally, the number of visas delivered for the Hajj 2000 was obtained from Saudi embassies, and information was collected in France on the observance of the specific prophylactic measures for pilgrims and their household contacts.N.meningitidis infections reported to the national surveillance system in France during 1995–1999. The same comparison was performed for the UK. The probability of dying from the outbreak strain was estimated for France and the UK by using a logistic regression model. In addition to the outcome (death), variables included in the model were age and having an epidemic case versus a national surveillance case. Consecutively, the age-adjusted relative risks (RR) of dying from the epidemic were estimated from the odd ratios by the log link function.As cases occurred predominantly in France and the UK, only these two countries were considered in further analysis. The case-fatality rate (CFR) observed in cases from France was compared with the CFR of all To evaluate the impact of the French control measures implemented on April 8, we compared the number of cases in France with those in the UK before and after April 8 for a) pilgrim and household contacts, and b) out-of-household contacts and those for whom no contact with a pilgrim was identified. The ratio of cases in France after and before the intervention was compared with the same ratio in the UK. An effective intervention would be expected to result in a measure of impact less than one, and vice versa. Confidence intervals were determined by Poisson regression. The p-values were estimated by Fisher’s exact test.The descriptive analysis was carried out with Epi Info . The logistic regression was performed with Stata version 6.0 . Data on cases reported during 1995–1999 were issued from the Public Health Laboratory Service in the UK and from the Institut de Veille Sanitaire in France.N.meningitidis serogroup W135 serotype 2a subtype P1.2,5. Six probable cases were diagnosed by soluble antigen detection (one case) or PCR (five cases). Microbiologic examination of bacterial isolates was described elsewhere From March 18 to July 31, 2000, some 90 cases of serogroup W135 meningococcal disease were ascertained from nine European countries  . QuestioThe Hajj 2000 was held March 15–18, 2000 (week 11). The peak of the outbreak was rapidly reached in week 14, two weeks after the first return of pilgrims from Mecca. Since that time, the number of cases regularly decreased . Of 90 cTwelve cases (13%) were in pilgrims , 31 (34%) in household contacts of a pilgrim, and 21 (23%) in contacts outside the household; for 26 cases (29%), no pilgrim contact was identified.A total of 19,749 pilgrims from the UK and 19,100 from France participated in the Hajj 2000. Eight cases of meningococcal disease occurred in the UK and four in France, giving incidence rates in the pilgrim population of 41 and 21/100,000, respectively. No cases occurred in pilgrims from other countries, although Germany had 18,000 pilgrims and the Netherlands had 4,500 pilgrims. For Finland, Sweden, Belgium, Switzerland, and Norway, no data were available on visas delivered.Pilgrims were affected first : the peak of cases occurred in week 13, household contacts cases peaked in week 14, out-of-household contact cases peaked in week 16, and cases with unknown or no identified contact with a pilgrim peaked in week 19 .Forty-seven (54%) of the 87 patients whose sex was known were female. Fifty-one (65%) of the 78 nonpilgrim patients were <5 years of age. The median age of the pilgrim patients was 51 years; for nonpilgrims, it was 2 years. Information on clinical features was available for 69 cases, including meningitis (30 cases), septicemia (23 cases), or both (16 cases). Arthritis (six cases), osteomyelitis (one), and pneumonia (one) were also reported from patients with septicemia. Purpura fulminans was reported in 12 cases. Thirteen patients had underlying long-term diseases, but none had preexisting immunodeficiency. In the UK, 75% of patients were of Indian ethnicity; in France, the majority (74%) were North African.N. meningitidis in France from 1995 to 1999. The age-adjusted RR of dying during the outbreak was 1.26, . In the UK, 8 (19.0%) of 42 patients died, compared with 862 (8.3%) of 10,448 observed for meningococcal disease due to all serogroups of N.meningitidis in the UK during1995–1999. The age-adjusted RR was 1.99 . There was no evidence that the RR in France differed from the RR in the UK (p=0.55).Fourteen patients died  . In FranThe median length of stay in Saudi Arabia for pilgrim case-patients was 21 days (range 14–40). The median interval between the beginning of the Hajj and the date of onset of disease was 16 days (range 11–29); the median interval between return from the pilgrimage and the onset of disease was 2 days (range 0–8). Among cases with pilgrim contact, the median interval between return of the pilgrim from Mecca and onset of disease was 8 days for household contacts and 6.5 days for out-of-household contacts (p=0.58). Further characteristics of pilgrim cases and cases by type of contact with a pilgrim are shown in Regarding the assessment of the French recommendations, the comparison between France and the UK of the ratio of cases after and before interventions were put in place was 2.43 for pilgrims and their household contacts. This result of >1 yields no evidence that the French measures had a positive impact in preventing cases in pilgrims and their household contacts (p=0.27) . The ratThis report is the first description of a European meningitis outbreak involving nine countries. An international coordination group was set up within 2 weeks after the outbreak was recognized in Europe, followed by an early warning alert to all the European countries. This facilitated information sharing, standardization of the case definition, and implementation of a standardized questionnaire for the investigation. The willingness of participant countries led to a satisfactory completion rate for reports, allowing a precise description of the outbreak throughout Europe. However, case ascertainment may have varied in different countries since some countries identified cases through microbiologic findings only and some through clinical as well as microbiologic findings.N.meningitidis W135 of the same serotype and subtype has been documented since 1979, but pulsed-field gel electrophoresis and the sulfadiazine resistance of the W135 isolates indicate that the outbreak was probably due to a new strain of W135 meningococci In 1987, an outbreak of meningococcal disease linked to the Hajj was described in several European countries, but the description was limited to pilgrim cases and a few secondary cases . In the N. meningitidis W135 was considered a minor serogroup, of little clinical importance N. meningitidis W135 represented 2.6% to 4% of all reported N. meningitidis in the UK, France, and the United States  and two cases per year in France . The outbreak strain belonged to the ET-37 complex, which is mainly composed of serogroup C N. meningitidis in these two countries. Thus, the outbreak strain appears to be of similar virulence to N. meningitidis serogroups that normally cause meningococcal disease in the UK and France. CFRs have been shown to be linked with age and to increase among very young and older people Although the CFRs in cases from France and the UK were high, the age-adjusted RRs of dying during the outbreak were not significantly higher than those observed in the routine surveillance of meningococcal disease due to all serogroup of N. meningitidis W135 resistant to rifampicin had been isolated at the NRC. For the prevention of cases among pilgrims and household contacts, the <1 ratio between France and the UK indicated that the measure had no impact in preventing cases in pilgrims and pilgrims’ household in France. This might be due to the delay of 2 weeks between the first return of pilgrims and the release of the measure, an interval during which transmission of the pathogenic strain occurred inside the pilgrims’ households.Methods used to evaluate the impact of the specific control measures implemented in France intended to take into account differences in meningococcal disease incidence rates between the two countries and potential differences in pilgrims’ initial carriage of the outbreak strain at their return from Mecca. Since the number of cases in each group  was low, the power of the test did not allow identification of the difference of impact between them. Defining other groups could not have allowed conclusions to be drawn, since the number would have also been low. Information collected from the only manufacturer of rifampicin in France indicated that the total number of doses distributed to pharmacies represented only half the doses needed to treat the target group , indicating that compliance with the recommendations was low. In addition, all those who were provided treatment may not have taken it effectively, although compliance would not be expected to be a major problem with only 2 days of medication. As of the end of March 2001 in France, no cases were reported in persons who had taken rifampicin and no strain of N. meningitidis are sent to the reference laboratory in France and the UK. However, data for cases of W135, 2a P1.2,5 obtained from national reference laboratories in France and the UK for September 2000–February 2001 indicated that 13 cases were reported in the UK and 9 in France, suggesting that there was no long-lasting effect of the measure and that immunity to the strain was probably increasing in the population The absence of significant impact of the measure to limit the diffusion of the pathogenic strain to the out-of-household contacts and persons with no contacts identified may be explained either by the very small number of cases considered or by potential misclassification. Cases in the general population may also have been underestimated in comparison with the likely high case ascertainment in pilgrims. However, such underestimates are unlikely since virtually all invasive strains of N. meningitidis W135 2a P1.2,5 occurred in Hajj pilgrims and their contacts in 2001; most cases were from Saudi Arabia and the UK. During the period March 28–June 29, 2001, 10 cases of meningococcal disease due to W135 2a P1.2,5 were reported to the NRC in France (0 deaths) and 25 in the UK (8 deaths)  –25. Sinc


The aim of this study was to evaluate a method based on a single accelerometer for the assessment of gait symmetry and regularity in subjects wearing lower limb prostheses.Ten transfemoral amputees and ten healthy control subjects were studied. For the purpose of this study, subjects wore a triaxial accelerometer on their thorax, and foot insoles. Subjects were asked to walk straight ahead for 70 m at their natural speed, and at a lower and faster speed. Indices of step and stride regularity  were obtained by the autocorrelation coefficients computed from the three acceleration components. Step and stride durations were calculated from the plantar pressure data and were used to compute two reference indices (SI1 and SI2) for step and stride regularity.R2 up to 0.74), and Ad2 well correlates with SI2 (R2 up to 0.52). A ROC analysis showed that Ad1 and Ad2 has generally a good sensitivity and specificity in classifying amputee's walking trial, as having a normal or a pathologic step or stride regularity as defined by means of the reference indices SI1 and SI2. In particular, the antero-posterior component of Ad1 and the vertical component of Ad2 had a sensitivity of 90.6% and 87.2%, and a specificity of 92.3% and 81.8%, respectively.Regression analysis showed that both Ad1 well correlates with SI1 (The use of a simple accelerometer, whose components can be analyzed by the autocorrelation function method, is adequate for the assessment of gait symmetry and regularity in transfemoral amputees. Symmetry and regularity of walking are two important aspects in gait analysis. Symmetry is related to similarity of contralateral steps, whereas regularity is related to similarity of consecutive strides. Both symmetry and regularity of gait are usually impaired in subjects wearing lower limb prostheses -3. The oIn such a perspective, the availability of an easy-to-use, portable system capable of measuring the degree of gait symmetry and regularity may provide important contributions for the treatment of lower limb amputees, both in the clinical practice supporting professional caregivers , and for home-care practice to support the patient for self-rating (for example in the home environment or during activities of daily life).In this scenario, to facilitate the use of the system by both practitioners and patients, both in the hospital and in independent life, the device must implement the following features: low-cost, high-comfort, easy-mounting and low-maintenance requirements. For this purpose, the use of inertial sensors appears the most convenient choice, similarly to what has been done in other contexts, and only partially for lower-limb amputees -15, withFrom the on-board intelligence viewpoint, the development of a portable system for automatic detection of gait symmetry and regularity requires the selection of signal processing algorithms optimized for moderate processing resources consumption.The aim of this study was therefore to assess the suitability of a method based on a single accelerometer and on the computation of the acceleration autocorrelation function , to measTen AMPs, all wearing a lower-limb prosthesis with the same kind of electronically controlled knee  were recruited at the INAIL Prostheses Centre  for the study. All of them were confident walkers, since they had used mechanical prostheses for several years before using the electronically controlled knee, and by the time of measurements they had completed the training period with the C-leg. Ten healthy subjects were also studied as control group (CTRLs). Even if the control subjects resulted slightly younger than the amputees, they were, as the amputees, in the adult range of age, making the two groups suitable for the methodological validation of our approach. All participants were male and provided informed consent before data collection started. Further details on the two groups of subjects are presented in Table 2), one gyroscope  and one magnetometer, though in this study only the acceleration signals were considered. The sensing unit was placed on the thorax at the xiphoid process and fixed to the body through adhesive tape over an elastic bandage. Acceleration data were acquired with respect to the sensor's technical reference frame, which is certified by the manufacturer as being aligned along the MTx box borders with an error less than 3 degrees. The sensitive axes of the accelerometer were manually aligned along the anatomical vertical (V) axis , and medio-lateral (ML) and antero-posterior (AP) axes. The sensing unit was connected to the XSENS data logger, which transmitted the data to a PC via Bluetooth.Accelerometric data were acquired by means of an XSENS inertial sensing unit . The sensing unit consists of a small case of 58 × 58 × 22 mm (WxLxH) weighing 50 g only. This includes some triaxial sensors: one accelerometer  of proper size, based on capacitive sensor technology. Each insole provides up to 99 plantar pressure measurement spots. The Novel equipment was chosen since it is commonly used in the clinical practice, it has been widely validated in the literature ,18 and iA device was used to synchronize the acquisition from the XSENS and the Novel equipment . The SyncBox was connected to the Novel data logger, and it received a clock signal from the XSENS data logger, that acted as master in the acquisition. A picture of the set-up is shown in Figure st order, cut-off frequency = 28 Hz) before digitalising the accelerometric signals. Data processing and analyses were performed in Matlab .All the data were acquired at the sampling frequency of 100 Hz. Each MTx applied an anti-aliasing hardware filter  was computed by the following equation:Gait symmetry and regularity indices were computed on the basis of the unbiased autocorrelation coefficients, according to the method proposed by Moe-Nilssen and Helbostad . BrieflyN is the number of samples and m is the time lag expressed as number of samples.where m) on each of the acceleration signals derived from the triaxial accelerometer during the gait tests. For each component we excluded from the analysis the samples related to the first and last five steps, to avoid transitional phases of gait initiation and termination.We computed Ad(m), Ad1 coefficient, expresses the regularity of the acceleration between consecutive steps of the subject. This can be interpreted as a measure of the symmetry between steps performed by the prosthetic and the sound leg (or between left and right leg in CTRLs). The second peak of Ad(m), Ad2 coefficient, expresses the regularity of consecutive strides. Higher Ad1 (Ad2) values reflect higher step (stride) regularity. After normalization to the zero-lag component Ad(0) the maximum possible value for Ad1 and Ad2 is 1.The first peak of Ad or vanishes (toe-off). For each subject the force threshold indicating presence of foot contact was fixed to 10% of the mean vertical force maintained in orthostatic position for three seconds .For each gait test, from the duration of steps and strides measured with the pressure insoles two reference indices of gait symmetry and regularity were calculated, firstly for each couple of consecutive steps and strides, and then averaged over the entire gait test.For the regularity of steps (i.e. symmetry between legs) the following expression was used:STEP_R and TSTEP_L are the time duration of right and left step , respectively.where TSimilarly, for the regularity of strides the following expression was used:STRIDE_R and TSTRIDE_L are the time duration of a stride started with the right and left leg, respectively.where Ti) and SI2(i) were then averaged over the entire gait test to obtain the final values of SI1 and SI2:SI1(N (M) is the number of couples of steps (strides) in the gait test.where Such averaged values were assumed characteristics of the test and the corresponding standard deviations resulted negligible.Although there is no unique index, in the scientific literature, accepted as reference for the computation of symmetry, expressions like SI1 and SI2 were widely used . Also, STo validate the indices computed from the accelerometer through the autocorrelation analysis, the relation between Ad1 and SI1, and between Ad2 and SI2, were evaluated by means of univariate and multivariate regression analyses.To see how well the symmetry and regularity indices could detect differences between AMPs and CTRLs, an ANOVA was carried out, with Repeated Measures to take into account the repeated tests for each subject, and with automatic corrections for violations of sphericity. A P value less than 0.05 was assumed for statistical significance. Results were reported as mean ± SE.We performed a ROC analysis to measure the sensitivity and specificity of Ad1 (Ad2) in detecting a subject with "normal" or "pathologic" gait symmetry (regularity) during a test. For this purpose, a 5-step process was followed, here described for Ad1: 1) The SI1 values of all the tests in the CTRLs were displayed as a box & whisker plot; 2) Symmetry was assumed "normal" when the range of SI1 values was within the whiskers (1.5 times the interquartile range), and "pathologic" when outside the whiskers; 3) The SI1 values of all the tests in the AMPs were then considered, and each AMP's test was classified as featuring a "normal" or "pathologic" symmetry based on the previous definition: this was assumed as the reference classification for the AMPs' tests; 4) Each Ad1 value of the AMPs' tests was included in the "normal" or "pathologic" category according to the reference classification: we thus obtained two distributions of Ad1 values (for each of the three Ad1 indices); 5) Through ROC analysis on these two distributions, we determined the Ad1 threshold that maximises the correct classification of AMPs having "normal" or "pathologic" symmetry during a test.Similar steps were performed with SI2 and Ad2 for stride regularity.ML values are always negative, both in AMP and in CTRL, since they correspond to the lateral trunk acceleration along the right-left directions . However, the absolute values were considered for the analyses. The patterns for the two subjects also show that the AMP's values for Ad1 and Ad2 are generally lower than CTRL's, for all the directions. Ad1 seems in general more different between the two subjects than Ad2.Representative patterns of the autocorrelation function computed from the three components of the acceleration signals in an AMP and in a CTRL are shown in Figure AP , and between SI2 and Ad2V . Therefore, any one of the three Ad1 indices may be considered a good surrogate of SI1 for the assessment of step regularity, and the same states for Ad2 indices for the assessment of stride regularity. Values of R2 (and corresponding P) for all the indices are listed in captions of Figures Figures R2 reaching the value of 0.776. As for Ad2 indices, in multivariate regression analysis only Ad2V was a significant covariate of SI2, whereas Ad2ML and Ad2AP were not.Analysis of covariance showed that, for each index, there was no difference in the regression lines related to the three different walking speeds. Through the multivariate regression analysis, we found that any one of the three Ad1 indices contributes to explain the variability of SI1, i.e. all three indices were significant covariates (P < 0.016), with V and SI1, and the same for Ad1ML. Conversely, a significant correlation was found between Ad1AP and SI1 . Furthermore a significant correlation in SI2 was found with all the accelerometry-based indices, the best correlation being with Ad2V . Similarly, in CTRLs, no significant correlation was found between Ad1V or Ad1ML and SI1. For Ad1AP a significant though weak correlation was found with SI1 . Again, SI2 was significantly correlated with all the accelerometry-based indices, the best correlation being with Ad2V .Regression analyses were carried out, with AMPs and CTRLs treated separately in the analysis. In AMPs, no significant correlation was found between Ad1Mean values of all the indices in the two groups are shown in the bar graphs of Figure By means of ROC analysis, two subjects among CTRLs were found having impaired walking tests in terms of gait symmetry assessed by SI1. Conversely, three subjects among AMPs had some normal walking tests. As for gait regularity assessed by SI2, only one CTRL had one impaired walking test, whereas all the AMPs had one or more normal walking tests. Table Since the time of use of the C-leg varied within a wide range (2 months to 7 years), the presence of a correlation between the duration of use and the gait performance was investigated, but no significant correlation was detected.The aim of this study was to evaluate the appropriateness of a method based on the use of a single triaxial trunk accelerometer for the assessment of symmetry and regularity of gait in unilateral, transfemoral amputees. The interest for such measures is justified by their potential role in developing a portable automated device that may be able to evaluate the patient's gait features. In fact, one of the main characteristic that a portable, easy to use device to monitor gait features should have, is unobtrusive sensing units. Inertial sensors, such are accelerometers, are ideal candidate for such purpose. In this view, the methods and results presented in this study represent a step forward in the development of a potentially stand-alone portable system, that may act as a "virtual gait trainer" with the potentials of providing a summary score of walking ability in terms of gait symmetry and regularity during training, and, possibly, of alerting the therapist or the patient in the case of worsening of these gait features (biofeedback approach). The system could then be used even out of the clinics or rehabilitation institutes, allowing more frequent and prolonged training and rehabilitative therapy.To these purposes, it was important to select a method for gait symmetry/regularity estimation that is particularly simple, both in terms of equipment and of computational requirements. In fact, if many approaches are possible for the estimation of gait regularity or gait variability ,23, the overall gait performance, in terms of symmetry and regularity, since it requires a large number of consecutive steps to supply a reliable estimate of performance. This approach is indeed in accordance with the concept of task-oriented training, which has been recently confirmed as more appropriate than, e.g., single muscle or single body segment rehabilitation, when a specific motor function needs to be restored [The proposed algorithm may provide information to the user regarding the restored -26. A derestored  or opticrestored . Such syThe analysis of gait symmetry and regularity in subjects wearing lower limb prostheses has been performed in previous studies. However, only few studies included healthy control subjects ,2. In 11, 11 uniAs far as cross-validation of the two measurement systems is concerned, we found that Ad1 and Ad2 indices computed from the acceleration signals were well correlated with SI1 and SI2, hence the simple and inexpensive approach based on the use of a single accelerometer may be adequate to estimate gait symmetry and regularity in transfemoral amputees. To our knowledge, only a few studies used inertial sensors to evaluate gait in subjects with lower limb prosthesis -15, and In the regression analysis, a possible limitation might be due to the inclusion of the data from all the repetitions for each subject. However, since the regression was performed on two measures both acquired during different tests, the independency between the samples remained despite the fact than more than one test resulted from the same subject.As for the computation of Ad1 and Ad2 indices, comparison with other studies was possible only in relation to the control group. In , where tIt is worth noting that the difference in Ad1 between AMPs and CTRLs was more marked than in Ad2 , and this is in agreement with what the physiotherapist expects. In fact, Ad1 represents the regularity of consecutive steps, and most likely in a subject wearing a unilateral prosthesis the right and left steps are different. Thus, it is reasonable that the differences compared with the CTRLs are more evident in the step regularity rather than in the stride regularity that may be still quite regular even in the amputees. As evidence, differences in SI2 between and AMPs and CTRLs were found statistically different, but appear clinically irrelevant.R2 values) differs between components. Similar results were found for Ad2 and SI2. However, no significant correlation was found between Ad1V or Ad1ML and SI1 in the single AMP and CTRL groups. In AMPs, that may indicate the existence of compensatory trunk asymmetry to regain some degree of gait symmetry, and that may reflect in relatively high SI1 values, differently to Ad1 values that are generally low. In CTRLs, a relation between Ad1 indices and SI1 may not be possible to demonstrate, because of lack of dispersion in the data. In all the subjects, it must also be noted that the correlation between Ad1 and SI1 (and similarly for Ad2 and SI2), even if significant, showed a slope of the regression line far from 1, i.e. they have much different range: SI1 and SI2 have in fact much narrower ranges compared to Ad indices. This was particularly observed in SI1 for the control subjects.In the analysis of AMPs and CTRLs grouped together, we found that each component of Ad1  correlates with SI1, even if the degree of correlation /mean for the step, and similarly for the stride), and the main findings of the study were confirmed.Even if there is not a standard reference method for the calculation of the symmetry indices  our resuAP and Ad2v appear to be the best compromise between specificity and sensitivity for general uses, even though the 100% specificity for Ad2AP may be appealing when the amount of false positives is a major concern.The sensitivity and specificity of Ad1 and Ad2 further support their use in the clinical practice. In particular, Ad1AP), and regularity by the coefficient at the second dominant period computed along the vertical axis (Ad2v). The use of the simple, low-cost accelerometry-based system will allow for early detection of asymmetric and irregular walking patterns; it will possibly be beneficial in the correction of these alterations to prevent related comorbidities, with potential wide penetration of this approach both in the clinical practice, and, on a future perspective, for home-based rehabilitation.We studied gait performance in a homogeneous group of prosthesis-aided patients, and we compared the symmetry and regularity of their gait with that of a population of control subjects. We found that a simple accelerometer, placed on the thorax at the xiphoid process may be adequate for the assessment of gait symmetry and regularity. Symmetry can be best assessed by the autocorrelation coefficient at the first dominant period computed from the acceleration along the anteroposterior axis (Ad1The authors declare that they have no competing interests.AT has made substantial contributions to analysis and interpretation of data and has been involved in drafting the manuscript. MR has made substantial contributions to acquisition, analysis and interpretation of data. LR has made substantial contributions to analysis and interpretation of data and has been involved in revising the manuscript. AGC has made substantial contributions to conception and design, analysis and interpretation of data, and has been involved in revising the manuscript. LC has made substantial contributions to conception and design of the study and has been involved in revising the manuscript.All authors read and approved the final manuscript.


We expect that further efforts on modeling and experimental evaluation would contribute to diagnosing patients with metabolic diseases such as osteoporosis and chronic kidney diseases, and developing FGF23-linked treatment strategies.Fibroblast growth factor 23 (FGF23) has recently been identified as a critical regulatory factor in phosphate (P) metabolism. Although the exact molecular mechanism of FGF23 synthesis through sensing the concentration of P is yet to be determined, experimental and clinical data indicate the influential role of FGF23 in P and calcium (Ca) homeostasis. Here, we extended our previous mathematical model in calcium regulation and examined the conceivable roles of FGF23 in mineral metabolism. We assumed that the level of FGF23 was controlled through the concentrations of P and calcitriol in serum, and its actions such as lowering of the renal threshold for P, inhibition of the production of calcitriol in the kidney tubule, and inhibition of the production of parathyroid hormone (PTH) were included. Comparisons between the models with and without FGF23 demonstrate a complex interplay of FGF23 with calcitriol and PTH. In consistent with the model, our  The FGF family consists of 22 members for various functions in embryonic development, cellular proliferation and differentiation, tissue repair, and tumor growth and invasion. FGF23 has recently been demonstrated to represent a critical circulating hormone involved in phosphate metabolism. It is an approximately 32-kD (251 amino acids) protein and its N-terminal region contains the FGF homology domain.Understanding the mechanism of Ca and P metabolism requires multiscale mathematical modeling. Behaviors of this highly interactive, nonlinear process can hardly be predicted by simple intuition. FGF23 is primarily synthesized in bone but its site of action is the kidney. Although genetic and biochemical analyses are essential to identify regulatory mechanisms, those approaches alone will not be sufficient to quantitatively evaluate the key processes and develop effective therapeutic interventions for patients undergoing hemodialysis and patients with metabolic disorders. Various mathematical models have been developed for calcium homeostasis.in vitro experiments to evaluate the mRNA expression level of FGF23 using MLO-A5 osteocyte-like cells.In this study, we included the actions of FGF23 to a previously published mathematical model of calcium and phosphate metabolismThe present work with the action of FGF23 is an extension of a previously published model without FGF23.Considered in the previous model were the functional compartments, which represented the secretory mass of the parathyroid glands, the pools of transporting intestinal translocators for Ca and P, the pools of renal tubular translocators for Ca and P, and the pool of the tubular 1-hydroxilating enzyme . With the exception of the parathyroid gland secretory mass that was assumed to remain constant, the dynamics of the pools were modeled with the first-order nonlinear, diffusional, differential equations. A modeling principle was that the instantaneous rate of variation of the pool size was equal to the sum of the influxes minus the sum of the effluxes. Thus, the dynamical exchange processes in each of the compartments was represented by a time derivative of 8 pools in a general form:Q = size of the pool of the state variable x (e.g. Ca in bone); J(x)influx = influxes of variable x ; and J(x)outflux = outfluxes (e.g. urinary output of Ca). These 8 pools included 3 pools of Ca in bone, extracellular space (serum), and intestine, 4 pools of P in bone, serum, intracellular space, and kidney, and 1 pool of PTH in parathyroid grand. No independent state variables were considered for Ca in kidney or intracellular space, or for P in intestine (see details in 8 and where QFGF23 (pg), was modeled:In addition to dynamical alterations of three molecular regulators  in the previous model, the dynamical alteration of FGF23 was added in the current model as a mineral metabolism regulator . The totJFGF23 = rate of FGF23 secretion (pmole/hr), and δ = rate factor for FGF23 degradation (0.8 h−1). Since the secretion of FGF23 is reported to be modulated by the serum concentrations of P and calcitriol  and  represented the maximum rates and Michaelis constants in Michaelis-Menten kinetics, respectively.in which P (pathway V). In each pathway, the effect of FGF23 was modeled in a form:Three effects of FGF23 were considered in the extended model: inhibition of the production of calcitriol in the kidney tubule (pathway III), inhibition of the production of PTH by the parathyroid glands (pathway IV), and lowering of the renal threshold for Vmax = maximum rate in Michaelis-Menten kinetics; KM.M. = affinity for the FGF23 receptor; and XFGF23 = serum concentration of FGF23. The former was used for stimulatory effects (pathway V) and the latter for inhibitory (pathways III and IV).in which Prior to numerical simulations, we identified the reference states (steady-states), in which the state variables were kept constant. Using stepwise perturbations to the selected reference state variables, we evaluated the transient responses for 0–100 h, and a set of new steady states at 2000 h after an onset of perturbations. In order to avoid an impulsive jerk, stepwise perturbation was given using an exponential function in the initial perturbation phase with a time constant of 1 h. The key parameters employed in the study are listed .10(PO4)6(OH)2] and examined the effects of hydroxylapatite in the culture medium on the FGF23 mRNA level. In the second experiment, cells were grown with and without a pharmacological agent Using MLO-A5 osteocyte-like cells, we conducted two experiments for investigation of FGF23 mRNA expression. In the first experiment, cells were grown either in the presence or absence of hydroxylapatite [Ca6 cells were seeded on the 3D collagen matrix  and cultured in αMEM medium containing 5% fetal bovine serum, 5% bovine serum and antibiotics  for two days. Deposition of hypdroxylapatite was conducted by immersion of the collagen matrix in 500 mM Na2HPO4 solution for 5 min followed by rinsing in water and immersion in 500 mM CaCl2 solution for 5 min.In both experiments, approximately 1 × 10Total RNA was harvested using RNeasy mini kits (Qiagen), and reverse transcription was conducted with high capacity cDNA reverse transcription kits (Applied Biosystems).The mRNA level of GAPDH was used as an internal control. In evaluation of the effect of hydroxylapatite on FGF23 expression, the GAPDH mRNA levels in the two samples were compared and the ratio (GAPDH level in hydroxylapatite/GAPDH level in control) was calculated. The level of FGF23 mRNA in hydroxylapatite was then divided by this ratio, and the mean value of the FGF23 mRNA level in control was set to 1. In evaluation of the effect of pertussis toxin on FGF23 expression, the same normalization procedure based on the GAPDH mRNA levels was taken in which the FGF23 mRNA level for the sample with 0 ng/ml pertussis toxin was set to 1. Experiments were conducted three times.Based on the above mentioned formulation of the expression and actions of FGF23, we first evaluated the effects of the stepwise increase in the secretion of FGF23 on the regulatory and homeostatic variables such as the concentrations of PTH, calcitriol, Ca, and P in serum as well as the absorption and excretion amounts of Ca and P. In the transient responses (0–100 h) to an increase in FGF23 from 30 to 260 pg/ml, those variables were decreased with varying temporal behaviors . The conIn order to characterize the chronic effects of the increase in FGF23, we evaluated the pseudo steady-state values (2000 h) . The varTo evaluate the effects of FGF23 actions in mineral metabolism, we next evaluated the responses of the state variables in the presence and absence of FGF23 actions after a stepwise increase in the phosphate intake. As predicted in the responses to the stepwise FGF23 increase in Consistent with the transient responses, those variables  at 2000 h after the onset of the increased phosphate intake in diet from 2 to 20 mM/h were significantly lowered in the presence of FGF23 . The obsThe effects of FGF23 in response to the increased uptake of Ca were opposite to those in response to the elevated uptake of P, since the stepwise increase in Ca uptake lowered the concentration of FGF23 in serum . AlthougNumerical simulations were also conducted in response to a stepwise increase in two regulatory factors , and the pseudo steady-state values (2000 h) in the presence and absence of FGF23 were evaluated. Although the synthesis of FGF23 is affected by calcitriol and not by PTH in the current model, the stepwise perturbations in both calcitriol and PTH altered the level of FGF23. First, the increase in calcitriol upregulated the level of FGF23 , while tNeither perturbation did not show any clear effects on the absorption of Ca in intestine, but they changed the absorption and excretion of P. In accordance with the predicted increase in FGF23, the stepwise input of calcitriol decreased the intestinal uptake and the urinary output of P. In concert to the simulated decrease in FGF23, on the other hand, the stepwise elevation of PTH increased the intestinal absorption and the urinary excretion of P.The FGF23 mRNA level was unregulated in the culture that was rich in Ca and phosphate (hydroxylapatite). Quantitative real-time PCR showed that the mRNA level of FGF23 was elevated 8.7 fold in the matrix deposited with hydroxylapatite . Furtherin vitro, although hydroxylapatite is a compound consisting of calcium and phosphate. Furthermore, similarly to a calcium sensor,The current study presented mathematical formulation of the metabolism of Ca and P including the actions of FGF23. Prior to the discovery of FGF23, the calcium-PTH-calcitriol axis has been considered a primary regulatory pathway in mineral metabolism. The major function of this axis is maintenance of the serum calcium level, while its role in the serum phosphate level is treated secondarily.The described model allows prediction of the concentrations of FGF23, calcitriol, PTH, Ca, and P in serum together with the intestinal absorption and the urinary excretion of Ca and P in response to varying perturbations. The FGF23 elevation is considered to reduce production of calcitriol in the kidney and decrease absorption of calcium and phosphate from the intestine.in vivo, supporting the assumption of the model that FGF23 directly regulates PTH expression.ex vivo rat parathyroid cultures, it is reported that FGF23 suppresses expression and secretion of PTH.22The current model should be further refined using experimental and clinical data regarding the upstream and downstream events for expression of FGF23. Furthermore, the parameter values in Physiological data from FGF23 mutations can be also used for validation of the role of FGF23. The FGF23 gene was identified by its mutations associated with autosomal dominant hypophosphatemic rickets, which is an inherited phosphate wasting disorder.In summary, the described mathematical model allows us evaluation of the dynamical metabolic processes in Ca and P by considering the regulatory actions of calcitriol, PTH, and FGF23. Biological experimentation indicated a potential role of G-linked protein receptors in regulating expression of FGF23 in bone cells. Further evaluations are necessary to refine the model. The results herein support that the described model-based approach together with biological verification is useful for characterization of the dynamical metabolic responses, which is indispensable for development of quantitative treatment strategies for patients with metabolic disorders.


The study was conducted to know how the consumption of relatively large amounts of dairy products by adult Fulani affected the TFA content of their serum phospholipids. Blood samples were collected from 22 male and 29 female Fulani, aged 35–60 years, who were living in rural areas of Gombe state in northeastern Nigeria. The total serum phospholipid fraction was isolated, and its fatty acid composition was determined. Surprisingly, vaccenic acid was not detected, and three other TFAs—18:1-t6, 18:1-t9, and 18:2-t9, t12—together accounted for only 0.16% of the total fatty acid. The mean serum total cholesterol, LDL-C, and triglyceride concentrations of the subjects were within the normal range for populations in developed countries; however, at 32 mg/dL, the mean serum HDL-C concentration of the Fulani males was slightly below the lower limit of the reference range. No correlations were observed between the total TFA percentage or that of the three individual TFAs and any of the parameters of the serum lipid profile. These findings indicate that, with respect to TFAs at least, the fatty acid pattern of the serum phospholipids of Fulani pastoralists does not reflect the high TFA content of their traditional diet. Despite the consumption of rumenic acid-rich dairy products, for unknown reasons, the semi-nomadic Fulani manage to maintain a low level of TFAs in their blood and a relatively healthful serum lipid profile. While the mechanism that accounts for this disconnect between the consumption of TFAs by Fulani pastoralists and the proportion of TFAs in their serum phospholipids is obscure, possibilities include discrimination against rumenic acid during the process of triglyceride synthesis and chylomicron synthesis in the intestine and the preferential oxidation of TFAs by Fulani the people compared to other ethnic groups.The Fulani are semi-nomadic pastoralists of West Africa whose diet, culture, and economy are centred on cattle. Previous studies have shown that the Fulani of northern Nigeria derive 50% of their total calories from fat and 30% of their calories from milk, cheese, yogurt, and butter oil that contain significant amounts of  Since dic acid) , we woulic acid)  would seWe, therefore, determined the fatty acid composition of the total serum phospholipid fraction of 29 Fulani women and 22 Fulani men living in the vicinity of Gombe in northeastern Nigeria. Since the fatty acid composition of human plasma phospholipids reflects recent dietary fatty acid intake –12, we hHealthy Fulani (Fulbe) men (n=22) and women (n=29) aged over 18 years were recruited consecutively over a five-day period from among relatives accompanying patients to the General Medicine Clinic at the Federal Medical Centre-Gombe in Gombe. Gombe, the capital of Gombe state, is located in northeastern Nigeria and has a population of approximately 270,000. The major occupations of the Fulani people in the region are farming and rearing of livestocks. The exclusion criteria included use of alcohol or tobacco. Information on age, weight, and height was recorded. A fasting blood sample was obtained for the analysis of serum lipids.A stadiometer accurate to 0.25 cm and a battery-operated scale  accurate to 0.2 kg were used for determineing, respectively, the height and weight of each subject. Blood pressure was measured using a nylon cuff and latex inflation system . Fat-free mass (FFM) and body fat (BF) were estimated by bioelectrical impedance analysis conducted using a portable analyzer  as described elsewhere –16. Reacet al. , palmitelaidate (C16:1-t9), petroselaidate (18:1-t6), elaidate (C18:1-t9), vaccenate (C18:1-t11), conjugated linoleate (CLA), linoelaidate , and eicosenoate (C20:1-t11). The coefficient of variation for the various fatty acid values ranged from 1.5% to 5%.Total serum lipids were extracted using a minor modification of the Folch method . BrieflyDescriptive statistics, group comparisons, and correlations were performed using the Number Cruncher statistical software (Version 2001) . Since body-composition parameters and serum lipid concentrations are known to be gender-dependent, data for male and female subjects were analyzed separately. A p value of 0.05 was considered significant.The Ethics Committee of the Federal Medical Centre-Gombe and the Human Research Review Committee of the University of New Mexico Health Sciences Center, Albuquerque, NM, USA, approved the study. Informed consent was obtained from each subject after the purpose and requirements of the study were explained in English or Hausa, the predominant language used in the area.2 respectively) was confirmed by the results of bioelectrical impedance analysis of their body composition: men had 13.8% fat, and women had 26.5% fat. Although the mean (systolic/diastolic) blood pressure of both male (137/84 mmHg) and female subjects (137/82 mmHg) indicated borderline hypertension, the mean phase angle of both the sets of subjects were in the range of values reported for individuals whose overall health is good  years, and 29 women aged 35–60 (mean 47.6) years . The lea5.5 years is good , 22.The concentration of total cholesterol was higher in women than in men  was not detected. The t-6 and t-9 isomers of oleic acid accounted for approximately two-thirds of the total TFAs whereas the di-unsaturated TFA linolaidic acid  accounted for the other one-third. The most common conjugated linoleic acid present in cow's milk–rumenic acid  was not detected in the serum phospholipids of either the male or female subjects. Significant differences in the proportions of individual fatty acids in the serum phospholipids of the male and female subjects were not observed, except for cis-vaccenic acid (18:1n-7) and arachidic acid (20:1) (d (20:1) . CollectThe percentages of the two essential fatty acids—linoleic acid and α-linolenic acid—were 19.9% and 0.18% respectively. The proportions of two other nutritionally-important polyunsaturated fatty acids—arachidonic acid (14.1%) and docosahexaenoic acid (3.14%)—in the serum phospholipids of the Fulani adults in the present study were similar to those we reported in an earlier study of Fulani adults in northern Nigeria  and by ot6 plus 18:1-t9, or the percentage of 18:2-t9, t12 versus total cholesterol—HDL-C, LDL-C, or TG—in either the male or female subjects. No correlations were observed between any of the anthropometric parameters with TFAs or elements of the lipid profile.We were interested in knowing if there were relationships between the proportion of TFAs in the serum phospholipids of the subjects in our study and the serum levels of total cholesterol and certain lipoproteins that are risk factors for cardiovascular diseases. When adjusted for age and gender, no significant correlations were observed among the total percentage of the three TFAs, the percentage of 18:1-t11)  , 8, we elsewhere .It has been well-documented in numerous studies in different parts of the world that the TC and LDL-C levels of an individual correlate positively with the weight percentage of TFAs in his or her serum phospholipid fraction and inversely with the HDL-C level. Although the TFA proportions in the serum phospholipids of the Fulani adults in our study were low compared to those of other populations worldwide, we were, nevertheless, still interested in inquiring if there were relationship between the TFA content of the phospholipids of the Fulani men and women and the various components of their plasma lipid profiles. In fact, no such correlations were observed. It is puzzling why TFAs would be so under-represented in the fatty acid profile of the serum phospholipids of the Fulani pastoralists whose culture, economy, and diet are centred on cattle and dairy products.Since few studies have addressed the question of possible differences in the synthesis and catabolism of TFAs between ethnic groups and populations, we can only speculate that one or more steps in the metabolism of dietary TFAs by the relatively homogenous Fulani might differ compared to fatty acid pathways of other ethnic groups and account our finding of unexpectedly low levels of TFAs in the serum phospholipids of the Fulani pastoralists. Candidate enzymes and metabolic processes that could conceivably play a role in accounting for the finding of low levels of TFAs in the serum phospholipids of the Fulani include: chylomicron synthesis and secretion in the intestine, the specificity of lipoprotein lipase, the specificity of transporters that facilitate the uptake of fatty acids across the plasma membrane of cells, the activity of acyltransferases involved in synthesis of triglycerides, and the rate at which TFAs are oxidized by the various beta-oxidation pathways. Perhaps, the Fulani people absorb TFAs poorly from the intestine and those TFAs that are absorbed are rapidly taken up by cells and either preferentially oxidized or stored as triglycerides.A fourth possibility has to do with the unusually low (estimated) fluidity of the serum phospholipids of the Fulani. In a previous study of the Fulani population of northern Nigeria , we founThe results of the present study shed light on the question of why the milk-fat of the Fulani women contains so little TFAs. Our recent study that included 41 lactating Fulani women in northern Nigeria  found thOur experience over the past 15 years in northern Nigeria informs us that the diets of the rural Fulani people in Plateau state and Gombe state where our studies were conducted tend to vary little from year to year. Nevertheless, a major limitation of the present study was that, since we did not analyze the diets of the Fulani subjects, we can only assume that their diets were similar to those of the Fulani men and women who participated in our previous studies in which dietary analysis was performed , 5. The et al. and Chardigny et al. recently reported that a daily intake of 10–12 g of ruminant TFAs is required to raise the plasma total cholesterol and LDL-C concentrations, which far exceeds our estimates of the amount of TFAs consumed by the Fulani pastoralists in northern Nigeria  sources contained in western diets do not contribute importantly to the risk of coronary heart disease . Our finFuture studies should be aimed at assessing the metabolic machinery of the Fulani population, particularly their ability to incorporate dietary TFAs into chylomicrons and to clear TFAs from the blood circulation. Comparative studies of the ability of Fulani and other ethnic groups to synthesize triglycerides and oxidize TFAs should also be encouraged.This study was supported by a Minority International Research Training grant from the National Institutes of Health, USA.


In animals mtDNA inheritance is maternal except in certain molluscan bivalve species which have a paternally inherited mitochondrial genome (genome M) along with the standard maternal one (genome F). Normally, the paternal genome occurs in the male gonad, but it can be often found, as a minority, in somatic tissues of males and females. This may happen in two ways. One is through "sperm mtDNA leakage" into somatic tissues, a deviation from the normal situation in which the sperm mtDNA vanishes in females or ends up exclusively in the germ line of males. The other is through "egg heteroplasmy", when the egg contains, in small quantities, the paternal genome in addition to maternal genome.M. galloprovincialis. Presence of the M genome was rarer in the foot of females than males. The M genome in the sperm and in the foot of males was identical.To test the two hypotheses, we compared the sequences of one of the most variable domains of the M molecule in a somatic tissue (foot) and in the sperm of ten male and in the foot of ten female individuals of Given that the surveyed region differs from individual to individual, the identity of the M genome in the foot and the sperm of males supports strongly the hypothesis that, at least for the tissue examined, the presence of the M genome is due to sperm mtDNA leakage. Mytilus species, where DUI has been studied extensively, the sperm carries on average five mitochondria , thus producing a profile that distinguishes this genome from the F and the M genomes.In addition to the F and M genomes, M genome ,25. ThesM genome , which iM genome ,24. We uTaq polymerase was used in all polymerase chain reactions (PCR). PCR amplifications were carried out in 25-50- μl reaction volumes containing ~50-100 ng of template DNA, 0.5-1 μM of each primer, 200 μM of each dNTP, 2-2.5 mM MgCl2, and 0.625-1.25 units Taq polymerase in the buffer supplied by the company. The reactions were heated initially at 94°C for 2 min and then incubated at 94°C for 20-35 sec, 56-58°C (depending on the Tm of the primer pair) for 30-35 sec and 72°C for 13-110 sec (depending on the length of the amplified product) for 35-40 cycles and 72°C for 5 min for a final extension. The amplified products were visualized on a 1% agarose gel.Promega Escherichia coli DH5α cells. Recombinant plasmid DNA from one positive clone from each transformation was isolated using standard procedure and sequenced commercially as mentioned above.DNA from PCR products was recovered using the PureLink Quick Gel Extraction Kit (Invitrogen) and the NucleoSpin Extract II PCR purification kit (Macherey-Nagel) according to the company's instructions. The purified PCR products were sequenced directly or after cloning from both directions using a commercial outlet . DNA fragments were cloned in pGEM-T vector (Promega) or pTZ57R/T vector (Fermentas) following the procedure provided by the supplier in The DNA sample of the foot of one male was accidentally damaged, thus this male was not entered in the sequence comparison. To have a comparable number of M sequences from both genders we returned to the females that did not spawn and amplified for the M genome in foot preparations. We got 12 positive reactions out of the 70 females that we screened for this purpose and sequenced the product from eight of them taken at random. This gave us a set of 30 M-type sequences for comparison: 10 from the foot of 10 different females, 10 from the foot of 10 different males and 10 from the sperm of these same males. All sequences obtained were deposited in GenBank under the Accession Numbers HM027601-HM027630.M. galloprovincialis/M. edulis currently available in the literature [The length of the obtained sequences varied from 658 to 693 bp. This is due to several indels at a poly(A) stretch that occur in the first variable domain VD1) of the control region (CR) of the M genome, specifically between nucleotide positions 207 and 232 (reference sequence AY363687 of the first complete M-type sequence reported ). This sterature ,26-28, c of the cSequences were aligned on the basis of nucleotide similarity using the default parameters of ClustalX v. 1.83 , followeMytilus galloprovincialis 36 spawned: 17 males and 19 females. Total DNA was isolated from the foot and sperm of these males and from the foot of females. The PCR assay for the C-genome was negative in all samples. All foot samples, from either female or male individuals, were positive for the F-genome, but no sperm sample tested positive for this genome. The M-specific test was positive in two female foot preparations out of 19 (or 10% of cases) and in 11 male foot preparations out of 17 (or 65%). The frequency of occurrence of the M genome in the foot tissue is statistically higher in males . This result agrees with that by Garrido-Ramos et al. [M. edulis. Further comparison of the two studies is not possible because of the different ways of detection of the M genome they used.Out of 424 individuals of s et al. , who obs16S-rRNA and the CD fragments are shown. The most striking observation is that in each of the ten males the sequence from the foot and the sequence from the sperm were identical. Among the 30 sequences one was encountered 5 times , one 4 times , one tree times , 7 twice each in a different male and 4 once each in a different female. This provides a rough estimate, the best we can have on the basis of our sample size, of haplotype frequencies under the hypothesis that the foot and sperm haplotype in a male are independently derived. For the hypothesis of egg heteroplasmy to be compatible with our data we would require that the male's father and the male's maternal grand-father happened to have the same sequence. The probability, p, that this may occur for any single male is p = (5/30)2 + (4/30)2 + (3/30)2 + 7(2/30)2 + 4(1/30)2 = 0.091. The probability, P, that this will happen for all ten males is P = (0.091)10 = 4 × 10-11, an extremely small number. We may confidently conclude that the presence of the M genome in the foot of males is due to sperm mtDNA leakage. On the basis of this conclusion, the set of independently derived sequences is reduced to 20. The new probability for obtaining the same sequence in two draws is h = 11(1/20)2 + 3(3/20)2 = 0.095. This is not very different from the one obtained when the two sequences in a male were assumed to be independently derived, and this is due to the high diversity of the VD1 region.To obtain an equal number of M-positive foot samples from both genders we extracted DNA from the foot of females that did not spawn (see Methods). The complete set of 30 sequences we obtained  are given in Additional file For females we cannot have direct evidence that the M genome found in the foot is that of their father because we do not have an independent way of knowing what the father's M genome was, as we do have in males. This could be done only in females with a known male parent. Even though several studies of DUI have used pair-matings ,31,32, nMytilus edulis  and of Mytilus galloprovincialis (in the second study) was treated with MitoTracker Green FM, a fluorescent stain that binds to the outer surface of mitochondria, and used to fertilize eggs. The sperm mitochondria were observed microscopically in the embryo up to the 4-cell stage . The eggs used in these experiments were derived from mothers that were known from previous studies to produce daughters either exclusively or in very high frequency (daughter-biased mothers) or from mothers that produced sons in high frequency (son-biased mothers). The researchers found that in embryos from daughter-biased mothers the sperm mitochondria dispersed randomly in both blastomeres at the 2-cell stage or in all four blastomeres at the 4-cell stage and that the distribution pattern was random. Thus, in female embryos, the sperm mitochondria appear to be randomly mixed with the egg mitochondria, which vastly outnumber them, and are either entirely lost or become barely detectable minorities in the somatic tissues of adult females. In contrast, in embryos from son-biased mothers the five sperm mitochondria formed a condensed mass that was found in the larger cell resulting from the first or second egg division. The researchers suggest that this is a developmental mechanism through which the sperm mtDNA is delivered in the male's primordial germ cells. Thus in males sperm mitochondria appear to follow a regimented developmental process, the result of which is that the male gonad becomes mainly or exclusively occupied by the sperm mtDNA (M-type), whereas the somatic tissues contain mainly the egg mtDNA (F-type). We say mainly, not wholly, because according to the results of several studies [Our results can be discussed in the context of the studies by Cao et al. , Obata & studies -19 the MOur study is the first to compare the M genome that is always found in the gonad of a male and the M genome that is occasionally found in the somatic tissues of the same male (but see ) and leaThe authors declare that they have no competing interests.EK carried out the experiments and drafted the manuscript. EZ and GCR supervised the work, participated in the experimental design and helped in manuscript preparation. All authors contributed to data interpretation and analysis, and have read and approved the manuscript.The M-type sequences found in the foot (m_ft) and the sperm (m_sp) of ten males and the foot of ten females (f_ft). The sequences include the full length of VD1 and only the nucleotide of the variable sites of the amplified parts of the 16S-rRNA gene and CD. The A-rich stretch of VD1 is highlighted. Dots imply nucleotide identity with the sequence given at the top and dashes indicate deletions. The alignment position numbers are given above the sequence.Click here for file


The clinical study included 30 patients with complicated cutaneous haemangioma . The patients were studied regarding the age group, sex, site of lesion, size of lesion, and the percentage of regression after treatment with steroid.The age ranged from three months to six years, there were 20 female patients and 10 male patients. We used local injection of diluted triamcinolone 4 mg with 5 ml. 0.9% NACI , injected through 23-guage syringe under local or general anaesthesia every two weeks for six to eight sessions depending on the severity of the case, followed by a local pressure dressing. We measured the size of the lesion before each session and recorded the regression of the lesion. The patients were followed up for two years. Haemangioma commonly presents in infants and children, most commonly in females, especially in the head and neck and are usually of a small size. It regresses if the treatment is started earlier. Haemangiomas are immature rests of vasoformative tissue that demonstrate angioblastic proliferation and regression and represent the most common vascular tumour of childhood. InfantilThirty patients with complicated haemangioma  of different age groups collected through simple random sampling in outpatient cases were treated with local injection of diluted triamcinolone 4 mg with 5 ml normal saline in multiple sessions, two weeks apart Figure ,b. The aOf the 30 patients that visited the outpatient clinic, 20 were females while 10 patients were males.The age of presentation to the clinic was: 10 patients < one year, 10 patients from one year - < four years, and 10 patients from four years - < six years.The most common site of haemangioma in these patients was head and neck in about 51%, the second most common site was the trunk (33%), followed by the extremities (13%), and the genitalia (3%).We found also that 10 patients the size of cutaneous hemangioma was (1-2cm), 8 patients was (2-3cm), 7 patients was (3-4cm),& 5 patients was (4-5cm), as shown in the After treatment we found that the 60% of the cutaneous haemangiomas regressed in the age group between one-<two years old, in the two to four years age group, they regressed in about 40%, and in >six years old, the regression was seen in about 25% as shown in We found that only three patients suffered from complication of steroid treatment, moon facies in two and systemic infection in one.et al., who found that 65.3% were infants and children.[In 30 patients with cutaneous haemangioma, we found that the most common age group presenting with haemangiomas was that of infants and children (three months - six years). This deduction agrees with the study of Winter The female to male ratio was 2:1, and is similar to the study of Garzon M who found that haemangiomas occur in females three times more than males.Haemangiomas presented in the head and neck region in about 51%, trunk (33%), extremities (13%), and genitalia (3%). This agrees with the study conducted by Mullkin and Glowacki, who found that 60% of haemangiomas are in the head and neck area, 25% in the trunk, and 15% in the extremities.Like the Garzon study we also et al., who found that the age of initiation of treatment is the most important factor affecting the response to treatment.[After treatment with local steroid the percentage of regression was found to be 60% when we started the treatment in the early period (three months - <two years), 40% from two - <four years and 25% from four – six years. This is similar to the study of Akyus reatment.


This work describes the immobilization of purine and pyrimidine bases and immobilization/hybridization of synthetic oligonucleotides on graphite electrodes modified with poly(4-methoxyphenethylamine) produced in acid medium. The immobilization of adenine, guanine, cytosine and thymine on these modified electrodes was efficient, producing characteristic peaks. Another relevant observation is that, according to the literature, pyrimidine bases, cytosine and thymine are more difficult to detect. However, when immobilized onto the poly(4-methoxyphenethylamine), a significant increase in the magnitude of the current was obtained. The observation of the hybridization between the poly(GA) probe and its complementary, poly(CT) target, was possible by monitoring the guanosine and adenosine peaks or through methylene blue indicator, using differential pulse voltammetry. Hybridization results in a decrease of the peak current of guanosine and adenosine or the signal of methylene blue accumulated on the modified electrode surface. The hybridization with the complementary target was also investigated by electrochemical impedance spectroscopy. The results showed a significant modification in the Nyquist plot, after addition of the complementary target, with increase of the charge transference resistance. Electrodes chemically modified by polymeric films have attracted considerable interest over the past two decades due to their potential application in electrochromic devices, electrocatalysis, electroanalysis and in the design of molecular electronic devices –4. ElectThe manipulation of the molecular composition of the electrode aims at improving sensitivity, selectivity and/or stability allowing for tailoring its response to meet analytical needs .Electropolymerization of aromatic compounds has been investigated –9. PreviThe immobilization of biomolecular probes on an appropriate substrate is an important process since the sensitivity, the detection resolution and the reproducibility are significantly affected by the immobilization. In general, the surface of the substrate and/or terminus of DNA has been modified for stable immobilization of DNA probes .in situ preparation of electrodes coated with a conducting film of poly(4-aminophenol) ) equivalent circuit where Rs describes the solution resistance, Rct the charge transfer resistance, Q1 and Q2 represent the double layer capacitance (due to the interface between the electrode and the electrolytic solution), ZT represents the transmissive impedance. Studies were carried at a potential of +0.4V for electrode modified with polymeric film and + 0.9V for electrode modified with polymeric film with biomolecules immobilized at the surface.The experimental data were fitted to a RAs shown in The electrochemical behavior of the graphite electrode electrochemically treated in a 4-methoxyphenethylamine solution (pH 0.5) is shown in λ: +0.7V).The main oxidation peaks of the graphite electrode modified in a solution of 4-methoxyphenethylamine are located at +0.45V and +0.65V while the reduction peaks are observed at +0.25V and +0.38V. This result indicates the formation of an electroactive polymer derived from 4-methoxyphenethylamine onto the graphite surface. The peaks separation does not mean that the electron transfer is irreversible but instead, is the consequence of a certain degree of resistance of the polymer as supported by the slopping current at the switching potential  is shown in The cyclic voltammogram, in 0.1 mol L4 solution , prepared in acid medium, presents an increase in the electrochemical response of the redox pair, when compared to the bare graphite electrode, indicating the modification of the graphite electrode, in agreement with the results of the modified electrode analyzed in HClOsolution . A signiPurines: adenine and guanine, and pyrimidines, cytosine and thymine, were immobilized applying drops at room temperature onto the surface of modified electrodes prepared in acid pH .Guanine, adenine, thymine and cytosine irreversibly oxidize on the modified graphite to produce oxidation peaks in acetate buffer.The oxidation potentials of the immobilized bases onto the modified electrode are gathered in The pyrimidine bases, cytosine and thymine are more difficult to detect . HoweverIt is well known that large biomolecules can bind to polymers by adsorption . ImmobilHeterooligomers of two DNA nucleobases present a significant electrochemical response. The immobilization of a 16-mer DNA sequence on a graphite electrode without film and after modification with poly(4-methoxyphenethylamine) is shown in The hybridization experiments were carried out in incubated solutions containing the complementary oligonucleotide. The peak currents of guanosine and adenine gradually decreased after 15 minutes of incubation in phosphate or acetate buffer, in agreement with Oliveira-Brett and col. , who repAnother reason to the higher current values obtained for single strand DNA is that the latter presents higher proximity and a higher degree of adsorption onto the electrode surface, due to its higher conformational flexibility , facilitOthers experiments were carried out using an electrode modified with film/poly(GA) and an electrode modified with film/poly(GA):poly(CT) as transducers and methylene blue as electroactive indicator .Methylene blue interacts in different forms with the simple strand DNA and the double strand DNA, therefore governing its signal of reduction. This fact reflects the hybridization extension with the immobilized probe . EvidencThe decrease in the values of the reduction current is governed by the specific interactions between methylene blue and free guanine, with lesser accessibility of methylene blue to guanine in hybridized DNA . The eleThe hybridization effect was also characterized using other techniques besides cyclic and differential pulse voltammetry. Electrochemical impedance spectroscopy (EIS) is proposed as the transduction principle for the detection of DNA complementary strands .T component.−1 phosphate buffer.−2–10−3 attest the good quality of the data. Q is defined as the double layer capacitance, Y0 is defined as the Q absolute value, n1, n2 and B is the phase angle displacement.The chi-square values of the Kramers-Kronig test of the order of 10s) is reasonably constant, centered around 14.3 ± 1.3. This is a reasonable value considering the electrolyte composition. The slight dispersion of the Rs–values is acceptable and is obviously due to the difficulty to position the electrodes at exactly the same distance between experiments.Some interesting features came out of the data gathered in 1-values close 0.5 of the Q1R1 combination supports Q1 behaves very similarly to a faradaic behavior. The n2-values values present values between 0.5 and 1.0 suggesting a behavior between capacitance and Warburg.Two (QR) serial combinations, with an additional T component for the film/poly(GA):poly(CT) system, were required to fit the results, suggesting that two different film regions contribute to the impedance response. To take into account the frequency dispersion, due to roughness/porosity of the systems, it was necessary to use two constant phase elements, Q, instead of pure capacitor elements. However, the n1 and Q2 values for the film+poly(GA) and film+poly(GA):poly(CT) with the data for the film supports an increase in the surface area after the application of poly(GA) and poly(GA):poly(CT). The application of the latter, however, results in approximately 10 to 15× higher enhancement. This is a reasonable result. The model for the morphology of the electroactive layer that emerges from the results supports the existence of two regions: a more internal region, close to the graphite surface, described by the R1 and Q1 data, and a more external region after the poly(CT) is applied described by the Q2 R2 data. The modest increase in Q1 is due the presence of poly(GA) and its rather similar value with the Q1-value for poly(GA):poly(CT) suggests that the poly(GA) strand is adsorbed closely to but not parallel to the graphite surface. The significant increase of Q2 compared to Q1 suggests that the CT strand is adsorbed, with its elements in close contact with the poly(GA) strand (hybridization), increasing the distance of the double strand formed with the electrode surface, due to the lower conformational flexibility, resulting in a decrease of the charge transfer  increases the R2ct compared to the R1ct-value, consistent with the oxidation of more distant oxidable sites.Comparison of the Qsfer see . This coThe data presented in this work showed that poly(4-methoxyphenethylamine) is an efficient matrix for purines and pyrimidines immobilization. Differential pulse voltammetry and electrochemical impedance spectroscopy have been used to characterize the immobilization and hybridization process of nitrogenated bases and/or oligonucleotide.All DNA bases are electroactive and give a significant electrochemical response on modified electrodes, in the absence  or in the presence (oligonucleotide) of phosphate groups onto a graphite electrode modified with polymeric films.The results support the oligonucleotide hybridization with the complementary target. Additionally, the interaction between DNA molecules and small biomolecules (methylene blue) can be observed through differential pulse voltammetry and electrochemical impedance spectroscopy. Based on the response of the indicator, the complementary DNA sequence produces a decrease in the current signal, result in agreement with the literature. The oligonucleotide hybridization with the complementary target, analyzed by electrochemical impedance spectroscopy, showed a significant modification in the Nyquist plot upon addition of the complementary target with increase of the charge transference resistance.The combination of a graphite electrode with poly(4-methoxyphenethylamine) is a promising strategy for the DNA probe immobilization and other biological recognition elements.