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Neisseria meningitidis is one of the most frequent causes of meningitis and septicemia worldwide [1, 2], being not only responsible for 1020% of specific meningococcal - related mortality but also the cause of (particularly pediatric) long - term morbidity as it leads to permanent neurological sequelae and disabilities in an additional 20% [35]. Furthermore, the pediatric mortality rate among children with sepsis is over 20% [6, 7]. The incidence of meningococcal meningitis is greatest amongst children, adolescents, and adults aged up to 29 years, but young children are the most susceptible . The risk of n. meningitidis infection is particularly high in some regions of the world but, despite the introduction of innovations in health care, morbidity and mortality rates are high in both developed and undeveloped countries, and prevention is therefore a priority . The bacteria colonise the nasopharyngeal tract of human hosts and are spread from subject to subject via air droplets . Transmission rates vary and are also related to individual risk factors such as age and/or underlying medical and social conditions (e.g., primary or secondary immunodeficiencies, a history of travel, and overcrowded living condition). Twelve different serogroups are known, but most invasive meningococcal diseases are caused by one of the six capsular groups a, b, c, w, x, and y. a number of excellent conjugate vaccines against serogroups a, c, w, and y have been licensed, and the introduction of conjugate meningococcal c vaccine (menc) has led to a rapid and sustained reduction in the incidence of invasive menc disease across all age groups in italy [8, 9]. However, a vaccine against capsular group b (menb), which has now become responsible for most cases in italy and the rest of the world [7, 9], has long eluded vaccinologists, particularly because of the problems associated with the b polysaccharide [1012]. Unlike the highly immunogenic polysaccharides of serogroups a, c, w, and y, the serogroup b polysaccharidic capsule contains a polysialic acid whose antigenic structure resembles the cell surface glycoproteins of human neurological tissue, and this has proved to be a formidable challenge . The new protein - based vaccine against menb (4cmenb; bexsero, novartis vaccines and diagnostics, siena, italy) has now overcome this barrier by using a cocktail of four main immunogenic components: two recombinant fusion proteins (neisseria heparin - binding antigen [nhba - gna1030] and factor h binding protein [fhbp - gna2091]), recombinant neisseria adhesion a (nada), and detergent - treated outer membrane vesicles (omvs) derived from the nz98/254 new zealand meningococcal outbreak strain in which porin a (pora 1.4) is the major immunodominant antigen . These components were identified using reverse vaccinology, a technique that analyses the whole bacterial genome in order to predict meningococcal antigens (exposed on the pathogen's surface or secreted) that can act as vaccine targets . Nhba is a surface b - barrel lipoprotein that binds to the anticoagulant heparin and induces protective immunity in human hosts [1517], and fhbp is a surface - exposed protein that allows binding exclusively to human fh, mediates host serum resistance, and induces bactericidal antibodies upon host detection [15, 19]. Nada is a surface adhesin and invasin whose interactions with abundantly expressed human heat shock protein 90 (hsp90) also induce bactericidal antibodies [21, 22]. Finally, pora (one of the two b - barrel porin proteins produced by n. meningitides) assists in opsonophagocytic activity and is also involved in host actin reorganisation during infection, which depends on its ability to nucleate actin filaments . This four - component meningococcal serogroup b vaccine (4cmenb), the first successful vaccine against the endemic form of this cause of serious bacterial meningitis and septicemia, has been in development for almost 20 years and has recently been approved for the active immunisation of subjects aged 2 months [9, 10] by licensing authorities in europe, canada, and australia . A bivalent fhbp recombinant vaccine (also known as lp2086; trumenba, pfizer inc ., philadelphia, pa, usa) has been developed since 2006 and has now been approved by the us food and drug administration for use in 10-to-25-year olds . This vaccine appeared safe in a phase 3 study in approximately 5,600 healthy individuals 10 to 25 years of age and immunogenic and safe when coadministered with routine meningococcal a, c, y, and w and tetanus, diphtheria, and pertussis (tdap) vaccines in a phase 2 study in more than 2,600 healthy individuals 10 to 12 years of age . Studies on this vaccine are ongoing in europe and approval from european medicines agency is expected in 2017 . The aim of this review is to discuss the immunogenicity, safety, and tolerability of 4cmenb vaccine in infants and toddlers, and the efficacy of different vaccination strategies . An important challenge for the licensing of 4cmenb vaccine was the difficulty in showing its activity against epidemiologically relevant strains of n. meningitidis [27, 28]. After its development and phases i and ii trials, some large - scale randomised phase iii studies were planned in order to assess its efficacy and describe adverse reactions, but due to the rarity of the diseases caused by n. meningitides serotype b (which have annual rates of 0.55 per 100,000 people) laboratory - based methods were developed with the aim of predicting the vaccine's effectiveness and coverage . In europe, the vaccine was licensed on the basis of a correlate of protection calculated using a titre of human serum bactericidal activity (hsba) that is present in convalescent patients which was shown to be protective in us army recruits . Hsba assay is a recognised in vitro surrogate for evaluating protective immunity against n. meningitidis, and an adequate response is a crucial criterion for licensing vaccines against serogroup b meningococci . In order to justify the inclusion of each antigen in the formulation, it is necessary to run four hsba assays on each serum sample, each using a n. meningitidis strain expressing the target antigen independently from the others in order to evaluate immunogenicity of each component of the vaccine, for example, a meningococcal strain that uniquely expresses nada but not factor - h - binding protein or neisseria heparin - binding antigen . Subsequent phase iii studies of 4cmenb in children used a more conservative titre of 5, which ensures that the level is> 4 with 95% confidence taking into account within - assay variability . The safety and immunogenicity of 4cmenb vaccine has been studied when administered at the same time as other routine infant vaccines (diphtheria, tetanus, acellular pertussis, inactivated poliovirus, hepatitis b, haemophilus influenzae type b [dtap - ipv - hepb / hib], and 7-valent pneumococcal conjugate vaccine [pcv7]), and it has been found that the antibody responses to the routine vaccines are equivalent to those observed when the routine vaccines are given alone in the case of all of the antigens except for the pertactin component of acellular pertussis and pneumococcal serotype 6b . However, this laboratory observation seems to be of no clinical significance, and published data also suggest that the incidence of pneumococcal disease due to serotype 6b is low in the countries in which pcv7 vaccination is used . Other studies have investigated the persistence of bactericidal antibodies in young children after primary immunisation and the level of immunogenicity after a preschool booster [29, 30]. The levels of bactericidal antibodies after primary 4cmenb vaccination at the ages of two, four, six, and 12 months had waned when measured at 4044 months, but an anamnestic response was observed following a booster dose given at the age of 4044 months . Similarly, bactericidal antibody levels in infants who originally received 4cmenb during late infancy (6, 8, and 12 months) had also waned when measured at the age of 40 months but, once again, there was an anamnestic response to a booster dose given at 40 months . The participating infants were observed for 30 minutes after each vaccine administration, and their parents were given a diary card on which to record the occurrence and severity of solicited local (i.e., injection site tenderness, erythema, induration, and swelling) and systemic reactions (i.e., changes in eating habits, sleepiness, vomiting, diarrhea, irritability, unusual crying, rash, and increased / decreased body temperature) and any other adverse events, during the following seven days . The rates of local and systemic reactions were similar to those seen following other routine infant and early childhood vaccinations, but injection site pain was consistently reported more frequently, especially by older children . Fever was more frequent in the children who received 4cmenb together with other routine infant vaccines . It mainly occurred during the first 24 hours after administration but, as in case of other vaccines, it has been found that the prophylactic administration of paracetamol before and 46 hours after vaccination significantly reduces postvaccination fever without affecting immunological responses [31, 32]. The pivotal and phase iib studies found that the most frequently reported local reaction of tenderness affected 87% of the 4cmenb injection sites, 80% of the dtap- ipv - hepb / hib sites, and 79% of the pcv7 sites when all three vaccines were administered together . The frequency of reported tenderness after dtap - ipv - hepb / hib and pcv7 injections when they were administered without 4cmenb was respectively 59% and 53%, whereas when dtap - ipv - hepb / hib and pcv7 injections were administered with 4cmenb it was respectively 68% and 62% . The reported rates of local reactions to 4cmenb were slightly higher than those related to routine vaccines, but the majority were transient, most intense on the day after vaccination, and resolved within a week . Although the systemic reactions that occurred when 4cmenb was administered concomitantly with routine vaccines cannot be specifically attributed to one or other of the vaccines, it is possible to assess the overall profile . The occurrence of 4cmenb - related seizures is rare: the combined data of infant studies including> 20,000 vaccinations in the primary 4cmenb study arm indicate an overall rate 0.1 febrile seizures/1000 vaccinations on the day of vaccination or the day after and no events in the control arm . They are similarly rare in toddlers: 0.4 events/1000 vaccinations (95% confidence interval (ci): 0.051.46) after a total of 11,000 4cmenb vaccinations administered with or without routine vaccines, as against 0.3 event/1000 visits (95% ci: 0.041.05) in the case of those receiving routine vaccines alone [10, 33]. Six suspected cases of kawasaki disease reported during the course of two infant studies (four in the pivotal trial and two in the phase iib study) were evaluated by an independent external expert panel in order to assess whether they were true kawasaki cases and whether they were vaccine related . Analysis of the kawasaki cases indicates an annual incidence of 72/100,000 person - years (95% ci: 23169) after 4cmenb vaccinations, as against 56/100,000 person - years (95% ci: 1311) after routine vaccinations alone . The overall data from different studies indicate that the frequency of febrile seizures, the incidence of kawasaki disease, and the proportion of infants using antipyretics are similar to those observed during clinical licensure programmes . However, as the number of exposed infants is still too small to exclude any relationship with rare adverse events, further postmarketing surveillance is necessary . The new 4cmenb vaccine may not protect against all invasive meningococcal b strains because the antigens included in the vaccine are expressed by only some of the strains in circulation . However, it is not known what protection 4cmenb vaccine provides against invasive meningococcal disease (imd) because it depends on the vaccine antigens expressed by the meningococcal strains in any given geographical area and their cross - reactivity with the antigens included in the vaccine . Epidemiological and microbiological data regarding the circulating meningococcal strains are important in order to predict the theoretical coverage provided by 4cmenb vaccine and assess its impact on disease burden . Further postmarketing surveillance will allow a more precise estimate of the effectiveness of 4cmenb . However, although an hsba can be used to demonstrate whether the vaccine induces antibodies capable of killing meningococcal strains, the presence of four antigens means that it is more complex than in the case of other meningococcal vaccines (i.e., menc). Moreover, the genetic diversity of serogroup b strains means that not all of them have the genes coding for each of the antigens, and their expression may vary over time or from place to place . For these reasons, the meningococcal antigen typing system (mats) is used to measure bacterial antigen expression in order to predict whether bactericidal serum is capable of killing particular strains [3436]. This method is characterised by both phenotypic and genotypic analyses: the expression of the individual antigens that cross - react with the corresponding vaccine antigen is quantified using polyclonal antibodies against nhba, nada, and fhbp in an enzyme - linked immunosorbent assay (elisa), and dna sequence homology to the variable region sequence of the vaccine strain pora gene is assessed, in order to estimate coverage in a specific region [32, 33]. It has not yet been proved that there is a correlation between the mats results and real vaccination coverage, but the predicted protection based on the expression of at least one matched antigen ranges from 73% to 87% . The mats has been applied to isolates of 1,052 menb strains causing imd in europe submitted to reference laboratories in france, germany, italy, norway, and the uk between 2007 and 2008, and the analysis demonstrated estimated efficacy values ranging from 73% in the uk to 87% in italy . Furthermore, a study conducted in canada between 2006 and 2009 analysed 157 menb isolates collected from children and adults with imd and found that the potential coverage of 4cmenb vaccine was 7590% . On the basis of the mats elisa findings, the authors predicted that 66% of the circulating strains were covered by at least one vaccine antigen although none were covered by all four . A new meningococcal serotype x has recently been isolated in africa, against which no vaccine is currently available . However, some authors have recently used the mats and bactericidal assays of 11 serogroup x isolates taken from nine african and two french patients and found that 4cmenb vaccine could have a good coverage against the strains from africa but not those from france . In regions where meningococcal strains are appropriately monitored, the mats can evaluate the real effectiveness of 4cmenb vaccine, and the development of changes in the menb serogroup over time . It is also a very useful means of monitoring the emergence of new menb mutants due to selective vaccination pressure . Although the development of a meningococcal b serogroup vaccine was slow and difficult, the new bexsero 4cmenb vaccine has recently been licensed in the eu, australia, and canada . The introduction of any new vaccine is never easy, but it is especially complex in this case . The success of a vaccination programme is based on both cost - effectiveness and public acceptance and although meningococcal b infection is a cause for concern in the general population, the acceptability of the vaccine by parents is influenced by worries concerning its potential side effects, its real effectiveness, and the consequences of its coadministration with other routine vaccines in terms of the number of injections and possible immunological interference . For example, its acceptance may be reduced by the fact that it has been associated with increased rates of fever when coadministered with the routine vaccinations provided during infancy on the basis of the national immunisation schedules . One recent study of parental attitudes to 4cmenb showed that 82.5% of the interviewees wanted their children vaccinated . The most frequent concerns were side effects including fever (41.3%) and adequate vaccine testing (11.7%), but 26% of the parents said that they had no concerns . Moreover, as in the case of other vaccinations (e.g., hpv), the authors found that most parents (81.7%) were more likely to accept the vaccination if their immunisation providers recommended it . The vaccination schedule should take into account the age of the subjects most frequently affected by meningococcal disease and the epidemiology of meningococcal infections . One recent study aimed at defining the optimal age for administering 4cmenb to children has shown that the incidence is highest in those aged <5 years (particularly those in their in the first year of life, when deaths are more frequent) and, on the basis of this finding, the authors suggested that vaccination should be started in the first year of life, with a catch - up dose being given at the age of five years . Other recent studies have evaluated the cost - effectiveness of a potential menb vaccination programme [43, 44]. One study carried out in the netherlands estimated that an infant menb vaccination programme would prevent 14% of cases over the lifetime of a birth cohort and concluded that this was not cost - effective and although another study carried out in the uk estimated that routine vaccination would prevent 2756% of cases over the lifetime of a birth cohort, the authors also considered it not cost - effective . Finally, a recent study of the economic impact of menb vaccination in canada found that the menb vaccination programme exceeds the generally cost - effectiveness thresholds and therefore should not be considered economically advantageous . Nevertheless, 4cmenb is now being used in canada and, upon parents' request, also in all of the countries and it has been licensed and its use in at - risk populations has been implemented . It has also been announced that it may be introduced into the uk's routine infant immunisation schedule using a 2 + 1 regimen although only a 3 + 1 regimen with the concomitant use of paracetamol is currently licensed, and some other countries are reconsidering their cost - effectiveness calculations by also considering its possible impact on carrier status . With the availability of the bivalent rlp2086 vaccine for the adolescent age, it will be important to compare vaccination strategies that cover different age groups as well as to understand the impact of the two vaccines against imd overall, meningococcal disease due to serogroups different from menb and meningococcal carriage in the nasopharynx . N. meningitidis is still one of the major causes of sepsis and meningitis among children worldwide and is associated with a high mortality rate . Considerable efforts have therefore been made to prevent meningococcal disease by means of vaccination, and two effective conjugate vaccines have recently been licensed and led to good results (men c and menacyw). However, it proved to be very difficult to develop a vaccine against serogroup b because of the poor immunogenicity of its capsular polysaccharide, even when conjugated with a carrier that could also induce an autoimmune response, until experts used reverse vaccinology to identify appropriate antigenic recombinant proteins . The overall findings of various studies have shown that the administration of three doses of 4cmenb to young children (alone or with routine vaccines) does not interfere with immune responses, and most have found that its safety and tolerability are acceptable . However, its coadministration with other vaccines does lead to increased reactogenicity (particularly fever) and so such coadministration should be combined with paracetamol given both before and after vaccination . The new 4cmenb vaccine represents an important opportunity to fight pediatric imds, but its introduction should take into account the need to maintain the appropriate use of meningococcal conjugate vaccines that cover serogroups other than b, community opinion, and cost - effectiveness data . Moreover, it will be important to compare 4cmenb potential efficacy with that of bivalent rlp2086 vaccine . Finally, it is very important to continue surveillance in order to monitor the emergence of new meningococcal b strains in order to identify any that are not susceptible to 4cmenb.
Hepatitis c virus (hcv) infection is a leading cause of end - stage liver disease and hepatocellular carcinoma [1, 2]. Hcv - related liver disease is the most common indication for orthotopic liver transplantation in the usa and northern europe and for living donor liver transplantation (ldlt) in japan [3, 4, 5]. Unfortunately, liver transplantation is not a cure for hcv infection, and the occurrence of graft reinfection with hcv is universal, leading to progression of liver fibrosis and occasionally to graft loss at rates higher than in transplant patients not infected with hcv [5, 6]. In japan, because of long - standing legal difficulties associated with cadaveric donation, ldlt is the main type of liver transplantation for end - stage liver disease . The sustained virologic response (svr) rate was 40% in donors of ldlt treated with pegylated interferon (peginterferon) plus ribavirin, although adverse events were commonly observed even in nontransplanted patients receiving this treatment . Reported that the dose reduction rate and the discontinuation rate of peginterferon plus ribavirin treatment was 40 and 42%, respectively . Recent progress in the development of direct - acting antivirals (daas) against hcv has made it possible to eradicate hcv effectively and to shorten the treatment duration compared to the previous standard of care, i.e. Peginterferon plus ribavirin . Treatment with the hcv ns5b polymerase inhibitor sofosbuvir plus ribavirin for 12 weeks was shown to lead to high rates of svr (97%) in japanese patients infected with chronic hcv genotype 2 . Asian - pacific countries, including japan, account for 50% of all chronic hepatitis b virus (hbv) infection globally . Given the prevalence of hbv, ldlt from hepatitis b core antibody - positive donors to recipients was occasionally performed with passive immunization with hyperimmune hepatitis b immunoglobulin (hbig) plus nucleos(t)ide analogs . Here, we report on an ldlt recipient who was reinfected with hcv genotype 2a after receiving a graft from a hepatitis b core antibody - positive donor . Hcv genotype 2a was eradicated by a 12-week treatment with sofosbuvir plus ribavirin with hbig plus entecavir, one of the nucleos(t)ide analogs, for the successful prevention of hbv reappearance . A 66-year - old japanese woman developed liver failure due to cirrhosis and hcv genotype 2a infection . She was a treatment - nave patient, but her il28b rs8099917 was a favorable genotype (tt). Two years prior to ldlt, she has been diagnosed with liver cirrhosis due to hcv infection without liver biopsy . She had been infected with hcv after having received a blood transfusion during childbirth at the age of 30 years . At ldlt, she had peripheral edema with a meld (model for end - stage liver disease) score of 12 . Ldlt with a right liver graft from a hepatitis b core antibody - positive donor was performed in february 2015 . Five months after ldlt, the hcv rna level was 5.8 log iu / ml, and she was diagnosed with graft reinfection with hcv genotype 2a . Combination treatment with 400 mg daily of sofosbuvir and 400 mg daily of ribavirin was commenced . Her height, body weight, and body mass index were 1.56 m, 50 kg, and 20.5, respectively . Her laboratory data before treatment are shown in table 1 . The hcv rna level before treatment was 5.8 log iu / ml . After ldlt, she received triple immunosuppressive therapy consisting of tacrolimus (3 mg daily), mycophenolate mofetil, and basiliximab with passive immunization with hbig and 0.5 mg daily of entecavir . Four weeks after initiating the combination treatment with sofosbuvir and ribavirin, hcv rna levels were undetectable . She completed this treatment for 12 weeks and achieved svr at 24 weeks following the termination of this treatment (svr24) (fig . 1). There was no evidence of hbv reactivation . Because her hemoglobin level was 12.2 g / dl before the commencement of this treatment, an oral iron preparation was also started . After 2 months of treatment, her hemoglobin level fell to 10.8 mg / dl . Then, the dose of ribavirin was decreased to 200 mg daily, and her hemoglobin level improved to 12.8 g / dl (fig ., she did not develop evidence of bone marrow suppression, such as observed in peginterferon - plus - ribavirin treatment . No serious adverse events were observed . During treatment, the trough level of tacrolimus remained stable . We presented a female patient with living donor - related graft reinfection with hcv genotype 2a who was treated with a combination of sofosbuvir and ribavirin for 12 weeks . Although several daas have drug - drug interactions, no changes in this patient's drug protocol, which included immunosuppressants and combination treatment of sofosbuvir plus ribavirin, were necessary in this case . Peginterferon plus ribavirin with or without daas may be attempted, but the use of peginterferon is restricted by severe side effects and inadequate efficacy . In addition, interferon has an immune - mediated cytotoxicity, occasionally causing allograft dysfunction . Interferon - free therapy is a viable treatment option and improves treatment efficacy [15, 16, 17, 18, 19, 20]. This case suggests that daas are well tolerated and effective for the eradication of hcv from post - liver transplantation patients . In the present case of ldlt, graft reinfection with hcv genotype 2 the patient achieved svr24, although she had anemia as an adverse event, and anti - hcv treatment was continued without blood transfusion . Curry et al . Reported that common adverse events of combination treatment with sofosbuvir and ribavirin were fatigue (in 38% of patients), headache (23%), and anemia (21%) after liver transplantation . Charlton et al . Reported that the most common adverse events were fatigue (30%), diarrhea (28%), headache (25%), and anemia (20%) during a 24-week combination treatment course of sofosbuvir plus ribavirin after liver transplantation . In the present study, the patient received a liver graft from a hepatitis b core antibody - positive donor, and, in general, hbv from these donors is transmitted to recipients at a high rate . Therefore, passive immunization with hbig plus treatment with entecavir were provided to prevent hbv reactivation . Curing hcv infection with daas in hbv / hcv coinfection and monitoring for hbv reactivation should be performed, because daas against hcv do not have any effect on hbv replication, unlike interferon . In the present case, the patient was treated with hbig and entecavir, and there was no evidence of hbv reactivation . Recently, more effective regimens with daas against hcv have been reported [22, 23]. These regimens may make it possible to shorten the duration of treatment and to make it easier to achieve svr . The use of interferon - free regimens is possible for the eradication of hcv in post - ldlt patients with grafts from hepatitis b core antibody - positive donors, as was demonstrated by our patient, who was successfully treated with hbig and entecavir to prevent hbv reactivation . In conclusion, 12-week treatment with sofosbuvir plus ribavirin is relatively safe and highly effective for the eradication of hcv genotype 2 in ldlt patients . The other authors declare that there is no conflict of interest regarding the publication of this manuscript.
Cutaneous leishmaniasis (cl) is endemic in more than 70 countries in the world (1). The available drugs for leishmaniasis treatment are expensive and toxic, cause to severe side effects and there is an increasing incidence of drug resistance (2, 3). Therefore there is a need for an effective vaccine to control and prevent from this disease . However, in spite of intensive efforts during the past decades, only a few first vaccines generation of whole killed leishmania reached to phase 3 of clinical trials . It seems that the reason of this failure is mainly due to lack of an appropriate adjuvant (2). An inoculation of live virulent parasites known as leishmanization (lz) was practiced in several countries including iran and uzbekistan, due to safety concerns and difficulties in standardization of the injected parasites, lz practice was stopped in different countries except in uzbekistan (5, 6). A large number of adjutants and delivery systems like bcg, g - csf, il-12, cpg oligonucleotides, plga microspheres and liposomes have been used to potentiate the immune response against leishmania antigens in animal models (3, 4, 6 - 8). Mycobacterium bovis -bacillus calmette - gurin (bcg) has been used in field trial as an immunoadjuvant against different forms of leishmaniasis . Vaccination with autoclaved l. major (alm) mixed with bcg was found to be safe but did not induce significant protective immune response in healthy volunteers against cutaneous leishmaniasis (11, 12); furthermore in several studies m. bovis bcg inoculation induced autoimmune reactions (13). The only vaccine adjuvant that is approved by fda is alum (aluminum - based mineral salt) (14). The major limitations of alum is its poor inducer to elicit cell - mediated immunity and t helper 1 (th1) responses that are required to protect against intracellular pathogens (15, 16). Opioids have significant role in the modulation of th 1/th 2 balances (17). Naltrexone (ntx), as opioid antagonist, is capable to block -opioid receptors and reduces the positive reinforcing effects of opioids (19, 20). Naltrexone can enhance lymphocyte proliferation and shift the immune response toward a th1 pattern (21). Furthermore, that ntx reduce viral replication and inhibit tumor growth (19, 22, 23). Ntx is a long - acting opioid receptor antagonist that is approved by the fda as a prescription drug and is widely used for treating alcohol and opiate addiction (20). In the current study, we tested the immunogenicity potential of naltrexone alone or in mixture with alum as an adjuvant against heat - killed l. major promastigotes in the susceptible balb / c mice . Female balb / c mice (68 weeks old) were purchased from the razi vaccine and serum research institute of iran . Experiments were performed in accordance with the animal care and use protocol of urmia university of medical sciences . The l. major strain mrho / ir/75/er was provided from school of public health, university of tehran . Promastigotes of l. major were harvested from stationary growth phase by centrifugation (2000 g, 20 min, 4c), washed 3 times with cold pbs (ph 7.2) and homogenized with glass - glass homogenizer followed by autoclaving . Total protein content of the autoclaved antigen was determined by bradford method (each dose containing 40 g / ml of protein parasite). Female balb / c mice were divided into five groups (10 mice per group). Each group was divided to two subgroups, five mice in each subgroup: one subgroup was evaluated for lymphocyte proliferation and shift the immune response to th1/th2 and the second subgroup was assessed for delayed type hypersensitivity (dth) and challenge by the parasite.the alum ntx mixture was prepared by thoroughly mixing 50 l of pbs containing neltrexone (sigma, germany) at a concentration of 6 mg/ kg with 50 l of alum (aluminum phosphate, sigma). Balb / c mice were immunized subcutaneously three times at 2-week intervals with one of the followings: group vac (50 l ag + 100 l pbs /mouse), group al vac (50 l ag + 50 l al + 50 l pbs /mouse), group ntx vac (50 l ag + 50 l ntx + 50 l pbs /mouse), group al - ntx vac (50 l ag + 50 l al + 50 l ntx /mouse) and control mice received 150 l of pbs . Two weeks after the last immunization, the spleens were aseptically removed (five mice from each group) and separately homogenized in incomplete rpmi 1640 (gibco - brl). Erythrocytes were lysed with ammonium chloride (0.9%) and the splenocytes were washed three times with incomplete rpmi 1640 . The cell concentration was adjusted to 1 10 cells / ml in complete rpmi (ccm) containing 10% fbs (gibco - brl), 2 mm l - glutamine, 100 g / ml streptomycin and 100 iu / ml penicillin . One hundred microliters of diluted cell suspensions were dispensed into 96-well flat - bottom culture plates . Five l of the antigen suspension was added to each well and the volume was adjusted to 200 l with ccm . Control wells were made with 100 l of diluted cell suspension from the same mouse and 100 l of ccm . Each mouse s splenocytes were plated in duplicate . Lymphocyte proliferation was measured by an mtt assay (thiazolyl blue tetrazolium bromide, sigma, germany) after 48 hours incubation in 37c and 5% co2 . Spleen cells were removed and cultured as above and after 72 h of culture the levels of cytokines ifn- and il-5 in the culture supernatant were measured by commercial kit (elisa pro kit for mouse ifn- il-5, mabtech). Leishmania major promastigotes were collected at the stationary phase by centrifugation, washed three times with cold pbs and diluted with pbs to have 1 10 promastigotes per ml . Three weeks after the last immunization, dth was assessed by sc injection of 50 l from this promastigote crude lysate into left footpad . As a control, four weeks after last immunization, all groups (5 mice per group) were challenged subcutaneously in the tail base of the mice with 1 10 stationary phase promastigotes . Lesion development and survival rates of the vaccinated and control mice were weekly monitored after the parasite challenge . The l. major strain mrho / ir/75/er was provided from school of public health, university of tehran . Promastigotes of l. major were harvested from stationary growth phase by centrifugation (2000 g, 20 min, 4c), washed 3 times with cold pbs (ph 7.2) and homogenized with glass - glass homogenizer followed by autoclaving . Total protein content of the autoclaved antigen was determined by bradford method (each dose containing 40 g / ml of protein parasite). Female balb / c mice were divided into five groups (10 mice per group). Each group was divided to two subgroups, five mice in each subgroup: one subgroup was evaluated for lymphocyte proliferation and shift the immune response to th1/th2 and the second subgroup was assessed for delayed type hypersensitivity (dth) and challenge by the parasite.the alum ntx mixture was prepared by thoroughly mixing 50 l of pbs containing neltrexone (sigma, germany) at a concentration of 6 mg/ kg with 50 l of alum (aluminum phosphate, sigma). Balb / c mice were immunized subcutaneously three times at 2-week intervals with one of the followings: group vac (50 l ag + 100 l pbs /mouse), group al vac (50 l ag + 50 l al + 50 l pbs /mouse), group ntx vac (50 l ag + 50 l ntx + 50 l pbs /mouse), group al - ntx vac (50 l ag + 50 l al + 50 l ntx /mouse) and control mice received 150 l of pbs . Two weeks after the last immunization, the spleens were aseptically removed (five mice from each group) and separately homogenized in incomplete rpmi 1640 (gibco - brl). Erythrocytes were lysed with ammonium chloride (0.9%) and the splenocytes were washed three times with incomplete rpmi 1640 . The cell concentration was adjusted to 1 10 cells / ml in complete rpmi (ccm) containing 10% fbs (gibco - brl), 2 mm l - glutamine, 100 g / ml streptomycin and 100 iu / ml penicillin . One hundred microliters of diluted cell suspensions were dispensed into 96-well flat - bottom culture plates . Five l of the antigen suspension was added to each well and the volume was adjusted to 200 l with ccm . Control wells were made with 100 l of diluted cell suspension from the same mouse and 100 l of ccm . Each mouse s splenocytes were plated in duplicate . Lymphocyte proliferation was measured by an mtt assay (thiazolyl blue tetrazolium bromide, sigma, germany) after 48 hours incubation in 37c and 5% co2 . Spleen cells were removed and cultured as above and after 72 h of culture the levels of cytokines ifn- and il-5 in the culture supernatant were measured by commercial kit (elisa pro kit for mouse ifn- il-5, mabtech). Leishmania major promastigotes were collected at the stationary phase by centrifugation, washed three times with cold pbs and diluted with pbs to have 1 10 promastigotes per ml . Three weeks after the last immunization, dth was assessed by sc injection of 50 l from this promastigote crude lysate into left footpad . As a control, four weeks after last immunization, all groups (5 mice per group) were challenged subcutaneously in the tail base of the mice with 1 10 stationary phase promastigotes . Lesion development and survival rates of the vaccinated and control mice were weekly monitored after the parasite challenge . 1, the mean level of mtt in the mice immunized with al ntx vac and ntx vac induced significantly higher lymphocyte proliferation comparing to the control group (p <0.001and p <0.01, respectively). Spleen cell proliferation with al ntx vac (p <0.001) was significantly higher than al- vac and vac groups . Furthermore lymphocyte proliferation was significantly higher in mice immunized with the ntx vac compared with mice that received antigen alone (p <0.05). There was no significant difference in lymphocyte proliferation between other groups . As it is shown in fig . 2, the supernatant of splenocytes of mice immunized with al ntx vac or ntx vac showed significantly the highest (p <0.01) level of ifn- compared with the other groups . The mice of ntx- vac group produced the highest amounts of il-5 but ifn- to il-5 ratio was higher in al - ntx- vac group, none of them proofed as significant (table 1). To assess cell - mediated responses to parasite antigens in vivo the results of dth showed that different groups of immunized mice induced a stronger dth response than pbs control group (fig 3); however, the difference was not statistically significant except for the al ntx vac group (p <0 . Stationary phase promastigotes of l.major were injected subcutaneously at the tail base of 5 mice from each group, an injection site was analyzed over a period of 10 weeks . There was no significant difference between immunized and control groups of mice, during 10 weeks after challenge with l. major (data not shown). 1, the mean level of mtt in the mice immunized with al ntx vac and ntx vac induced significantly higher lymphocyte proliferation comparing to the control group (p <0.001and p <0.01, respectively). Spleen cell proliferation with al ntx vac (p <0.001) was significantly higher than al- vac and vac groups . Furthermore lymphocyte proliferation was significantly higher in mice immunized with the ntx vac compared with mice that received antigen alone (p <0.05). 2, the supernatant of splenocytes of mice immunized with al ntx vac or ntx vac showed significantly the highest (p <0.01) level of ifn- compared with the other groups . The mice of ntx- vac group produced the highest amounts of il-5 but ifn- to il-5 ratio was higher in al - ntx- vac group, none of them proofed as significant (table 1). To assess cell - mediated responses to parasite antigens in vivo, the dth reaction was measured 72 h post infection . The results of dth showed that different groups of immunized mice induced a stronger dth response than pbs control group (fig 3); however, the difference was not statistically significant except for the al ntx vac group (p <0 . 01). Stationary phase promastigotes of l.major were injected subcutaneously at the tail base of 5 mice from each group, an injection site was analyzed over a period of 10 weeks . There was no significant difference between immunized and control groups of mice, during 10 weeks after challenge with l. major (data not shown). In this study, alum naltrexone as a th1 immunostimulatory adjuvant has been used for the first time with autoclaved l. major to elicit a protective immune response challenge with the parasite in susceptible balb / c mice . Our selection to test naltrexone in this paper was based on reports of the literature indicating that has been effective in shifting immune responses to some antigens from a th2 response to th1 immune responses (21 - 23). Brown et al . Suggested that low - dose naltrexone presents a safe and promising approach to prevention and/or treatment of many autoimmune diseases and cancer variants, as well as potentially various viral (aids) and neurological diseases (multiple sclerosis) that are exacerbated by compromised immunity (22). Moreover, naltrexone was reported to stimulate ifn- production and induced effective immune responses against fibrosarcoma tumor and led to a significant inhibition of tumor growth in balb / c mice (23). The finding of the current study about adjuvant activity of naltrexone and previous studies about adjuvant activity of naloxone (which are structurally similar pure opiate receptor antagonists) and propranolol (24 - 28), emphasizes that the local microenvironment at the time of up taking and processing an antigen by apcs has a key role in the fate of subsequent acquired immune response against infection (29, 30). One important mechanism for adjuvant activity of naltrexone is blocking the opioid receptors which accelerates local inflammation via direct effect on monocytes, macrophages and dendritic cells (31, 32). Ifn- secreted by t cells, plays a key role in protection against intracellular infectious agents and is the cytokine primarily used as a marker for the existence of th1 immune responses . Cytokine analysis revealed that the administration of leishmania proteins with al ntx or ntx induced high levels of th1 cytokine (ifn-). Furthermore, the spleens of mice immunized with al ntx vac showed significantly the highest ifn- /il-5 ratio compared with the other immunized groups which is an important correlate of immune protection against l. major . These results are in agreement with other studies showing that ntx induce th1 type of immune response (23, 32). Ntx can shift the immune response toward a th1 pattern while alum is a th2-type adjuvant . However, despite the opposing effects of these adjuvants on skew the immune system toward a th1/th2 response, our results show that co - administration of naltrexone with alum more skewed the immune response in balb / c mice towards a th1-type than administration of naltrexone alone . Evaluation of lymphocyte proliferation response revealed that immunization of mice with al ntx vac stimulate the proliferation of spleen cells significantly moreover footpad swelling after infectious with l. major was measured and the results demonstrated that al ntx vac elicited strong dth responses . Unexpectedly, immunization of balb / c mice with different groups and challenging with l. major did not show any significant differences in the death rate until 10 weeks after infection . Immunization with the alum naltrexone mixture as an adjuvant, in combination with the autoclaved l. major promastigotes antigens, can enhance cellular immunity and shift the immune responses to a th1 pattern . To our awareness, this study is the first evaluation for using an alum naltrexone mixture as an adjuvant for vaccination . Therefore, follow - up studies are needed to elucidate the effect of naltrexone on the mice immune system and to examine adjuvant activity of naltrexone when combined with vaccines against other microorganisms.
Lithium ion batteries are the power source of choice for most mobile electronic devices . These systems generally work using the insertion and removal of lithium ions into host materials, resulting in redox and structural changes during the electrochemical cycling . Lifepo4 adopts the olivine structure type ((mg, fe)2sio4, orthorhombic),with feo6 corner - linked octahedra in the bc plane and lio6 octahedra forming edge - sharing chains on the b axis . Hence the li ions reside within 1d channels, allowing their extraction and insertion during charge and discharge via the reaction shown in figure 1 . Equation for charge and discharge of lifepo4 and structural diagrams of the lifepo4 and fepo4 active materials with iron atoms shown in orange, small gray phosphorus atoms, large blue lithium atoms, and red oxygen atoms . Both structures adopt space group pnma, with lattice parameters of a = 10.3290(3), b = 6.0065(2), and c = 4.6908(2) for lifepo4 and a = 9.8142(2), b = 5.7893(2), and c = 4.7820(2) for fepo4 . The discharge profile is characterized by a flat plateau at 3.45 v vs li . This flat potential discharge reaction is characteristic of the coexistence of two phases, lifepo4 and fepo4, each having a narrow compositional stability range in contrast with nonstoichiometric electrode materials such as lixcoo2 that generally show sloping profiles . The first in situ x - ray diffraction (xrd) study of lifepo4 was presented by andersson et al . Using a coffee bag this cell can be placed directly in the x - ray beam and diffraction is observed in transmission mode through the coffee bag . The study clearly showed the phase change reaction and monitored the growth of the heterosite (fepo4) phase as the triphylite (lifepo4) phase diminishes during charge, with the reverse occurring on discharge . The intensities of the peaks were found to be in good agreement with those anticipated from the charge passed during cycling . Several other designs for in situ diffraction studies have since been reported including a recent study that characterized a meta stable new phase formation at high rates in large particle size lifepo4 . Since some of the first commercial uses of lifepo4 have emerged for high power applications such as power tools, many different preparations of lifepo4 have been explored to improve the material s performance in order to allow for practical use at high rates . These have focused on control of its particle size, doping on the li and fe site, and various coating methodologies . These strategies have been largely successful on the particle and agglomerate levels, reducing solid state diffusion times, phase boundary strain, and electronic resistance . Given the above successes in improving the discharge of single particles and agglomerates such that intraparticle equilibration processes are not rate limiting, attention has recently focused on the effects of ion transport restrictions in composite electrodes containing dispersed active material, an electron conducting additive, and electrolyte . In the case where electronic conductivity is not rate limiting, discharge should begin at the electrode / separator interface where ion transport restrictions are at a minimum . With nonstoichiometric electrode materials, where the equilibrium potential decreases continuously with discharge, the progression of discharge from the front to the back of the electrode can be described by ambipolar diffusion of lithium ions and electrons according to the delevie description of a porous electrode . Here the active material is represented by a series of capacitances distributed along the electrode thickness . These are connected to the bulk electrolyte through the infused electrolyte within the pores, and to the current collector via the electron conducting additive, so that parts of an electrode that are at different states of charge are continuously equilibrated during discharge . The interface between charged and discharged material is diffuse, and further diffusion occurs after the current has been switched off . Importantly, the driving force for ambipolar diffusion is the increase in the potential with the state of charge . This is notably absent in the case of stoichiometric materials such as fepo4/lifepo4 where the potential is constant for most of the composition range, as shown by a long plateau in the discharge curve . Ambipolar (ion - electron) diffusion should not occur in these materials; instead, the interface between charged and discharged parts of the electrode should be linked directly to the passage of charge and should stop moving once the current stops despite the sharp change in the average concentration of lithium . We recently described this phenomenon as the sharp discharge front (sdf) effect, and supported our hypothesis with electrochemical discharge data that responded to changes of electrolyte conductivity and salt diffusion as predicted, but previously had no direct evidence for the distribution of discharged material within the electrode . The above example cites just one case among many where a direct observation of the profile of the extent of discharge with depth into the electrode thickness could provide valuable insight into the discharge process and verification of simulated discharge phase profiles . In situ neutron depth profiling can resolve variations in lithium concentration through the thickness of an electrode via the energy profile of particles formed as a result of neutron capture by li . Cross - sectional imaging by neutron absorption or tem can also provide valuable information on variations through the electrode . The use of x - ray diffraction allows direct observation of the phase distribution of the active materials during charge and discharge . This information is different from the lithium distribution, which would include any lithium in the electrolyte, and can be collected rapidly in situ providing the possibility of time resolution . Variation of the incidence angle provides a depth sensitivity as absorption of x - ray photons results in a limited path length so with low incident angles the diffraction signal comes largely from close to the surface . The lifepo4/fepo4 system provides a model composite electrode, which should provide a sharp and controllable discharge front . Positive electrodes for in situ cycling were formed by mixing appropriate amounts of carbon - coated lifepo4 (hydro - quebec) and acetylene black conducting additive (shawinigan black), then mixing in a polytetrafluoroethylene (ptfe) binder (6c n, dupont). The resulting mixture was calendared to a controlled film thickness of 100 m and punched to produce circular electrodes with a diameter of 8 mm . The electrodes were dried overnight at 120 c under vacuum, before being transferred to an argon - filled glovebox . Two compositions of lifepo4 electrodes were used for the testing: (a) 50% lifepo4, 40% acetylene black, and 10% ptfe by mass and (b) 25% lifepo4, 60% acetylene black, and 15% ptfe by mass . Currents were calculated to achieve complete charge or discharge in a fixed time period based on theoretical capacity, e.g. C/2 = complete charge or discharge in 2 h and 2c = 0.5 h. scanning electron microscopy (sem) used a jeol jsm-6500 fegsem with 15 kv accelerating voltage and secondary electron imaging powders or whole electrodes were mounted on conducting carbon tape and imaged without any further coating . Initial xrd patterns were collected with a bruker d2 phaser (cu k x - rays) and rietveld refinement of the data used the gsas package . The electrochemical cell used for the in situ work is based on the commonly used swagelok cell design and therefore consists largely of cheap, commercially available parts . This cell can be constructed readily in any laboratory and does not require the use of specialized equipment or the use of toxic beryllium metal windows . The few bespoke parts used in its assembly are easily fabricated with use of basic tools . Furthermore, it is simple to assemble and clean after use, and the positive current collector, which doubles as the x - ray window, is disposable and easily replaced . The cell consists of three main sections as shown schematically in figure 2a and as a photograph in figure 2b: (1) a 12 m thick aluminum foil acts as the positive current collector . This was attached to a stainless steel washer with black silicone rubber adhesive (loctite type 5910this was found to be inert in the environment of an operational cell). The washer was similarly attached to the swagelok nut used to seal the cell . (2) a 12.7 mm diameter stainless steel piston was used as the negative current collector . This was placed under tension with a steel spring held in place with a 12.7 mm diameter stainless steel rod, the bottom of which was machined to fit into a standard goniometer head for mounting onto the beamline . (3) ptfe sheaths, ferrules, and a nut sealing the bottom of the cell were used to avoid short - circuits, including during handling of the cell . (a) schematic and (b) image of the electrochemical cell used in the in situ xrd studies . The layered battery assemblies consisted of a composite positive electrode, two electrolyte - soaked 12.7 mm diameter separators (whatman gf / f grade glass fiber with eight drops of 1.0 mol dm lipf6 in 1:1 ethylene carbonate / dimethyl carbonate (novolyte technologies)) and a lithium metal negative electrode formed by compressing lithium (99.9%, aldrich) into a hemispherical mold and punching to a diameter of 11 mm . The shaped lithium negative electrode allowed an even pressure to be applied across the diameter of the stack, countering the effect of deformation of the aluminum window under pressure and allowing a similar electrochemical performance to be achieved to that observed with the same electrode material in a standard swagelok cell with flat electrodes . To study the electrode composition as a function of depth and state of charge the diffraction geometry shown schematically in figure 3 was used . The sample was mounted on the hexapod stage at beamline i07 of the diamond light source with use of an insulating mount and visually centered in the xy plane such that the highest point on the curved surface of the window was aligned with the center of the diffractometer cirles . The x - ray beam height was approximately 80 m and a scan in the vertical direction monitoring the direct beam intensity was used to position the sample such that the beam center was level with the top of the 100 m thick positive electrode . Hence 40 m of the beam passed through the back of the sample with 0 incidence angle and 60% of the electrode (the part facing the separator and negative electrode) was not contributing to the observed xrd pattern . The sample was then tilted to increase the incidence angle and the penetration depth into the electrode . Additional information about sample mounting on the beamline is included in the supporting information . Schematic showing the effect of changing the angle of incidence on the penetration of the 80 m high x - ray beam into the positive electrode at 0 (left) and 4 (right) angle of incidence . In situ xrd patterns were collected with 20 kev beam energy (= 0.620) and an exposure time of 1 s using a pilatus 100k area detector at a camera length of 497 mm such that a 7 range could be collected in a single frame with high resolution . This restricted the accessible 2 range but facilitated fast acquisition times so that the phase change reaction could be observed at high rates . Data were continuously collected with a series of different incidence angles between 0 and 6, although ultimately the analysis focused on data collected at 0 and 4. collecting 1 s patterns at 0, 0.5, 1, 2, 3, 4, and 6 incidence angles resulted in a 20 s cycle of measurements (patterns could be recorded with acceptable quality in 0.1 s but the sample position adjustment to effect the different angles of incidence was the rate limiting step). The peak width was between 0.05 and 0.06 at both incidence angles hence the variation in peak width with incident angles was not found to be significant . With a battery performing to theoretical capacity at our fastest rate of 20c under these conditions 9 patterns could be collected at each angle during the 3 min discharge . The peak heights of the most intense bragg reflections for lifepo4 at 15.7 and fepo4 at 16.3 were then used to provide a measure of the electrode phase composition at each angle of incidence . This structural change was correlated to the state of charge by using time stamps in both the electrochemical and xrd data files . The peak heights were extracted by using a matlab macro with a baseline correction . Strictly peak area is proportional to the phase fraction, but since peak widths of the lifepo4 and fepo4 phases were similar in any given pattern the intensity was taken as a good indication of the relative phase contents . Commercially sourced lifepo4 was used in this work to take advantage of its optimized performance, achieving a capacity of approximately 150 ma h g with a good cycle life and rate capability . Powder xrd studies showed it to contain single phase lifepo4 (supporting information, figure s1). Rietveld refinement by using the standard triphylite model in space group pnma resulted in a good fit with a = 10.32227(12), b = 6.00341(6), and c = 4.69092(7), similar values to those described in the literature . To obtain a good fit it was necessary to employ a preferred orientation parameter allowing for a small increase in intensity along 010 (march dollase preferential orientation ratio of 0.9105(12) along 010). The sem (figure s1, supporting information) showed well - formed crystallites that are slightly elongated along one axis and with an approximate size of 200 nm . Since preferred orientation is only being observed along one axis it is likely that the large flat face observable on some crystallites is becoming aligned with the xrd sample surface during sample preparation and that this face is the {010} plane of the crystallites . Figure 4 shows xrd patterns obtained during the slow (c/2) charge and discharge of a lifepo4 half cell . The electrochemical response is characteristic of the coexistence of two phases with a flat plateau observed in the voltage capacity profile during charge and discharge . In the fully discharged (or as - constructed) state the characteristic triphylite lifepo4 phase can be clearly identified and as expected after charging (due to the near theoretical capacity extraction) a complete conversion to the heterosite fepo4 phase was observed . In the partially charged or discharged condition we can clearly observe a mixture of these two phases in the diffraction data with the phase contributions to the pattern corresponding to the state of charge (specifically peaks at 16.2 and 13.7 corresponding to lifepo4 and those at 15.7 and 13.6 pertaining to fepo4). It can be observed that peak overlap is quite limited due to the narrow xrd reflections and the significant differences in lattice parameters between the lifepo4 and fepo4 phases . Charge and discharge curve for lifepo4 at a rate of c/2 with stacked diffraction patterns (4 incident angle) showing the linked structural changes between the lifepo4 and fepo4 phases . A significant enhancement in the 020 reflection relative to the expected intensity distribution based on the literature powder patterns is observed in both lifepo4 and fepo4, a larger elongation of the 020 reflections than observed in the powder pattern of the starting material (figure s1, supporting information). Here the calendaring process used to make the electrodes is likely to have induced this orientational effect . Importantly the degree of 020 preferred orientation was observed to be very similar in lifepo4 and fepo4 due to the topotactic transformation between them . The discharge performance of the batteries constructed for in situ testing using 25% and 50% active material with rates between 2 and 20 c is summarized in figure 5b, d (a constant charging rate of 2c was used for all experiments irrespective of the discharge rate to ensure the condition of the electrode at the start of discharge was as similar as possible). At relatively slow rates of discharge a characteristic flat discharge plateau was observed around 3.45 v vs li in both cases . As the rate was increased the discharge changes to a negative gradient linear profile as noted in our previous publication and explained by an ohmic potential drop in the electrolyte within the composite electrode to the nearest delithiated particle, increasing with the distance from the separator to the discharge front . Discharges at rates greater than 5 c showed sharp end points at capacities well short of those obtained at low rates, similar to our previous work where an explanation was given in terms of severe lithium salt polarization at high rates due to a low lithium ion transference number . For electrodes containing only 25% and 50% lifepo4 (used in this work to ensure that x - rays could pass from the front to the back of the electrode without total absorption during the penetration path length) the capacity was well maintained up until rates of around 10c (figure 5a, c), with a slight reduction in capacity easily explained by premature termination of the discharge resulting from an arbitrary choice of potential limit that did not account for the discussed increase in ir drop . At rates greater than 10c we observed that sharp end points are reached which are premature of that anticipated entirely from ir drop and were hence consistent with the electrolyte limitation discussed earlier . The good retention of capacity at high rates observed in these electrodes results from a reduced amount of lifepo4; we chose relatively dilute amounts of active material to ensure that x - rays could pass from the front to the back of the electrode without total absorption during the penetration path length . If we assume that at the highest rates the electrodes are discharging under the sdf model discussed in our previous work then we can calculate effective diffusion coefficients of lithium ions in both electrodes using eq 1.1where dod is the degree of discharge, f is c rate, [lix]0 is the concentration of salt ions in the electrode, t is the transport number (assumed to be 0.3 in this case), [li] is the concentration of li ions stored in the active material within the electrode, d is the diffusion coefficient, and l is the thickness of the electrode . The effective diffusion coefficients for the salt in these structures were found to be 1.7 10 and 2.2 10 m s for the 25% and 50% electrodes, respectively . These values are significantly larger than we reported for an electrode with 75% active material in our previous work (10) and can be explained by increased porosity and a lower tortuosity of the diffusion paths . Electrochemical performance of 25% (a, b) and 50% lifepo4 (c, d) electrodes shown as electrode capacity retention as a function of rate (left) and discharge capacity vs potential (right). Slow charging rates and constant voltage top up periods were used to ensure full charging of the battery . Based on the electrochemical performance an almost complete discharge of the cell at all rates is likely for the electrode containing 25% active material and therefore relatively small concentration gradients should be observed . For the 50% active material electrode much larger gradients are likely to be observed, especially at high rates . During the electrochemical measurements presented in figure 5, xrd patterns were collected at a number of incidence angles to probe the structural changes as a function of state of discharge rate . Our analysis focuses on data collected at 0 and 4. grazing incidence xrd is widely used to increase the effective sample thickness in the study of thin films . Applying this technique to battery electrodes can provide an effective method to profile any differences in phase behavior as a function of depth in the electrode by varying the proportion of the signal that is scattered from the side on which the beam impinges . The absorption of x - ray photons is a significant consideration in this geometry as with a 4 incidence angle the path length through the electrode will be increased from the 100 m electrode thickness to 1400 m . Based on calculated x - ray absorption characteristics of the electrode components (supporting information, table s1) only about 6% of photons are expected to reach the front face of the electrode so the observed phase concentrations will be significantly biased toward contributions from material close to the current collector . The battery could be assembled the other way up to reverse this bias, but that geometry would also contain a compromise in that the beam would have to pass through the negative electrode and the separator, which would increase the contribution to the diffraction patterns from these components . Importantly the absorption profiles of the two electrode compositions discussed herein are similar (table s1, supporting information) and so direct comparisons between their behavior can be made, while keeping in mind the bias in the data toward the back of the electrodes . The intensity of the 020 reflection of fepo4 is approximately equal to that of the 020 + 211 reflections of lifepo4 in an equimolar mixture of these phases, hence the intensities of these reflections were used as a semiquantitative measure of the content of the relevant phase . Figure 6 shows the variation in the phase fraction of fepo4 based on the intensity of the 020 reflection during discharge at various rates . As expected the intensity changes occur over shorter time periods as the discharge rate is increased . With 25% active material in the electrode the intensity profile at all rates is similar using a 0 or 4 incidence angle . It is striking, however, that these profiles diverge with 50% active material and that the observed intensity of the fepo4 020 reflection is stronger with 4 incidence angle than it is with 0 incidence angle at all rates above 4c . This divergence shows that the back of the electrode is undergoing less discharge at these rates than the region being sampled closer to the front of the electrode . The divergence is largest at 8c and 10c, and is observed to decrease again at 20c . This indicates that the region of the electrode with the largest variation in composition is moving further from the side of the electrode from which the x - ray is impinging and hence closer to the electrolyte - soaked separator . The variation in fepo4 phase fraction expressed as the fepo4 020 peak height (relative to the combined heights of the fepo4 020 and the lifepo4 020 + 211) with time during cell discharge for electrodes prepared with 50% and 25% lifepo4 and linear fits to the data . Figure 7a, b shows the gradients of the linear fits to the data in figure 6 plotted versus current density and c rate for both the 25% and 50% electrodes, respectively . This plot emphasizes the divergence in the observed intensity of the fepo4 020 reflection of the 50% active material electrode during fast discharge . A particular strength of our approach of rapidly collecting a series of incidence angles is that these data were collected on a single electrode so are directly comparable . The rate of change of the intensity of the fepo4 020 reflection during discharge as a function of current density and c rate for (a) 25% and (b) 50% lifepo4-containing electrodes . Points circled in red highlight those which indicate significant concentration gradients within the electrode . The variations in phase behavior that we have observed during fast discharge are consistent with the effects described earlier based on observations from the electrochemical performance . In the electrode containing 25% active material the electrochemical data show only a slight increase in the negative gradient of the discharge plateau indicating that some small ionic diffusion gradients may exist in the electrode resulting in some preferential discharge of material near the bulk electrolyte as observed in figure 7a (a notable deviation from this trend is seen at 20c which may be due to a low number of data points increasing the bias from experimental scatter). In the 50% electrode, little or no variation was seen in the gradients at the front and back of the electrode with rates of 4c or slower . However, at elevated rates there is a sharp deviation in the rate of change between the front and back of the electrode suggesting that a significantly different limitation is controlling the electrode composition . This effect is consistent with the salt concentration polarization aspect of our previously reported sdf model, and originates from the insufficient concentration of lithium ions in the electrolyte stored within the electrode (see table 1). During the discharge of the battery lithium ions must be transported from the bulk electrolyte to the active material by migration and diffusion . At slow rates these mass transfer processes are sufficient to allow complete discharge of the battery, but at higher rates they are severely limiting . Calculated assuming that the solid material is approximately 80% of the volume and that the free space (filled with electrolyte) is 20% (approximated from ref (48)). Densities were taken as 3.6, 2, and 2.2 g cm for lifepo4, carbon black, and ptfe, respectively . It was also assumed that changes in composition of the active material did not change the packing of the solids and therefore the occupied volume . The concentration of the lipf6 in the electrolyte solution used was 1 m. in the flat discharge plateau region of lifepo4 the discharge reaction can be written as: the fepo4 and lifepo4 phases coexist within the electrode structure, so if one region of the electrode has an insufficient supply of lithium ions then a slower discharge will occur in one region and a faster rate of discharge in another . At high rates of discharge lithium ions this is followed by a mass transport process driven by diffusion from the bulk electrolyte . The lithium ions thus transported will react with the first particles of electrode material encountered which will be in the region of the electrode facing the separator . Hence this region will fully discharge and the electrode region near the current collector will be undercharged (as observed at high rates in the 50% active material electrode). This is shown schematically in figure 8; when there is no limitation on the electrolyte during discharge, an even concentration in the electrode can be seen and when an electrolyte limitation is in effect, a preferential discharge occurs at the electrode close to the bulk electrolyte and results in an incomplete discharge . Schematic showing how the discharge proceeds in an electrode where there are no limitations on the discharge from the electrolyte and also where there is a severe limitation as a result of insufficient transport of li ions (usually resulting from a high ratio of lithium ion vacancies to lithium ions in solution and when the electrode is discharged at high rates). This schematic negates the inclusion of any conductive additive or binder and assumes no electronic limitations . During the in situ cycling experiments described above, in which the electrodes were cycled sequentially at rates of 2, 4, 6, 8, 10, and 20 c, a reduction in the crystallinity of the active electrode was observed . Note that the peak widths of lifepo4 and fepo4 were observed to be similar in any given pattern throughout the study so this broadening does not affect the phase faction calculations presented above . However, xrd patterns recorded at the start of discharge at each rate for the 25% electrode do show a clear increase in peak width (figure . 9a). To check whether this breakdown could be caused by x - ray beam damage we recorded patterns at the end of a 2c discharge while the battery was under open circuit conditions for 40 min . These results showed no discernible degradation of the active material as a function of time exposed to the beam, and since none of our measurements exceeded 5 h total collection time it seems unlikely that beam damage is a significant factor . We also investigated the behavior using ex situ measurements of cells cycled with the same regime used during this in situ test, and two cells cycled at 10c and 2c for the same number of total cycles . A significant broadening of the fepo4 reflections was observed in the materials which were cycled at a range of different rates (figure 9b) but very little extra broadening was observed with either of the fixed cycling rates . Xrd patterns at the start of charge during a sequence of cycles at different rates (2c 20c), showing the increased peak width (a) and the full width half - maximum value of the fepo4 020 reflection in patterns recorded ex situ with a fresh electrode, an electrode cycled at a number of different rates (as per the left - hand image), an electrode cycled 10 times at 10c, and an electrode cycled 10 times at 2c (b). We also examined sem images of the electrodes before (figure 10a) and after (figure 10b) cycling using the multi - rate regime employed for the in situ measurements described above . No obvious breakdown in particle size can be seen in these images and we therefore suggest that the line broadening is a result of increasing disorder within the crystallites . This disordering seems to be limited to cases where multiple cycling rates have been applied . Sem images of electrodes containing 25% lifepo4 before (a) and after (b) cycling under the test regime employed in diamond . The results presented above have tracked the formation of concentration gradients within electrodes under high rates of discharge . The limitations seen are consistent with effects corresponding to an insufficient transport of li ions from the bulk electrolyte through the electrode structure as predicted by sdf theory . This confirms that the rate performance of many modern materials used in battery technologies is not a result of the intrinsic properties of the material itself but rather the matrix in which it is stored . In this study, this effect is observed in electrodes with relatively dilute amounts of active material where the x - ray absorption is not obscuring measurement . However, these gradients should be much more pronounced in electrodes with higher concentrations of active material in the electrode (such as those conventionally used in research laboratories with 75% active material) and thus the electrochemical rate performance of these systems will be significantly hindered . The effect should also be far more pronounced in electrode materials which have a much higher volumetric capacity, where the requirement of lithium ions by the material stored in the electrode during discharge will be much larger . This means that to realize the full rate potential of battery materials the following strategies should be considered: (1) dilution of the active material (which reduces the stored energy density); (2) reducing the electrode thickness; (3) increasing the concentration of lithium ions in the electrolyte: the electrolyte concentrations used herein are typical for conventional liquid electrolytes but there are recent reports of more concentrated electrolytes which retain high diffusion rates, these would allow an increase in the amount of lithium initially in the pores c.f . Values given in table 1; and (4) increasing the diffusion coefficient of lithium in the electrolyte . A new method for the in situ study of battery materials that allows for the visualization of concentration gradients formed in electrodes during discharge is introduced . A significant difference in the performance of the material dependent on whether it is near the bulk electrolyte or current collector is observed . At higher rates of discharge (> 10 c) the electrode material near the current collector changes at a much slower rate compared with the material close to the bulk electrolyte in cells containing a high concentration of lithium ion vacancies . It is believed that this effect is the major limitation in the rate performance of electrodes in conventionally prepared batteries . In parallel we observed a significant breakdown in crystallinity of the lifepo4 during the electrochemical measurements . It was shown that the breakdown is far more significant when the battery is cycled at a range of different rates rather than for the same number of cycles at either a high or low rate . Ideally, to obtain the maximum rate out of a battery material of a given particle size and ionic and electronic conductivity we need an electrolyte that can supply the ions at high rates . In some cases the particles of the electrode material may be significantly large or the ionic or electronic conductivities sufficiently small such that electrolyte is not limiting the discharge rate . Nevertheless, modern synthesis techniques such as sol gel and hydrothermal routes mean that synthesis of materials on the nanometre scale is routinely achieved which means that commonly the rate limiting step in the discharge of the battery material even with intrinsically poor electronic and ionic conductivities is the li ion transport through the electrolyte.
Tinea corporis (b35.6) caused by microsporum canis which is fungal species that causes numerous forms of disease . Though mostly well known for ringworm in pets, it is also known to infect humans . We will report about a case, 22-year - old female, residing in a village, with typical changes of a mycotic infection caused by m. canis . Dermatological description can be summarized with polymorphic erythematous, papulosquamous changes, erosions mainly on genital organ and spread to the thighs and lower abdomen which are accompanied with itching and burning . Diagnosis b35.6 was determined on the basis of clinical appearance complemented with anamnesis, microscopic examination and culture . Dermatophytosis (tinea) infections are fungal infections caused by dermatophytes - a group of fungi that invade and grow in dead keratin (1). Infection is limited to the dead layers of skin but encouraged by a damp and warm local environment . The infection can be transmitted to humans by anthropophilic (between people), geophilic (from soil) and zoophilic (from animals) spread (2). Tinea cruris is three times more common in men than in women because of the scrotal anatomy (3, 4). Itching, rash and nail discoloration are the most common symptoms of tinea infection (2). Tinea cruris, commonly referred to as jock itch, involves the medial aspect of the upper thighs (groin) (5, 6). Complications such as secondary infection (cellulitis and impetigo) can lead to symptoms (2). More unusually the lesions can appear as overlapping concentric circles (tinea imbricate) (7). Reactions to a dermatophyte infection may range from mild to severe as a consequence of the host s reactions to the metabolic products of the fungus, the virulence of the infecting strain or species, the anatomic location of the infection, and local environmental factors (8). Fungal transmission occurs through direct contact with infected persons, animals, soil or fomites . Zoophilic sources should be identified (if possible) and treated to prevent human reinfection (9). The classic presentation of tinea infection, known as ringworm, is a lesion with central clearing surrounded by an advancing, red, scaly, elevated border . Inflammation assists in colonization and may result in vesicles on the border of the affected area . The presentations of tinea infections range from mild scaling and erythema to severe inflammation with bacterial superinfection . The differential diagnosis: multiform erythema, annular granuloma, nummular dermatitis, rosea pityriasis, versicolor pityriasis, psoriasis, secondary syphilis, candidal intertrigo . We report on a case diagnosed not directly due to delay of visiting a doctor by the patient because of stigma and prejudice attributed to local environment, lack of experience of the family doctor and specialist regional dermatologist in right diagnosis and treatment . The case is a 22-year - old female, with mycotic infection in the genital area (figure 1). Female, 22 years old, lives in the village, hospitalized due to skin changes in the genital pubic part, thighs and lower abdomen, which are accompanied with extraordinary itching and burning . The changes had started much time before but she delayed her visit to the doctor to the point when her condition deteriorated with fever and temperature . The changes began with rash and itching presented in genitalia and then changes spread to areas around . In anamnesis, the patient denies that other members of the family have similar changes whereas she claims to own and have in family care a dog and a cat and no other domestic animals . She comes from a low level of socio - economic status . In dermatological description, changes are polymorphic, plaques in the pubic area with erythematous fluorescence, pustules, erosions, crusts and papulosquamous changes in the shape of circles in thighs and lower abdomen which are accompanied with intensive itching . The center of the circles is inactive while the surrounding is active with distinctive bordering line to the skin around . Due to scratching because of itching diagnosis was determined on the basis of clinical appearance, anamnesis, positive dermatophytes microscopic view and culture m. canis . The patient was treated with general and local antimycotics (terbinaphin 250 mg / day for 4 weeks), as well as with antibiotics according to sensitivity chart . From the anamnesis of the reported case is emphasized the continuous itching which never stopped for the whole time being described as terrible and which corresponds to the literature . Also, the humidity of the genital area itself, washing with water after each defecation and urination (islamic belief tradition), was a suitable ground for the development of the disease . The differential diagnosis was: multiform erythema, annular granuloma, nummular dermatitis, rosea pityriasis, versicolor pityriasis, psoriasis, secondary syphilis, candidal intertrigo but we were also based on clinical appearance, anamnesis complementing it with microscopic examination and fungal culture . We consider that the report on the mentioned case will be beneficial to family doctors so they can complement their professional experience with more extensive knowledge on clinical manifestation, diagnosis and differential diagnosis.
P. brasiliensis isolates and patient sera - five human p. brasiliensis isolates were included in this study, one belonging to the s1 species from botucatu, state of so paulo (sp), brazil (pb265), one of the ps3 species from colombia (epm83) and the other three belonging to p. lutzii from goinia, state of gois, brazil (pb01, pb8334 and pb66). The isolates were maintained on bbl mycosel agar (bd biosciences, sparks, md, usa) and transformed into the yeast phase on 4% glucose, 1% peptone, 0.5% yeast extract and 1% agar medium . Furthermore, the antigen obtained via filtration of a b-339 culture, considered the standard antigen for immunodiffusion assays, was added to the analyses; this isolate was maintained on potato agar medium and transformed into yeast on fava - netto agar . Regarding the sera from pcm patients, 71 samples were evaluated and divided regionally, as follows: 20 serum samples from botucatu, 20 from jundia and 31 from the central - west region of brazil . The sera from the botucatu region were stored in the serum bank at the laboratory of tropical diseases - mycology area of botucatu medical school of so paulo state university, whereas the samples from the jundia and central - west regions were stored in the adolfo lutz institute / sp collection . Preparation of culture - filtered antigen - the yeast cells from each fungal isolate were transferred to 10 slant cultures containing fava - netto agar . After five days of growth at 36c, the contents of the tubes were inoculated into 500 ml 2% dextrose, 1% neopeptone, 0.018% thiamine and 0.036% asparagine medium and incubated at 35c for 15 days with agitation at 100 rpm . G / l final concentration) for four days under the same conditions and then filtered through filter paper . The preparations were concentrated using polyethylene glycol until 10% of the initial volume was reached . The preparations were then dialysed against phosphate buffered saline (pbs) at ph 7.2 for three days at 4c . After this period, 1.0-ml aliquots of the samples were separated, lyophilised and, at the moment of use, resuspended in 20 l of pbs . Other methodologies were also tested to produce such antigens as the somatic antigen (franco et al . 1996) and the cell - free antigen (camargo et al . 1991), which, in our experience, proved to be less sensitive than the culture - filtered antigen in immunodiffusion assays (data not shown). Did assays - carefully cleaned slides were pre - coated with 1.0 ml 1% agar solution, allowed to dry at 60c for 24 h and then coated with 3.0 ml citrate agar solution (1% purified agar, 0.9% sodium chloride, 0.4% sodium citrate, 7.5% glycine or amino acetic acid and 0.01% thimerosal). The citrate agar was solidified at room temperature (rt) and the slides were stored in a humidified chamber . The medium on the slides was bored with the aid of a rosette - shaped mould containing one central and six peripheral holes, each equidistant from the central hole . The slides received 10 l antigen (central well), 10 l polyclonal anti- p. brasiliensis antibody, the positive reaction control and 10 l test serum (peripheral wells), and were incubated in a humidified chamber for 48 h at rt . After this period, the slides were incubated in 0.5% sodium citrate solution for 45 min and in saline solution for 18 h. the wells were then covered with 1% citrate agar and the slides were wrapped in humid filter paper and allowed to dry for 8 h at 60c . Staining was performed for 10 min with 0.4% schwartz starch solution and 10% glacial acetic acid or coomassie blue . Destaining was performed in 5% glacial acetic acid solution, twice for 10 min each . The presence of a precipitation line corresponded to an antigen - antibody pair and indicated a positive reaction . The most dubious results were subjected to one or, if necessary, two replicates . Sodium dodecyl sulphate - polyacrylamide gel electrophoresis (sds - page) - the gels and reagents for sds - page were prepared as previously described (laemmli 1970); a 10% resolving gel and 3% acrylamide stacking gel were used . After electrophoresis, the gels were stained with coomassie blue solution (0.1% coomassie blue, 45% methanol and 10% glacial acetic acid) and the excess stain was removed with destaining solution (10% glacial acetic acid and 10% methanol). Analysis of pbgp43 exon 2 selection pattern - the sequences of pb gp43 exon 2 available in genbank were employed: 10 haplotypes of p. lutzii (accessions: eu870212-eu870214, eu870217, eu870218, eu870220, eu870221 and eu870226-eu870228) (teixeira et al . 2009) and 11 from the other cryptic species (accessions: dq003724, dq003741, dq003744, dq003746, dq003748-dq003750, dq003771-dq003773 and dq003781) (matute et al . 2006) were used . The sequences were aligned using the software mega4 and the synonymous and nonsynonymous substitution rates (ds / dn) were calculated pairwise on the syn - scan website (gonzales et al . 2002) according to the method of nei - gojobori (nei & gojobori 1986), yielding a mean ds / dn value . Double agar gel immunodiffusion testthe results of did assays performed using 71 serum samples from the botucatu, jundia and central - west regions are shown in table i . Only 10 sera from the botucatu region reacted against the standard antigen agb-339 at different titrations . The remaining 10 sera corresponded to patients with active disease or confirmed pcm, but with no reactivity according to did . Of the five antigen preparations evaluated, the one obtained from the ps3 isolate, epm83 (agepm83), showed the highest reactivity index for the samples from the botucatu region . In addition to reacting with three sera that were non - reactive to the standard antigen, this antigen was capable of detecting the presence of antibodies in all samples reactive to b-339 . Agepm83 and agb-339 presented a substantial strength of agreement in the botucatu region, as represented by a kappa coefficient (k) of 0.7 (table ii). In contrast, agpb265 (s1) and agpb01 (p. lutzii) did not react with any of the 20 serum samples from the botucatu patients . The other two antigens produced from the p. lutzii isolates agpb8334 and agpb66 showed identical results (k = 1.0), reacting with the same eight sera among the 10 positive for agb-339 and with none of the 10 sera non - reactive to agb-339 . The strength of agreement between these antigens (agpb8334 and agpb66) and agb-339 was also substantial (k = 0.8). Table ireactivity profile shown by culture - filtered antigens in immunodiffusion assays against sera from patients from botucatu, jundia and central - west regions of brazil, reactive or not to the standard antigen agb-339reactivity n (%) agepm83agpb265agpb01agpb8334agpb66botucatureactive sera agb-339 (n = 10)10 (100)0 (0)0 (0)8 (80)8 (80)non - reactive sera agb-339 (n = 10)3 (30)0 (0)0 (0)0 (0)0 (0)jundiareactive sera agb-339 (n = 10)7 (70)2 (20)2 (20)2 (20)3 (30)non - reactive sera agb-339 (n = 10)2 (20)0 (0)3 (30)3 (30)3 (30)central - west regionreactive sera agb-339 (n = 11)4 (36)1 (9)0 (0)3 (27)4 (36)non - reactive sera agb-339 (n = 20)0 (0)0 (0)0 (0)0 (0)0 (0) table iidistribution of patients with active paracoccidioidomycosis from different regions as to the hierarchical weights of agreement in serological measuresorigen of the patientscomparisonsagreements (+ +) (- -)disagreements (+ -) (- +) kappa coefficientconfidence intervalstrength of agreementmc nemar s testbotucatub339vsepm83107030.700.40 - 1.00substantial0.08 b339vspb2650101000.000.00 - 0.44slight <0.01 b339vspb010101000.000.00 - 0.44slight <0.01 b339vspb8334810200.800.54 - 1.00substantial0.16 b339vspb66810200.800.54 - 1.00substantial0.16pb01vspb66012800.000.00 - 0.54slight <0.01pb01vspb8334012800.000.00 - 0.54slight <0.01pb66vspb8334812001.001.00 - 1.00perfect0.32pb01vsepm83070130.000.00 - 0.32slight <0.01pb66vsepm8387050.530.17 - 0.89moderate0.0254pb8334vsepm8387050.530.17 - 0.89moderate0.0254jundiab339vsepm8378320.500.12 - 0.88moderate0.65 b339vspb265210800.200.00 - 0.46slight <0.01 b339vspb0127830.000.00 - 0.28slight0.13 b339vspb833427830.000.00 - 0.28slight0.13 b339vspb6637730.000.00 - 0.40slight0.21pb01vspb66413120.620.23 - 1.00substantial0.5637pb01vspb8334515001.001.00 - 1.00perfect0.32pb66vspb8334413210.620.23 - 1.00substantial0.5637pb01vsepm83410150.370.00 - 0.79fair0.1025pb66vsepm8349250.270.00 - 0.71fair0.2568pb8334vsepm83410150.370.00 - 0.79fair0.1025central - west regionb339vsepm83420700.420.12 - 0.73moderate <0.01 b339vspb2651201000.110.00 - 0.32slight <0.01 b339vspb010201100.000.00 - 0.26slight0.01 b339vspb8334320800.330.03 - 0.62fair <0.01 b339vspb66420700.420.12 - 0.73moderate <0.01pb01vspb66027040.000.00 - 0.91slight0.0455pb01vspb8334028030.000.00 - 1.00slight0.0833pb66vspb8334327100.840.53 - 1.00high0.3173pb01vsepm83027040.000.00 - 0.90slight0.0455pb66vsepm83427001.001.00 - 1.00perfect0.32pb8334vsepm83327010.840.53 - 1.00high0.3173sodium dodecyl sulphate - polyacrylamide gel electrophoresis in 10% acrylamide with antigens filtered from culture of paracoccidioides brasiliensis stained with coomassie blue . Lines 1, 7: pageruler plus prestained protein ladder (fermentas); 2: epm83; 3: pb265; 4: pb01; 5: pb8334; 6: pb66 . Sodium dodecyl sulphate - polyacrylamide gel electrophoresis in 10% acrylamide with antigens filtered from culture of paracoccidioides brasiliensis stained with coomassie blue . Lines 1, 7: pageruler plus prestained protein ladder (fermentas); 2: epm83; 3: pb265; 4: pb01; 5: pb8334; 6: pb66 . Regarding the samples from the jundia region (10 sera reactive and 10 sera non - reactive to agb-339), again, agepm83 presented the highest reactivity rate among the five antigen preparations . This antigen reacted with seven of the 10 samples reactive to b-339 and with two of the 10 non - reactive samples . However, the k of these antigens was 0.5 (moderate), demonstrating a weaker agreement in comparison to their performance in the botucatu region . Agpb265 displayed an unsatisfactory performance, reacting with only two serum samples (20%) that were reactive to the standard antigen . A similar reactivity pattern was noted for the three antigens obtained from the p. lutzii isolates in sera reactive to the standard antigen: agpb01 (20%), agpb8334 (20%) and agpb66 (30%). These antigens also showed reactivity to three of the 10 samples negative for the standard antigen . The assessment of agepm83 performance against the 40 serum samples from the botucatu and jundia regions, considered to be endemic for p. brasiliensis, indicated that this antigen preparation was capable of discriminating among 22 samples, whereas agb-339 reacted with 20 . In addition, agepm83 reacted with five of the 20 (25%) sera that were non - reactive to the standard antigen . Interestingly, the p. lutzii antigens reacted with five of the serum samples from the jundia region, three of which had not shown reactivity against the antigen agb-339 . Considering the 31 serum samples from the central - west region of brazil, 11 showed serological reactivity against the b-339 antigen and none of the evaluated antigen preparations was capable of reacting with more than four of these 11 serum samples . Similarly, none of the 20 sera non - reactive to the standard antigen was reactive to any of these antigens . All of the antigens showed low k values when compared to the standard antigen . All the antigen preparations were also evaluated for specificity by a did assay against nine serum samples from patients with confirmed histoplasmosis and no cross - reaction was noted against the heterologous sera . Sds - page - among the antigen preparations, agepm83 had the most evident gp43 level by sds - page (figure) and also exhibited an intense band near 90 kda . The antigens obtained from the p. lutzii isolates showed several common bands between 100 - 60 kda . Agpb01 and agpb8334 showed additional bands at 43 kda, but of lower intensity than the signal for agepm83 . Analysis of ds / dn rate of pbgp43 exon 2 - exon 2 of the pb gp43 gene from the p. lutzii and p. brasiliensis groups had a ds / dn mean rate higher than 1.0 (supplementary data), indicating that the isolates in this region have undergone purifying selection and amino acid sequence conservation (nei & kumar 2000). However, the mean ds / dn ratio for the p. lutzii group (1.13) was lower than for the p. brasiliensis group (1.52), suggesting that the gp43 amino acid sequence is more variable among the p. lutzii isolates . Comparing the five antigen preparations, the antigen obtained from the epm83 isolate had a reactivity profile similar to the profile observed for the standard antigen (agb-339) against the serum samples from botucatu . An analysis of the antigenic profile of culture filtrates by sds - page indicated that agepm83 had a more evident band at 43 kda compared to the other antigens . This finding confirms the importance of the 43-kda glycoprotein in pcm immunodiagnosis (puccia et al . 1986), at least for regions in which the s1 and ps2 species occur (batista jr et al . It must be highlighted that the epm83 isolate belongs to the species ps3 and not to the species occurring in the botucatu and jundia regions . Regardless, ps3 isolates are known to be phylogenetically close to s1 (matute et al . 2006, theodoro et al . The reaction of agepm83, associated with high gp43 secretion, with sera non - reactive to the standard antigen in the botucatu and jundia regions suggests that this specific antigen may serve as an alternative when the standard antigen (agb-339) is not capable of detecting circulating anti- p. brasiliensis antibodies in the residents of regions in which the particular species s1 and ps2 occur . However, certain samples reactive to agb-339 from the jundia region did not show reactivity against agepm83, resulting in a moderate strength of agreement between these antigens in this region . This finding, associated with the low positivity of agepm83 reactivity with serum samples from the central - west region of brazil, indicates that it is not possible to use only a single antigen preparation to diagnose a disease caused by highly diverse agents, particularly in such a large country as brazil . Additionally, other authors have noted that an antigen produced from an isolate from the central - west region of brazil was capable of reacting with a larger number of sera from that region in comparison to the standard antigen from an isolate from sp (b-339). Nevertheless, when tested with sera from sp, the antigen from central - west of brazil did not produce satisfactory results (batista jr et al . These findings confirm the necessity of using different strains to produce antigens and indicate the epm83 isolate as a potential alternative that should be further studied . The present study confirmed the practical difficulties in producing antigens reactive to sera from patients from the central - west region of brazil, even against three isolates of p. lutzii, a genotype that appears to be the most prevalent in this region . This difficulty must be related to the greater diversity of gp43 in this species . Given its higher nonsynonymous mutation rate compared to the isolates of the p. brasiliensis group, the amino acid sequence of this glycoprotein must be more variable in p. lutzii isolates . Thus, patients from central - west of brazil would be sensitised by various gp43 forms, which could explain the difficulty in producing a single antigen reactive in all patients from this region . These facts cast doubt on the potential of gp43 as a universal antigen for all species in immunodiffusion assays, thus requiring a search for new alternatives . In addition to constituting a problem in serological diagnosis, the variability of this antigenic portion could represent an escape mechanism for avoiding host immune responses (matute et al . 1995), p. brasiliensis strains were observed to express variable quantities of gp43 (berzaghi et al . 2005). This result corroborates the difficulty in using this molecule for the universal serological diagnosis of pcm because a patient infected with an isolate that produces gp43 at low rates will not show antibodies specific for this antigen at detectable concentrations . Indeed, the determination of variations in the expression of genes encoding gp43 in p. brasiliensis is long - standing, but still unresolved problem . Although a large number of isolates from this group remain to be evaluated, our results suggest that isolates of the p. lutzii group generally have low gp43 production . Speciation events, however, are not sufficient to fully explain the variation in gp43 rates because not all the isolates of the s1, ps2 and ps3 species produce high quantities of this molecule . Eventual variations in the promoter region of the gp43 gene among the isolates and interference in posttranscriptional processes, such as alternative splicing and silencing by rna interference, can influence this variability . In summary, considering speciation within p. brasi - liensis is important for the serological diagnosis of the disease, particularly for the more distant genotypes represented by the p. lutzii group . Regarding the group represented by the s1 and ps3 genotypes, the most relevant factor in serological diagnosis is the expression of gp43, which is highly variable among isolates . Mutation pattern observed at pb gp43 exon 2 region in haplotypes of the paracoccidioides lutzii group seq 1 seq 2 sd nd s n ps pn ds dn ds / dn 01 7455 0.00 2.00 96.50 326.50 0.00 0.01 -0.00 0.01 * 01 84 0.00 1.00 96.67 326.33 0.00 0.00 -0.00 0.00 * 01 6810 0.00 3.00 96.50 326.50 0.00 0.01 -0.00 0.01 * 01 717 0.00 2.00 96.50 326.50 0.00 0.01 -0.00 0.01 * 01 3171 0.00 2.00 96.50 326.50 0.00 0.01 -0.00 0.01 * 01 189 1.00 1.00 96.17 326.83 0.01 0.00 0.01 0.00 3.42 01 218 0.00 2.00 96.67 326.33 0.00 0.01 -0.00 0.01 * 01 133 12.75 37.25 96.67 326.33 0.13 0.11 0.15 0.12 1.17 01 769 9.62 39.38 97.17 325.83 0.10 0.12 0.11 0.13 0.81 7455 84 0.00 1.00 96.83 326.17 0.00 0.00 -0.00 0.00 * 7455 6810 0.00 1.00 96.67 326.33 0.00 0.00 -0.00 0.00 * 7455 717 0.00 0.00 96.67 326.33 0.00 0.00 -0.00 -0.00 * 7455 3171 0.00 2.00 96.67 326.33 0.00 0.01 -0.00 0.01 * 7455 189 1.00 3.00 96.33 326.67 0.01 0.01 0.01 0.01 1.13 7455 218 0.00 2.00 96.83 326.17 0.00 0.01 -0.00 0.01 * 7455 133 12.75 37.25 96.83 326.17 0.13 0.11 0.14 0.12 1.17 7455 769 9.62 39.38 97.33 325.67 0.10 0.12 0.11 0.13 0.80 84 6810 0.00 2.00 96.83 326.17 0.00 0.01 -0.00 0.01 * 84 717 0.00 1.00 96.83 326.17 0.00 0.00 -0.00 0.00 * 84 3171 0.00 1.00 96.83 326.17 0.00 0.00 -0.00 0.00 * 84 189 1.00 2.00 96.50 326.50 0.01 0.01 0.01 0.01 1.70 84 218 0.00 1.00 97.00 326.00 0.00 0.00 -0.00 0.00 * 84 133 12.75 36.25 97.00 326.00 0.13 0.11 0.14 0.12 1.20 84 769 9.62 38.38 97.50 325.50 0.10 0.12 0.11 0.13 0.83 6810 717 0.00 1.00 96.67 326.33 0.00 0.00 -0.00 0.00 * 6810 3171 0.00 3.00 96.67 326.33 0.00 0.01 -0.00 0.01 * 6810 189 1.00 4.00 96.33 326.67 0.01 0.01 0.01 0.01 0.85 6810 218 0.00 3.00 96.83 326.17 0.00 0.01 -0.00 0.01 * 6810 133 12.75 38.25 96.83 326.17 0.13 0.12 0.14 0.13 1.14 6810 769 9.62 40.38 97.33 325.67 0.10 0.12 0.11 0.14 0.78 717 3171 0.00 2.00 96.67 326.33 0.00 0.01 -0.00 0.01 * 717 189 1.00 3.00 96.33 326.67 0.01 0.01 0.01 0.01 1.13 717 218 0.00 2.00 96.83 326.17 0.00 0.01 -0.00 0.01 * 717 133 12.75 37.25 96.83 326.17 0.13 0.11 0.14 0.12 1.17 717 769 9.62 39.38 97.33 325.67 0.10 0.12 0.11 0.13 0.80 3171 189 1.00 3.00 96.33 326.67 0.01 0.01 0.01 0.01 1.13 3171 218 0.00 2.00 96.83 326.17 0.00 0.01 -0.00 0.01 * 3171 133 12.75 37.25 96.83 326.17 0.13 0.11 0.14 0.12 1.17 3171 769 9.62 39.38 97.33 325.67 0.10 0.12 0.11 0.13 0.80 189 218 1.00 3.00 96.50 326.50 0.01 0.01 0.01 0.01 1.13 189 133 11.75 37.25 96.50 326.50 0.12 0.11 0.13 0.12 1.07 189 769 10.62 39.38 97.00 326.00 0.11 0.12 0.12 0.13 0.90 218 133 12.75 37.25 97.00 326.00 0.13 0.11 0.14 0.12 1.17 218 769 9.38 39.62 97.50 325.50 0.10 0.12 0.10 0.13 0.77 133 769 3.00 11.00 97.50 325.50 0.03 0.03 0.03 0.03 0.91 mean 4.19 15.09 96.80 326.20 0.04 0.05 0.05 0.05 1.13 dn: jukes - cantor correction for multiple values of proportion of observed nonsynonymous mutations (pn); ds: jukes - cantor correction for multiple values of proportion of observed synonymous mutations (ps); ds / dn: rate of synonymous mutations over nonsynonymous mutations; n: number of potential nonsynonymous substitutions; nd: number of observed nonsynonymous mutations; s: number of potential synonymous substitutions; sd: number of observed synonymous mutations . Asterisk means that ds is not significant due to the high similarity among sequences . Dn: jukes - cantor correction for multiple values of proportion of observed nonsynonymous mutations (pn); ds: jukes - cantor correction for multiple values of proportion of observed synonymous mutations (ps); ds / dn: rate of synonymous mutations over nonsynonymous mutations; n: number of potential nonsynonymous substitutions; nd: number of observed nonsynonymous mutations; s: number of potential synonymous substitutions; sd: number of observed synonymous mutations . Asterisk means that ds is not significant due to the high similarity among sequences . Mutation pattern observed at pb gp43 exon 2 region in haplotypes of the paracoccidioides brasiliensis group seq 1 seq 2 sd nd s n ps pn ds dn ds / dn a4 v3 1.00 5.00 97.83 325.17 0.01 0.02 0.01 0.02 0.66 a4 v2 2.00 11.00 97.83 325.17 0.02 0.03 0.02 0.03 0.60 a4 v1 2.00 5.00 97.83 325.17 0.02 0.02 0.02 0.02 1.33 a4 c1 0.00 4.00 97.83 325.17 0.00 0.01 -0.00 0.01 * a4 b26 1.00 10.00 97.83 325.17 0.01 0.03 0.01 0.03 0.33 a4 b25 0.00 1.00 98.00 325.00 0.00 0.00 -0.00 0.00 * a4 b23 2.00 11.00 97.50 325.50 0.02 0.03 0.02 0.03 0.60 a4 b21 0.00 3.00 97.83 325.17 0.00 0.01 -0.00 0.01 * a4 b18 0.00 5.00 97.83 325.17 0.00 0.02 -0.00 0.02 * a4 b1 1.00 1.00 98.00 325.00 0.01 0.00 0.01 0.00 3.33 v3 v2 3.00 8.00 97.33 325.67 0.03 0.02 0.03 0.02 1.26 v3 v1 1.00 0.00 97.33 325.67 0.01 0.00 0.01 -0.00 * v3 c1 1.00 1.00 97.33 325.67 0.01 0.00 0.01 0.00 3.36 v3 b26 2.00 7.00 97.33 325.67 0.02 0.02 0.02 0.02 0.96 v3 b25 1.00 4.00 97.50 325.50 0.01 0.01 0.01 0.01 0.83 v3 b23 3.00 8.00 97.00 326.00 0.03 0.02 0.03 0.02 1.27 v3 b21 1.00 2.00 97.33 325.67 0.01 0.01 0.01 0.01 1.68 v3 b18 1.00 2.00 97.33 325.67 0.01 0.01 0.01 0.01 1.68 v3 b1 2.00 4.00 97.50 325.50 0.02 0.01 0.02 0.01 1.68 v2 v1 4.00 8.00 97.33 325.67 0.04 0.02 0.04 0.02 1.69 v2 c1 2.00 7.00 97.33 325.67 0.02 0.02 0.02 0.02 0.96 v2 b26 1.00 1.00 97.33 325.67 0.01 0.00 0.01 0.00 3.36 v2 b25 2.00 10.00 97.50 325.50 0.02 0.03 0.02 0.03 0.66 v2 b23 0.00 1.00 97.00 326.00 0.00 0.00 -0.00 0.00 * v2 b21 2.00 8.00 97.33 325.67 0.02 0.02 0.02 0.02 0.83 v2 b18 2.00 8.00 97.33 325.67 0.02 0.02 0.02 0.02 0.83 v2 b1 3.00 10.00 97.50 325.50 0.03 0.03 0.03 0.03 1.00 v1 c1 2.00 1.00 97.33 325.67 0.02 0.00 0.02 0.00 6.77 v1 b26 3.00 7.00 97.33 325.67 0.03 0.02 0.03 0.02 1.44 v1 b25 2.00 4.00 97.50 325.50 0.02 0.01 0.02 0.01 1.68 v1 b23 4.00 8.00 97.00 326.00 0.04 0.02 0.04 0.02 1.70 v1 b21 2.00 2.00 97.33 325.67 0.02 0.01 0.02 0.01 3.38 v1 b18 2.00 2.00 97.33 325.67 0.02 0.01 0.02 0.01 3.38 v1 b1 3.00 4.00 97.50 325.50 0.03 0.01 0.03 0.01 2.54 c1 b26 1.00 6.00 97.33 325.67 0.01 0.02 0.01 0.02 0.55 c1 b25 0.00 3.00 97.50 325.50 0.00 0.01 -0.00 0.01 * c1 b23 2.00 7.00 97.00 326.00 0.02 0.02 0.02 0.02 0.96 c1 b21 0.00 1.00 97.33 325.67 0.00 0.00 -0.00 0.00 * c1 b18 0.00 1.00 97.33 325.67 0.00 0.00 -0.00 0.00 * c1 b1 1.00 3.00 97.50 325.50 0.01 0.01 0.01 0.01 1.11 b26 b25 1.00 9.00 97.50 325.50 0.01 0.03 0.01 0.03 0.37 b26 b23 1.00 1.00 97.00 326.00 0.01 0.00 0.01 0.00 3.38 b26 b21 1.00 7.00 97.33 325.67 0.01 0.02 0.01 0.02 0.47 b26 b18 1.00 7.00 97.33 325.67 0.01 0.02 0.01 0.02 0.47 b26 b1 2.00 9.00 97.50 325.50 0.02 0.03 0.02 0.03 0.74 b25 b23 2.00 10.00 97.17 325.83 0.02 0.03 0.02 0.03 0.67 b25 b21 0.00 2.00 97.50 325.50 0.00 0.01 -0.00 0.01 * b25 b18 0.00 4.00 97.50 325.50 0.00 0.01 -0.00 0.01 * b25 b1 1.00 0.00 97.67 325.33 0.01 0.00 0.01 -0.00 * b23 b21 2.00 8.00 97.00 326.00 0.02 0.02 0.02 0.02 0.84 b23 b18 2.00 8.00 97.00 326.00 0.02 0.02 0.02 0.02 0.84 b23 b1 3.00 10.00 97.17 325.83 0.03 0.03 0.03 0.03 1.01 b21 b18 0.00 2.00 97.33 325.67 0.00 0.01 -0.00 0.01 * b21 b1 1.00 2.00 97.50 325.50 0.01 0.01 0.01 0.01 1.67 b18 b1 1.00 4.00 97.50 325.50 0.01 0.01 0.01 0.01 0.83 mean 1.45 5.04 97.42 325.58 0.01 0.02 0.02 0.02 1.52 dn: jukes - cantor correction for multiple values of proportion of observed nonsynonymous mutations (pn); ds: jukes - cantor correction for multiple values of proportion of observed synonymous mutations (ps); ds / dn: rate of synonymous mutations over nonsynonymous mutations; n: number of potential nonsynonymous substitutions; nd: number of observed nonsynonymous mutations; s: number of potential synonymous substitutions; sd: number of observed synonymous mutations . Asterisk means that ds is not significant due to the high similarity among sequences . Dn: jukes - cantor correction for multiple values of proportion of observed nonsynonymous mutations (pn); ds: jukes - cantor correction for multiple values of proportion of observed synonymous mutations (ps); ds / dn: rate of synonymous mutations over nonsynonymous mutations; n: number of potential nonsynonymous substitutions; nd: number of observed nonsynonymous mutations; s: number of potential synonymous substitutions; sd: number of observed synonymous mutations . Asterisk means that ds is not significant due to the high similarity among sequences.
In september 2007, the american college of obstetricians and gynecologists (acog) in its committee's opinion recommended that labium minus operations can be performed to alter the size or shape (labioreduction) for the following medical indications: labial hypertrophy or asymmetrical labial growth secondary to congenital conditions, chronic irritation, or excessive androgenic hormones . Additionally, the acog committee opinion suggested that clinicians who receive request from patients for such procedures should discuss with the patient the reason for her request and perform an evaluation for any physical signs or symptoms that may indicate the need for surgical intervention . Also, acog viewed that women should be discouraged from cosmetic gynecologic surgery based upon variation of the anatomical appearance of female external genitalia . There are several surgical techniques that have been applied for labia minora labioplasty such as straightforward or partial amputation, central v - plasty (the wedge resection) and its modification, deepithelialized labioreduction, central wedge nymphectomy with a 90-degree z - plasty, inferior wedge resection and superior pedicle flap reconstruction, and laser labioplasty [28]. Existing surgical techniques of clinical applications for labioreduction of the labia minora had been detailed by ostrzenski elsewhere [9, 10]. Reviewing labium minus labioreduction techniques and performing some of those techniques, ostrzenski was guided to establish hypothesis that a surgical intervention for labium minus labioreduction should offer reduction of the height and length, should establish symmetry, should preserve natural color and contour of the labium minus, and should restore or create natural appearance of the labium minus frenulum (posterior edge of the fossa navicularis). In the observational prospective, multiple time case series clinical study, this hypothesis was tested, with study's objectives, to develop and to present a newly developed surgical intervention of labium minus labioreduction to determine applicability of this procedure, to evaluate aesthetic surgical outcomes, and to assess potential complications of this procedure . Today, woman's demands for labia minora labioplasty either for aesthetic motives or medical indication(s) are growing; therefore, such a new surgical intervention is very important not only for gynecologist but also for cosmetic - plastic surgeons, urologists, and general surgeons who perform labium minus labioreduction . Two out of three subjects presented with physical symptoms associated with the labium minus enlargements and one subject presented with dissatisfying appearance of her labia minora . The first subject was a 22-year - old caucasian woman, g0p0, unmarried, and a college student, who has been sexually active . The disproportionately protuberant labia minora were responsible for her symptoms such as persistent irritation leading to discomfort during physical activities and following voiding and defecation . Blood flow during menses significantly increased irritations and discomfort due to difficulties in maintaining personal hygiene related to enlargement of the labia minora . Also, she reported superficial dyspareunia, which was caused by twitching and inadvertently pulling the enlarged labia minora into the vaginal pool . Although she learned how to separate her labia minora enough to introduce a penis into her vagina to minimize superficial dyspareunia on insertion, during the act she could not control her discomfort caused by labia minora being brought into the vaginal introitus and the distal vagina . The second subject was a 27-year - old caucasian woman, g3 p3003, married, high school teacher, and sexually active . The symptoms included discomfort associated with rubbing while walking or wearing close - fitting underwear and superficial dyspareunia, which precludes her from reaching an orgasm during sexual intercourse . The third subject was a 22-year - old caucasian woman, g0p0, single, and a professional ballet dancer . This abnormality forced her to use specially designed compression underwear during her practices and performances . She does not report any physical discomfort when she does not wear compressive underwear on the vulvar area . However, when she wears it, she has significant discomfort, particularly, during her professional dancing . This condition caused a negative body image perception, which led to decreased self - confidence and a social phobia and anxiety . The subject requested to reduce the volume of both labia minora, which she has considered to be responsible for her deteriorated professional, social, and emotional well - beings . Aesthetic dissatisfaction with the subject's external genitalia led to social embracement and emotional disturbances . She felt extreme embarrassment not only during her professional dancing but also during her intimate life due to significant and disproportional overgrown of the labia minora . The subject requested to reduce the length and height and to create symmetrical and uniform appearance of the labia minora . All three women were subjected to newly developed fenestration labioplasty with inferior flap transposition under local infiltration . A search for the existing literatures was carried out from 1900 to may 2010, using medical subject headings (mesh) and keywords of fenestration labioplasty, fenestration labioreduction, cosmetic gynecology, labial reduction, labioreduction, labia minora labioplasty, labioplasty, labia minora procedures, female genitalia, labial hypertrophy, vaginal rejuvenation, vaginoplasty, designer vaginoplasty, designer vagina, and labium minus which were selected and used in a search on isi web of science (including conferences proceedings; 1950 pubmed), acognet, proquest, ovid, cochrane collection, the lancet on line collection, mdconsultant, new england journal of medicine, american college of physician on line resources, highwire journal, and citation index reference, and a manual search was utilized . An informed consent was structured in accordance with the existing recommendation of the american college of obstetricians and gynecologists . Additionally, all women authorized ostrzenski to use their clinical data and digital photo images of their genital organs for publishing in medical peer - reviewed journals . The procedure was executed under local anesthesia without conscious sedation . A thick layer of lidocaine - prilocaine (2.5%/2.5%) cream was applied to the labia minora and immediately adjacent areas bilaterally; the region was covered with sterile gauze for 1 hour, the last 30 minutes before procedures an ice pack was added to this area . Upon removing the ice pack, the remaining anesthetic cream was wiped off and the operative field was prepped with betadine solution . Half way between the posterior commissure and the upper part of the anus in the middle and the ischopubic ramus, just under the superficial transverse perineal muscle, 510 ml of plain 1% lidocaine was injected with the 27 g 1/2 inch needle and 10 cc syringes (terumo, elkton, md, usa) in one side for local anesthesia . The superficial part of the deep branch of the perineal nerve and the posterior labial nerves were infiltrated with one injection and provided with adequate local anesthesia for this procedures . Neither conscious sedation nor pudendal block was used . Upon determining the size of labia minora volume being reduced, the base of the lower margin of incision was determined and outlining of the amount of the tissues being removed was marked in the shape of a bicycle helmet within the anterior labial surface; see figures 1, 5, and 6 . Also, in this process the arch of the new labium was determined; see figure 1 . Shape was accomplished and excised, see figures 2, 5(b), and 6(a). By doing so, the labium was divided into two fragments: the superior strip was partially detached from the rest of the labium and the inferior part at the base of the labium minus . Immediately, the superior strip of the labium was sutured to the lower base edge of the labium minus . The anterior labial lamina and posterior labial lamina were sutured on both sites separately without suturing the erectile tissues between the labial laminae . Below the arch, the labium inferior flap is gradually reduced in the wedge shape (the proximal part of the flap being bigger and then the distal segment) gradually getting smaller and thinner to create natural look of the labia . The distal labium part of the inferior flap is modeled in the arch shape to meet in the midline with the opposite distal labium just above the posterior commissure . Such a tissue transposition creates the labium minus frenulum (posterior border of the fossa navicularis). The length of the inferior flap of the labium is trimmed and sutured in the same manner as presented above . The procedure was executed bilaterally in all the subjects; see figures 1, 2, 3, and 4 . Postoperatively, discomfort was controlled with external application of dermoplast, an antiseptic and pain relieving spray (medtech, jackson, wy, usa). The electronic and manual searches failed to identify fenestration labioreduction with inferior flap transposition or similar surgical intervention . Therefore, this presentation is the first description in the scientific - clinical literature of a fenestration labioreduction with inferior flap transposition technique . The disproportionately protuberant, enlarged, and asymmetrical labia minora were confirmed in each subject . All subjects reported feelings of decreased body image perception, being sexually inadequate and undesirable, and decreased self - image and confidence . Two women reported symptoms of persistent irritation leading to discomfort during physical activities, following voiding, defecation, and getting worse during menses as a result of difficulties in maintaining personal hygiene and reported superficial dyspareunia during sexual intercourse . The third subject presented with aesthetic dissatisfaction from her appearance of enlarged, asymmetrical labia minora . The newly developed operation of fenestration labioreduction with inferior flap transposition was applied without intraoperative, short- and long - term complications . In all subjects, the fenestration labioreduction with inferior flap transposition operation reduced the height and length, established symmetry, preserved natural color and contour of the labium minus, and restored or created natural appearance of the labium frenulum (posterior edge of the fossa navicularis). Postoperatively, medical and emotional symptoms and signs resolved; pleasing surgical outcomes exceeded subjects' aesthetic expectations . Additionally, body self - image and confidence improved meaningfully in all subjects . None of the subjects verbally reported feeling of regret and described reduction of the emotional tension, which was generated by conflict of being different and dilemma of feeling of being helpless . Social openness improved and intimate interaction increased, and their body image perception improved following the operation . There were no intraoperative, short-, or long - term complications recorded in all three subjects . The average time of surgery measured from the initial incision to completion of fenestration with inferior flap transposition was 36 minutes . All subjects engaged in vaginal sexual intercourse with their respective male partners 6 weeks following the surgery . This clinical study's results indicated that practitioners should look at the enlarged labia minora not only for physical symptoms (irritation, difficulties in maintaining personal hygiene, discomfort during physical activities, and discomfort during voiding or defecation) but also from the prospective of sexual dysfunction (pain during vaginal sexual intercourse) or emotional disturbances related to this condition (feeling inadequate, decreased feeling of body image perception, and being embarrassed socially). Therefore, not only clinical symptoms but also an aesthetic aspect plays significant roles in the labium minus enlargement . Implementation of fenestration labium minus labioreduction with inferior flap transposition demonstrates the use of clinical settings and eliminates potential for denuding the posterior vaginal introitus . Analyzing existing surgical techniques for labioreduction such as central v - plasty, central v - plasty, and central wedge nymphectomy with 90 z - plasty and w - plasty one can draw a conclusion that these surgical techniques will leave transverse single or multiple scars on longitudinal organ such as the labium minus [2, 4, 7]. An inferior wedge resection technique leaves completely denuded areas around posterior and lateral vaginal introitus, which lead to high superficial dyspareunia and unaccepted high rates of wound separations [5, 8]. Consequently, this technique will not only compromise aesthetic outcomes but also can be responsible for sexual dysfunction (superficial dyspareunia). Other labioreduction surgical interventions such as labial partial amputation, deepithelialized reduction labioplasty, inferior wedge resection and superior pedicle flap reconstruction will be appropriate for clinical implementations [2, 3, 6, 8]. Liao et al . And ostrzenski presented detailed evaluation of each surgical technique relating to labioreduction of the labia minora [9, 10, 13]. Differences between existing surgical procedures and the fenestration labioreduction with inferior flap transposition (flft) technique are significant . None of existing surgical interventions will encompass reduction of the height and length, established symmetry, preserved natural color and contour of the labia minora, and restored or created natural appearance of the labium frenulum (posterior edge of the fossa navicularis) in one procedure and only the fenestration labioreduction with inferior flap transposition technique incorporates all of them . When compared to partial labial amputation, flft preserved natural color and contour of the labium minus and restored or created natural appearance of the posterior edge of the fossa navicularis and partial amputation does not [2, 3]. Deepithelialized reduction labioplasty can only be offered for very thin and elongated labia minora, since it makes the labia minora much thicker or bulky at the base and flft will not do it . Deepithelialized reduction labioplasty can only reduce the height of the labia minora; flft will reduce both the height and the length of the labia minora . Deepithelialized reduction labioplasty will not restore or create the posterior boarder of the fossa navicularis and flft will . The inferior wedge resection and superior pedicle flap reconstruction will not restore or create the posterior boarder of the fossa navicularis and flft will . The inferior wedge resection and superior pedicle flap reconstruction will create unnatural appearance of the proximal labia connection and flft will not . The inferior wedge resection and superior pedicle flap reconstruction has tendency to stretch the superior pedicle flap and flft has not . The inferior wedge resection and superior pedicle flap reconstruction often creates undesirable permanent wrinkling and irregularity at the proximal approximation of the incision and the flft procedures are free of it . By all means, ostrzenski does not make any suggestion that flft is the only procedure that should be used in all cases . Therefore, the clinical judgment should be exercise, and all four relevant procedures (labial partial amputation, deepithelialized reduction labioplasty, and inferior wedge resection and superior pedicle flap reconstruction, and fenestration labioreduction with inferior flap transposition) should be taken into account and the surgical intervention which suits patient's needs should be selected . Small power of the study can be considered as a weakness; however, to test the established hypothesis the numbers of cases were sufficient to determine surgical applicability of flft . The importance of this study's results strongly suggest that not only aesthetic pleasing results can be accomplished in well - selected women by applying this surgical intervention but also the results imply that the clinical symptoms as well as emotional disturbances related to the enlarged labium minus can be eradicated . In this study group, fenestration labioreduction with inferior flap transposition surgical intervention can be executed effortlessly without complications and the method can be reproduced; aesthetically, the new operation achieves very pleasing results and the procedure improves physical-, emotional, and social well being.
The sporting career of an athlete depends not only on how soon he can return back to his sporting activity but also on the level of return to sports, with the least long - term complications . Rupture of anterior cruciate ligament (acl) results in a mechanically unstable joint, resulting in difficulty in athletic performance, increased risk of subsequent meniscal injury, and increased risk of early degenerative joint disease . Acl reconstruction is recommended in athletes to help restore knee stability for return to pivoting sports . Many different techniques using a variety of grafts with varying fixation techniques have continued to evolve to restore the stability to an acl - deficient knee . Numerous papers and meta - analyses have shown similar results by different graft materials using multiple graft fixation techniques [49]. However, the results on return to sports after acl reconstruction have varied [1012]. Although short - term evaluation is critical for assessment with regard to return to sports, an assessment 5 to 6 years after surgery is essential to determine the medium to long - term effect of surgery on maintaining knee joint stability, range of motion, restoring patient satisfaction while on field, returning to stressful pivoting sports, and development of complications if any . The ability to return to sports after acl reconstruction is governed not only by postoperative knee function but also by various other factors like social reasons, psychological impediments like fear of reinjury, and even monetary factors especially in sports persons of developing countries . There is a dearth of western literature regarding return to sports after surgery that is relevant to indian context making it difficult to counsel our patients regarding their eventual return to sports . To the best of our knowledge there is no study that evaluates the mid- to long - term results regarding return to sports after acl reconstruction in indian sports persons . The purpose of this study was to analyze the functional outcome in competitive level sports persons at 5 years after acl reconstruction . Our hypothesis was that reliable and sustainable results could be achieved over time using the arthroscopic technique of acl reconstruction . Additional goals were to assess function in the acl reconstructed knee, return to sports and level of sporting activity, patient satisfaction, identification of complications if any, and the factors or reasons in those who either stopped sports or showed a fall in their sporting levels . Between 2002 and 2005, records of 96 patients who underwent arthroscopic acl reconstruction by the single surgeon (first author) were procured . Patients with concomitant meniscal and chondral lesions were included whilst excluding those with multiligament injuries . 62 persons could be contacted out of whom 48 persons agreed to come for followup examination and interview . The mean age of our patients was 23.6 years (range 20.4 to 28.7 years). All were involved in competitive level sports at district and state level including 6 who were national level athletes . The sports played were wrestling (32 patients), kabaddi (8), athletics (6), and cricket (2). All had symptomatic and repeated episodes of instability despite conservative treatment and had wished to return to competitive sports that involved pivoting, cutting, and side stepping actions before proceeding to surgical procedure . All patients of our cohort underwent arthroscopic acl reconstruction using single incision transtibial technique by a single surgeon . Standard titanium interference screws were employed for fixation of patellar tendon graft with additional cortical screw post on the tibial side . For the hamstring grafts endobutton (smith & nephew, mass, usa) was used for fixation on the femoral side whilst using a biodegradable screw with tendon staple on the tibial side . 20 patients who had meniscal or chondral lesions or both were subgrouped and were compared with those patients who only underwent arthroscopic acl reconstruction the post operative program was standardized in all cases that involved quadriceps and hamstring isometric setting exercises, progressing to closed chain exercises and range of motion physiotherapy with the aim of regaining full range of motion by 6 weeks . Partial weight bearing was allowed at 3 - 4 weeks and light running on even ground, cycling, semi squats, and step exercises after 6 weeks . At 16 weeks, in addition to the strengthening exercises, sports - specific physiotherapy was instituted . Return to sports involving pivoting, cutting, or side stepping was permitted at 6 months after surgery if the patient had close to full range of motion and muscle strength . The patients were clinically examined and completed the subjective international knee documentation committee (ikdc) questionnaires, the lysholm knee form, and the tegner activity scale (tas). The ikdc subjective score is a questionnaire with different subjective factors such as symptoms, sports activities, and ability to function . The objective ikdc grading has 7 parameters related to the knee, reflecting both impairments and disability . The worst grading for the first three key parameters, that is, presence of effusion, knee range of motion, and ligament stability, determines the final ikdc grade . There are 4 grades a, b, c, and d implying, respectively, normal, nearly normal, abnormal, and severely abnormal . The lysholm knee score quantitates knee function, symptoms, and disability in a scale of 1 to 100 points, with 100 implying the best results and 1 the worst results . The tegner activity scale depicts the level of sporting activity and allows us to compare and document the preinjury activity level with the present activity level . All the patients in our cohort had preinjury tas level of 7 or more, which indicates that they were involved in competitive sports . At the time of review, they were asked whether they were still playing sports and whether they had returned to their preinjury levels of sporting activity . Return to sports was defined as returning to the same preinjury type and level of sports . Those patients who either stopped sports or showed a decrease in level of participation were asked to tell the reasons for the same . The group of patients who returned to the same level of sports was compared with the group of patients who either stopped sports completely or decreased their level of sporting activity . Statistical analyses using chi - square with yates' correction and one way analysis of variance (anova) test for independent samples were performed to compare results in patient groups to determine if the reasons for not returning to sports had any significant correlation to the documented objective and subjective scales . At 5-year followup, the mean lysholm score was 86.4 (sd = 8.8). 84.6% of patients had normal or nearly normal objective ikdc grade (a or b), while the remaining 15.4% had ikdc grade c (abnormal). The median preinjury tegner scale was 8 (sd = 1.1), and the median 5 years after acl reconstruction tegner scale was 7 (sd = 1.8). 8 patients out of the 48 that were reviewed at 5 years had left sports completely due to reasons other than sports . These included social reasons like marriage, getting into police and military services, and monetary reasons . Out of the remaining 40, 22 patients had returned to the preinjury levels of sports and 18 showed a decrease in their sporting levels . Of the 18 patients when asked for reasons for fall in sporting levels, 12 refered to fear of reinjuring the same or contra - lateral knee as the prime reason for the same . 6 patients refered to persisting knee pain, instability, annoying clicks, and numbness around the joint as reasons for a fall in their sporting abilities . Table 2 shows the results of various scores in the patient subgroups according to table 1 . We found that at 5-year followup, the subgroup of patients that had returned to preinjury level of sporting activity (45.8%) had the best scores this was in contrast to patients who showed a fall in their sporting levels because of painful and unstable knee (12.5%). Lysholm 74.3, subjective ikdc 64.6, and objective ikdc grade a and b 33.3% . Those patients who decreased their sporting levels due to fear of re - injuring their knee (same or contra - lateral) (25%) showed that they had intermediate scores lysholm 82.3, subjective ikdc 76.7, and ikdc grade a and b 75% . By statistical analyses, the difference in the outcome scores in the aforesaid three categories of patients was found to be statistically significant objective ikdc, subjective ikdc, and lysholm scale (p <0.05) (table 2). When we see their scores we find that lysholm was 88.9, subjective ikdc 86.4, and ikdc a and b 100% . They cited social reasons like marriage, monetary factors, and getting into police and military services as the main reasons for not continuing with sports despite having scores that were comparable with patients who returned to their preinjury levels of sporting levels (table 2). By this study we have reviewed the functional results at 5 years after arthroscopic acl reconstruction in a cohort of competitive level sports persons . The results that were quantitated by lysholm, subjective and objective ikdc, and tegner activity scale were comparable to those in previously published studies [15, 16]. In our study, subjective assessment with particular attention to return to sports and at what level was given more attention than objective findings, type of graft used, fixation method used, instrumented testing of joint stability, and investigations like roentogram . Noyes et al . Proposed the rule of thirds for chronic acl injury managed conservatively with rehabilitation and physiotherapy . They stated that one third of their patients resumed their previous recreational activities without reconstruction, one third managed by modifying their activity level and one third required reconstruction because of recurrent giving - way episodes even in day - to - day activities . . Showed in their followup of competitive hand ball players that 91% of players treated without reconstruction could return to their preinjury activity level compared to 58% in the reconstructed group ., however, found that at 6 years after acl injury, only 46% of their patients treated without reconstruction could return to preinjury sports . Kostogiannis et al . Indicated that many in their cohort who returned to sports at the same tegner level without reconstruction avoided contact sports as advised by the rehabilitation team . These kind of conflicting results in the literature create confusion in the mind of the attending surgeon who is counseling the injured sports person for acl reconstruction . However, the consensus rests on the suggestion that an athlete who wishes to return to his preinjury level should undergo reconstruction, especially competitive athletes or individuals engaging in pivoting sports [11, 12]. The literature is also full of a variety of grafts and fixation devices that are employed for arthroscopic acl reconstruction [49]. However most of them show similar results regarding stability, patient function, and final outcome . The median tas before injury in our patients was 8 and at five - year review it was 7 . This is comparable to that of 84.3 of matsumoto et al . And charlton et al . 84.6% of patients had normal or nearly normal objective ikdc grade (a or b). Return to sports is one of the most important outcome measures of a successful acl reconstructive procedure . In our study, 22 (45.8%) of the patients who underwent subjective and objective analyses at 5 years after their acl reconstruction had returned to their preinjury levels of sporting activities . In the literature the data for return to sports shows a wide variation51% (maletis et al . ), 53% (kvist et al . ), 65% (gobbi and francisco), 71.4% (smith et al . ), 92% (nakayama et al . ), and 100% (fabbriciani et al . ). The literature also shows that competitive level athletes are more likely to return to the same level of sports after acl reconstruction as compared to recreational level athletes . This may be one of the factors that account for a wide variation of percentage of return to sports as depicted in the literature . Report a 100% return of their cohort of 18 competitive level rugby players to the same level of sports after acl reconstruction at 6-month and at 2-year followup . The motivation to return to sports is very high especially in competitive sports persons after surgery . Reported that 81% of their patients who were competitive athletes returned to sports within 1 year of surgery . However, at mean followup at 43 months after surgery, this dropped to 71% of their initial cohort . Another interesting point was that 21.8% were still in sports despite major functional impairment in the operated knee . This study highlights the fact that a very high motivational factor may be the reason for a high return ration in competitive athletes . Also there is a significant fall in percentage when reviewed at 1 and at approximately 3 years after surgery . Thus assessment regarding return to sports should not only be a shortterm one but should also look at mid- to long - term results vis - visa return to sports . However, in our study of competitive athletes the return to sports was only 45.8%, due to reasons other than sports that include social reasons, monetary reasons, and fear of reinjury to the same or contra - lateral knee . In our study, if we exclude the 8 (16.6%) cases who had a stable symptom - free knee but had left sports due to social and other reasons, our results show that 55% of cases returned to preinjury levels of sports . But all these are western literature, where there are a dedicated team of sports - specific physiotherapists, a sports - specific psychologist to counsel the patients, and ample funding from government, and private sources to support the surgical costs, physiotherapy and rehabilitation of the injured sports person . Fear of reinjuring their knees and going through the surgery again, a prolonged period of physiotherapy, and remaining off the competitive field of sports proves to be a detrimental factor in the minds of our patients . This factor was found to be a major factor that led to fall in sporting levels in 12 of the 18 patients who showed a fall in their sporting levels at 5-year followup after arthroscopic acl reconstruction . The same has been observed by kvist et al . And lee et al . . Reported that 66.1% of their patients experienced fear of re - injury at 9.3 months . Reported that 72% of their patients who did not return to their preinjury levels of sporting activities feared instability . A striking point was that the majority (70%) of them had no objective knee instability . In our study, fear of re - injuring the same or contralateral knee was a major factor in 12 of the 18 cases who showed a fall in sporting activities at 5-year review . It has been observed also in our study where the results of various scores in the sub group of patients who had fear of re - injury were not poor but were intermediate (table 2) that is they were better than those who had a painful and unstable knee but worse than those who had returned to their preinjury sporting levels . Our study highlights an area that is often forgotten in the rehabilitation and evaluation after acl injury or reconstruction . No attempts are made to find the reasons for fear of disability to return to sports . Plausible factors that have not been evaluated are, for example, impaired knee proprioception and neuromuscular control possibly resulting in both decreased performance and increased fear of re - injury . The number of injured knee structures, objective knee stability, time between injury and acl reconstruction, and follow - up time are important factors that may influence performance . The long rehabilitation time and difficulties to regain a position in the sports team may affect motivation and cease the athlete's competitive career in favour of social and family life . Further prospective research combining assessments of psychological variables and functional tests is warranted in order to fully elucidate why patients return or not to their preinjury level and to fully establish the reasons . The tampa scale of kinesiophobia (tsk) has been used by kvist et al . To quantify the fear of re - injury due to physical activity . Their study reports a 53% return to preinjury level of sports after 3 to 4 years of acl reconstruction . A high score on tsk scale implying a greater fear of re - injury and pain correlated with patients who did not return to preinjury level of sports . The other group of 6 out of 18 patients who showed a fall in sporting levels pointed a painful and unstable knee as reasons for the same . This has been reflected very well in their outcome scores (table 2) which show poor subjective and objective scores . When we look at the three groups those who returned to preinjury level, those who showed a fall due to a painful, unstable knee, and those who showed a fall in levels due to fear of re - injury we find that the difference in the scores of the three groups was significant statistically . Possible factors that have been suggested for this are impaired knee proprioception and neuromuscular control leading to decreased performance and increased fear of re - injury . Our study highlights that the psychosocial issues that are relevant to the social milieu of the athlete are very important and affect the overall results of the surgery with respect to return to sports . Moreover, we found that there was a psychological fear in the mind of the athlete that his knee is weak and he can reinjure it more easily than the normal knee . The whole thing makes him afraid of rerupturing the graft as well as injuring the ligament in the contralateral knee as well . 8 of our patients left sports completely although on assessment their knees had good outcome scores (table 2). A thorough and in - depth counseling by the surgeon at the time of index surgery besides social and family support mechanisms including regular sport - specific physiotherapy and psycho therapy session prove to be of great help in this regard . Our study has limitations in the form of a short sample size and a high drop - out rate of followup at 5 years . A young, active population that undergoes this surgery has high relocation rates due to study and employment reasons . Long - term studies related to orthopedic sports medicine well document this problem of loss to follow - up . A national or regional level acl registry to follow up cases after surgery is called for in the current scenario . Despite the limitations, our study should prove useful to orthopedicians who operate and treat sports persons as they counsel them for surgery regarding the likelihood of eventual return to sports.
Ewing's sarcoma (ews) is an aggressive osteolytic tumor of bone and rapidly disseminates to other sites . It shares the histological features with other malignant small round cell tumors (msrct). Therefore the defining cytogenetic abnormality is a balanced translocation t(11;22) expressing the ews / friend leukemia integration-1 (fli-1) chimeric fusion protein . Metastatic ews should be diagnosed early as it is difficult to control and treatment needs a multimodality approach constituting chemotherapy, surgery and radiotherapy . The case we present here is a 14-year - old female child who presented with low backache since 1 year followed by right eye proptosis for 4 months and swelling over right side chest wall since 3 months [figure 1a]. Magnetic resonance imaging revealed involvement of posterior element of the sacrum with l5 vertebra and a presacral soft tissue mass measuring 11.8 cm 7.5 cm . Large abdominal soft tissue mass in pancreatic region measuring 6.2 cm 4.8 cm was seen indicating a possibility of nodal involvement . Venous invasion with tumor thrombus in left internal and common iliac veins and lower inferior vena cava was noticed . Fine needle aspiration smears from orbital mass were highly cellular showing cells arranged both in clusters and singly dispersed [figure 1c]. Cells were both light and dark types with a thin rim of pale cytoplasm and small round nuclei having small inconspicuous nucleoli . Rosette like structures, but no true rosettes were seen . Considering the clinical presentation, a provisional diagnosis of msrct possibly ews was given . Two tru - cut biopsy samples were taken from the same mass . From one sample, paraffin block was prepared . Histologically the tumor cells were arranged in irregular nests separated by dense fibrous stroma [figure 2a]. The cells were 1 - 2 times larger than small lymphocytes with scant eosinophilic or clear cytoplasm, centrally placed round nuclei with finely distributed chromatin and inconspicuous nucleoli [figure 2b]. Mitotic figures were few; necrosis scanty and rosettes were not seen indicating the absence of neural differentiation . The paraffin block was subjected to cd99 immunohistochemical marker and was strongly and diffusely positive [figure 2c]. Other sample was preserved in trizol reagent for molecular genetic analysis to look for ews - fli-1 translocation . Total ribonucleic acid (rna) was extracted from snap frozen tumor tissue using trizol reagent . Reverse transcription - polymerase chain reaction (rt - pcr) was performed with abl oligonucleotide primer to check for the quality of the rna isolated, which amplified a 300 bp product . The ews - fli-1 product was type 1 fusion (330 bp) [figure 2d]. (c) cytology of orbital mass (h and e 100) (a) photomicrograph showing small round cells in tissue section (h and e 100). Ews is a primary nonosteogenic malignant tumor of bone with peak incidence in second decade of life . We report the above case in a 14-year - old girl with involvement of l5 vertebra and sacral bone with metastasis to multiple sites at the time of presentation . Early and correct diagnosis of ews is essential for clinical management since it belongs to the group of primary msrct . This group includes neuroblastoma, lymphoma, desmoplastic small round cell tumor and rhabdomyosarcoma besides ews and peripheral neuroectodermal tumor where neural differentiation is seen in addition to all the features of ews . Since the morphologic features in all these tumors are more or less similar, one takes the help of special stains in addition to other ancillary techniques for differentiation of one from the other . Pas stain detects the presence of large amounts of intracellular glycogen, but it is a nonspecific finding since many childhood tumors contain it and up to 35% of ews may not contain detectable glycogen . Cd99 is a useful marker in the differential diagnosis of these tumors as 90% of ews / peripheral neuroectodermal tumors stain positive . Though neuroblastoma, the main tumor in the differential diagnosis is not positive for cd99, it is seen to be positive in other malignant small cell tumors such as small cell osteosarcoma, lymphoblastic lymphoma, mesenchymal chondrosarcoma, and alveolar rhabdomyosarcoma . The chromosome 22q12 breakpoints are clustered within a single gene designated ews and chromosome 11q24 are within a gene called fli-1 . This translocation results in the expression of an aberrant hybrid protein in which the n - terminal part of ews is linked to deoxyribonucleic acid binding domain (ets domain) of the fli-1 transcription factor . The ews - fli-1 protein is thought to be responsible for the origin of ews . Kelleher and thomas have suggested some promising therapeutics like ews - fli-1 gene silencing, growth factor - i receptor antagonists, mammalian target of rapamycin inhibition, kit oncoprotein targeting, etc . Although cytogenetic analysis can detect a wide variety of chromosomal translocations, it is time consuming, technically difficult and success rate is variable . Hence, we used the reverse transcription followed by pcr assay for detecting the presence and type of ews / fli-1 fusion transcript . The specific genetic alterations among other small round cell tumors can establish the genotypic diagnosis of these tumors . The most common site to which ews spreads, or metastasizes, is the lungs . Metastatic ewing's sarcoma is typically difficult to control, though patients with lung metastases have a better prognosis than patients with other distant metastases . Following diagnosis by tru - cut biopsy and cd99 immunopositivity (which was confirmed by rt - pcr analysis), our patient received three cycles of chemotherapy and no radiotherapy . After one cycle of chemotherapy, the right eye proptosis and swelling over the chest wall disappeared [figure 1b]. However after receiving next two cycles, she became bedridden and before receiving the 4 course of chemotherapy, the patient expired (within 1 year of diagnosis). This case exemplifies the idea that every new lesion in a patient with ews should be considered as a possible metastasis.
Most inhibitors occur at an early age and usually within the first 50 exposure days . As such however, it should be acknowledged that there are no clear data that demonstrate an increased risk of inhibitor when switching fviii concentrate in patients prior to 50 exposure days . Certain clinical scenarios have been associated with an increased risk of inhibitor development, and for some patients with haemophilia a, switching products requires careful consideration . Haemophilia patients with a history of inhibitors, including those in whom the inhibitor has been eradicated with immune tolerance induction (iti), may relapse and constitute a group of individuals at higher risk of inhibitor development . There may be similar concerns for haemophilia patients with a family history of inhibitor and/or a higher risk mutation in the f8 gene . As such, there may be reluctance on the part of the physician and the patient to consider switching products when they have been shown to be tolerant of their current therapeutic product . If such individuals are to have the opportunity to benefit from advances in therapy such as those with increased safety profiles or extended duration of action, they would need to consider switching products . In this situation, it should be noted that there is no evidence of increased risk of inhibitor development 6 . Lastly, intensive treatment (including surgery) is reported to be associated with an increased risk of inhibitor development 7 . As such, patients scheduled to have elective orthopaedic surgery should remain on their current product and switching in the intraoperative or early postoperative period should be avoided . However, for all patients, following discussions with patients or their caregivers, a product switch may be undertaken if there is a clinical need; there are no absolute contraindications for switching . For patients for whom product switching may be appropriate, a reluctance to switch products may be associated with concerns regarding the potential negative outcomes of such a switch . In addition, some patients with haemophilia often develop a strong psychological link with their current product 2 . To investigate patient concerns regarding switching, a semi - structured, non - random, brief, online survey was conducted using the web research platform surveymonkey. Participants from seven national haemophilia organisations (argentina, brazil, chile, santo domingo, mexico, nicaragua and spain) were informally invited (by e.r .) Through social media during 15 days in april 2013 . Survey participation was voluntary and a total of 46 participants (of whom 27.5% were parents of a child with haemophilia) anonymously completed the online survey (response rate 85%). Ethical standards for online behavioural research were strictly followed and all participants gave their electronic consent before taking the survey . Data were provided regarding haemophilia a (n = 37) and b (n = 9), of which the majority of patients had severe haemophilia (n = 27), and some patients had been diagnosed with an inhibitor (n = 9). Of note, 57% of the respondents believed that the probability of inhibitor development was high or very high when switching product . Moreover, when asked to list up to five concerns related to switching product, inhibitor development appeared first in the list (25.3% of respondents), followed by potential product side effects (21.8%), product effectiveness (17.2%), safety / purity (17.2%), and finally, product quality and longevity (4.6%). The original survey and complete report in spanish are available by email request to eduardo.remor@uam.es . To explore concerns regarding product switching by healthcare professionals, a recently conducted delphi consensus exercise was undertaken to canvass expert opinion on the topic 6 . Briefly, the delphi process is a structured group communication in which a complex problem is considered by a group of experts . The procedure usually begins with a face - to - face meeting to set the context of the communication, after which experts input their thoughts / opinions through several rounds of questions and answers . The delphi panel noted that currently available studies are often retrospective, characterised by a mixture of methodological approaches, and frequently lack appropriate control groups . Given this background, and the modest amount of data available on product switching, the delphi process provided an alternative approach to addressing the complex problem of assessing the risk of immunogenicity associated with product switching . The group addressed 14 separate items relating to the issue of product switching and the risk of inhibitor development and reached a high level of consensus on most items . They, too, concluded that much of current clinical practice regarding treatment switching in haemophilia was not based on evidence, but on the fear of developing an inhibitor 6 . Treatment - related factors, including treatment intensity as briefly mentioned above, therapeutic regimen (i.e. Prophylaxis vs. on - demand treatment) and product type have been proposed as possible influences on inhibitor development 712 . For example, a systematic literature review concluded that inhibitor incidence was lower in patients treated with one pd - fviii vs. those who had used multiple pd - fviii concentrates or a single rfviii product 12 . Although a more recent systematic review using multi - way analysis of variance concluded that source of concentrate did not significantly influence inhibitor development 11, suggestions of an increased incidence of inhibitor development and treatment with rfviii products, and also those with a b - domain deletion / modification, may continue to contribute to patient and physician reluctance to switch to new rfviii products . Three early studies in previously untreated patients (pups) suggested that the incidence of inhibitor development was less for those treated with pd - fviii than in patients treated with rfviii 1315 . However, significant differences in inhibitor development were only observed in two of these three studies 13,14 . In the uk study, inhibitors developed more frequently in patients initially treated with rfviii when compared with pd - fviii (p = 0.006) 13 . In a french cohort study, the risk of inhibitor development was reported to be higher in patients treated with rfviii than those treated with pd - fviii, regardless of other risk factors (e.g. F8 genotype, history of inhibitors in patients with a family history of haemophilia, age at first fviii infusion) 14 . However, in sweden, no significant increase in the incidence of inhibitors was reported for haemophilia a patients in the 1990s who were mainly treated with recombinant products (n = 10/48, total incidence 21%), as compared with the 1980s (n = 9/52, 17%), when patients received intermediate / high - purity plasma - derived concentrates 15 . The concerted action on neutralising antibodies in severe haemophilia a (canal) study was a retrospective, multi - centre cohort study designed to further describe the relationship between treatment and inhibitor development in 366 pups with severe haemophilia (residual fviii activity <2%) born between 1990 and 2000 9,16 . A total of 82 patients (26%) developed clinically relevant inhibitors; of 181 patients first treated with rfviii product, 53 (29%) developed inhibitors, while inhibitors were reported in 29 of the 135 (21%) patients treated with pd - fviii, and the relative risk (rr) of inhibitors in pd - fviii vs. rfviii products was 0.8 (95% confidence interval [ci], 0.51.3) 16 . In addition, switching between fviii products did not appear to increase the risk of inhibitor development (rr, 1.1; ci, 0.61.6). Hence, the canal study results do not support previous findings suggesting an increased risk of inhibitor development with rfviii products, nor that switching products may influence inhibitor development 16 . More recently, the potential influence of rfviii vs. pd - fviii product type on inhibitor development was also explored in the rodin (research of determinants of inhibitor development) study, which used data from the pednet registry that comprised 29 centres in europe, canada and israel . Overall, there was no difference in inhibitor risk between pd - fviii and rfviii products (adjusted hazard ratio 0.96; 95% ci, 0.621.49), and switching between different fviii products was not associated with an increased risk of inhibitor development (adjusted hazard ratio 0.99; 95% ci, 0.631.56). However, a significantly increased risk of inhibitor development was found to be associated with second - generation (produced in baby hamster kidney [bhk] cells) vs. third - generation full - length rfviii products (adjusted hazard ratio 1.60; 95% ci, 1.082.37) 7 . Although this latter finding is intriguing, there is no clear biological explanation for the difference in inhibitor development between second- vs. third - generation full - length rfviii . Concerns regarding a potential increase in immunogenicity associated with b - domain deleted rfviii were raised by an early italian study of previously treated patients (ptps) 17 . Of 25 low - risk ptps, one patient developed an inhibitor after switching from pd - fviii to b - domain - deleted rfviii 17 . Results from a more recent meta - analysis by aledort and colleagues of prospective clinical studies on product switching appeared to demonstrate an increased risk of inhibitor development with b - domain - deleted rfviii in ptps 8 . However, good results for meta - analyses come from inclusion of good data 18, and in this respect, of the two studies that contributed the most to the final odds ratio for the aledort meta - analysis, one consisted only of case reports 19 and the other contained only the prospective arm from the italian study 17 . The ongoing european haemophilia safety surveillance (euhass), a prospective adverse event reporting system, is exploring the incidence of inhibitors in pups and ptps and the potential factors that may be contributing to inhibitor development . Data reported from the first 2 years of the study, provided by 64 haemophilia centres from 27 european countries (caring for 22 242 patients), showed that the inhibitor rate in pups with severe haemophilia a was 25% overall, with a similar incidence of inhibitors in patients treated with rfviii (25%) as compared with those treated with pd - fviii (27%) 20 . For ptps, no significant difference was observed in inhibitor incidence between different rfviii products (including full - length and b - domain - deleted products) 20 . A more recent evaluation of data now available from the first 3 years of euhass has confirmed that in pups, there are no significant differences in inhibitor development between pd - fviii and rfviii products, or between different rfviii products 21 . Of note, the second - generation full - length rfviii product associated with increased inhibitor incidence in the rodin study was also one produced in bhk cells; together, these findings may lead to speculations that products may be associated with inhibitor development in pups . However, such speculation should be made with caution as many other variables may contribute to immunogenicity . Although early studies suggested a potential for increased inhibitor incidence in patients treated with rfviii and b - domain deleted product, the findings summarised above from canal, rodin and euhass do not provide support for these earlier suppositions . To date, studies on three national product switches (ireland, canada and the uk) have been published (table1) 2224 . While all three studies examined product switching, the product switches were different, and only the uk study investigated inhibitor incidence in both switchers and non - switchers . Summary of data from the three national product switches one patient had previously documented inhibitors, and one child who had been on prophylaxis with kogenate developed an inhibitor during intense therapy for treatment of an acute bleed . Patients remaining on kogenate. The difference in inhibitor incidence rates between switchers and non - switchers was not significant (p = 0.12). The national irish product switch resulted from a national tender process in 2006 in which all patients with haemophilia a changed their fviii treatment product en masse to a plasma and albumin - free recombinant full - length fviii product (advate) 22 . In this study, case records of irish ptps were retrospectively reviewed to evaluate the risk of inhibitor formation following this treatment switch . Only one of the 96 patients without a previous history of inhibitors developed an inhibitor following the switch . However, as this patient had only received three exposure days prior to the switch 22, the inhibitor might also have developed if the patient had remained on his previous treatment . In addition, there were no cases of recurrent inhibitor formation in any of 16 patients with previously documented inhibitors . The canadian national product switch surveillance study comprised 460 haemophilia a paediatric and adult patients from 17 canadian comprehensive haemophilia care centres, of whom 274 had evaluable data 24 . This study was conducted by the inhibitor subcommittee of the association of hemophilia clinic directors of canada to evaluate inhibitor development in patients with haemophilia a following the switch to a second - generation rfviii product (table1). An inhibitor was detected in four of the 274 (1.5%) evaluable patients at the time of the switch, but no additional patients with inhibitors were reported afterwards 24 . This finding highlights the importance of studying patients prospectively and testing for inhibitors before and after switching . A national tendering exercise conducted in the uk in 20092010 required half of patients receiving rfviii to change rfviii brands 23 . Based on the contractual requirements of the exercise, patients were randomly selected for switching in each local treatment centre . Centres were requested to test all patients for inhibitors prior to the switching date and 6-monthly thereafter . A total of 1217 patients with severe haemophilia a lacking an inhibitor history were analysed; 535 patients switched rfviii product and 682 patients did not . Four patients who switched, two from kogenate and two from advate, developed inhibitors; in two cases, the inhibitors were transient, while for the other two cases, patients were rapidly tolerised . The incidence of inhibitors reported (7.5/1000 treatment per years) did not significantly differ from the 5.31/1000 treatment year incidence observed during the 20-year period preceding the study (p = 0.24), although the study was underpowered . Of note, among the 682 non - switchers, one patient developed inhibitors . As these three studies employed different methodologies and studied heterogeneous patient populations, the findings of each cannot be directly compared, nor can the data be pooled for a combined analysis . However, all three studies suggest that switching is not associated with an increased risk of inhibitor formation relative to the very low background frequency of immunogenicity of these products, although the results are not conclusive and should be interpreted with caution . The implementation of prospective, controlled surveillance programmes on switching and not switching is imperative, as there is insufficient evidence currently available to support the development of clear best practice in addition, such surveillance programmes will provide researchers with the data needed to address the many unanswered questions regarding the patient - related and treatment - related factors that contribute to the risk of inhibitor development . Most importantly, for the full potential of surveillance programmes to be realised, all data on inhibitor development and products received should be submitted to a centralised, unbiased database to establish a baseline on the current inhibitor risk and include all new patients, regardless of which product they receive . This is the only way to ensure that the field is able to utilise all of the data in the service of our patients . Given the limitations of the existing evidence base, we can make a number of recommendations for the conduct of future studies on product switching . First, inhibitor testing should be performed before and after the switch to determine if any new inhibitors detected may be in association with switching to the new treatment . Similarly, inhibitor testing should also be performed before and after intensive treatment / surgery . Concerning routine patient care, educational materials addressing patients concerns about switching are needed because some of patients concerns are not supported by the actual evidence about the consequences of switching products . More importantly, physicians are encouraged to discuss directly with patients and parents their therapeutic approach and the other treatment options that are available before a more urgent need arises to consider switching . Doing so may increase patient satisfaction with treatment and foster more informed and positive attitudes when and if the need arises to address switching to a new product . In the future, it may become feasible in routine practice to calculate an inhibitor risk score and identify patients at high risk, thus aiding the evaluation of which patients to consider for switching treatments . Among patients with haemophilia (and their physicians), there is often a reluctance to switch factor concentrates because of concerns about increasing the risk of inhibitors . However, current evidence does not suggest that switching products significantly influences inhibitor development . With the forthcoming arrival of new haemophilia treatments, elena santagostino has received speaker fees for meetings organised by bayer, baxter, pfizer, csl behring, novo nordisk, biotest, kedrion, octapharma and grifols, acted as paid consultant for bayer, pfizer, csl behring, novo nordisk and grifols and has received unrestricted research grants from novo nordisk and pfizer . Victor jimnez - yuste has received reimbursement for attending symposia / congresses and/or honoraria for speaking and/or honoraria for consulting and/or funds for research from baxter, bayer, csl behring, grifols, novo nordisk, octapharma and pfizer . Thierry lambert has acted as a board member for baxter, bayer, csl behring, novo nordisk and pfizer . Rolf ljung has during the last five years received consultancy / speaker fees from novo nordisk, bayer, baxter and octapharma . Massimo morfini has served as a consultant and invited speaker for novo nordisk a / s, csl behring, wyeth / pfizer, baxter and bayer . Gnter auerswald, gary benson, gerry dolan and silva zupani alek have no conflict of interests to declare.
In view of growing awareness of the side - effects of some orthodox medicines, people are turning more towards alternative medicines with fewer and less - toxic side - effects, and homeopathy has become a major complementary and alternative medicine (cam) in many countries today . In homeopathy, microdoses of very high dilutions (potentized) of natural substances are generally preferred over mother tinctures (crude extracts) [2 - 4] for stronger and longer - effects . The initial drug substance is generally dissolved in an aqueous solution of ethanol (mostly 70%) and is potentized in gradual steps of dilution with agitation or succussion . On a centesimal scale, when 1 ml of mother tincture is diluted with 99 ml of an aqueous solution of ethanol (vehicle of drug) and given 10 mechanical jerks, potency 1c is produced . When 1 ml of 1c is again diluted with 99 ml of an aqueous solution of ethanol and given 10 jerks, the potency 2c is produced, and so on . Therefore, when the drug has attained potency 12c, it has been diluted to 10 (beyond avogadro s limit), and the existence of even a single molecule of the original drug substance becomes highly improbable . Although some researchers have demonstrated the existence of nanoparticles of the original drug in such ultra - highly diluted homeopathic drugs [5, 6], the efficacy is often questioned by rationalists, as the precise mechanism of drug action has still not been firmly established . Therefore, we became interested in the study of any perceivable differences in the actions between dilutions below avogadro s limit (6c, diluted 10 times) and above avogadro s limit (30c, diluted 10 times) in living cells, in vitro, and we attempted to understand the possible signalling pathway of their action . Non - small cell lung cancer (nsclc) is the most prevalent accounting for - 80% of all lung cancer cases . In addition, benzo[a]pyrene is one of the major polycyclic aromatic hydrocarbons found in cigarette smoke and is responsible for inducing lung tumours in smokers . Patients with lung cancer are commonly treated with conventional modalities including chemotherapy, radiation therapy, etc . Which also affect normal cells ., are now gaining importance in the cure / amelioration of many difficult- to - cure diseases including cancer . First, do the two potentized homeopathic drugs, condurango 6c and 30c show any ability to induce apoptosis in nsclc, and do they act via reactive oxygen species (ros) generation and mitochondrial membrane potential (mmp)-depolarization . Second, dose if condurango 30c have more apoptosis - inducing ability than condurango 6c . Nci - h460 (h460) human nsclc cells were procured from national centre for cell science (nccs), pune, india and cultured in rpmi-1640 media, supplemented with 10%-fetal bovine serum (fbs) and 1%-antibiotic - antimicotic solution . H460 cells (110 /well) were treated with different concentrations of condurango 6c and 30c (0.5l/100l media- 5l/100l media) and with placebos (successed 70% alcohol - vehicle for the drugs) as a control for 24 hours and 48 hours . The concentrations at which both the drugs showed nearly 50% cell death were determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assays . H460 cells were also treated with half maximal inhibitory concentration (ic50) doses of condurango 6c and 30c and were compared against placebo - treated and untreated cells . After 24 hours and 48 hours of treatment, the cells were observed and photographed under an inverted phase - contrast microscope (axiscope plus 2, zeiss, germany). For further confirmation of morphological changes, if any, a scanning electron microscopy (sem) study was done using an s530-hitachi scanning electron microscope . Reactive oxygen species (ros)-accumulation is generally known to occur at early hours of apoptosis . Cells were treated with condurango 6c and 30c (ic50 doses) and with their respective placebos for 2 hours, 6 hours, 12 hours, 18 hours, and 24 hours, to estimate the specific time - point at which maximum ros accumulated . After treatment, cells were incubated with 2,7-dichlorodihydrofluorescein diacetate (h2 dcfda) (5 mm) and ros was estimated by using fluorimetry (perkinelmer, usa). Fluorescence microscopy was done at the specific time - point(s) when ros - generation was maximum . The changes in mmp were recorded at 2 hours, 6 hours, 12 hours, 18 hours, and 24 hours of treatment with ic50 doses of both drugs were compared against drug - untreated cells by using rhodamine-123 and the cells were analyzed by using fluorescence microscope (leica, dmls). Changes in mmp were also measured using flowcytometry (facs, aria iii, bd bioscience) at the specific time - point(s) at which the mmp showed the maximum decrease . Cells were stained separately with 4,6-diamidino-2-phenylindole (dapi) (10gml) and acridine orange / ethidium bromide (ao / eb) (1mg / ml) to visualize changes in nuclear morphology . After 48 hours of treatment, stained cells were observed and photographed under a fluorescence microscope (leica, dmls). Dna - fragmentation was assayed using the conventional phenol / chloroform method and was visualized under a uv - transilluminator (ultracam digital imaging, genei, india). Dna strand breakage was analysed flowcytometrically by labeling the treated and the untreated cells with 5-bromo- 2-deoxyuridine 5-triphosphate (br - dutp) by using bd facs verse . The percent of cells in each phase (subg0/g1, g0/g1, s, and g2/m) was quantified flowcytometrically using bd facs verse . Rt - pcr analysis was done using primers of bax, bcl2, cytochrome - c, caspase-3 and gapdh . The cells were treated with ic50 dose of both drugs for 18 hours in the case of bax - bcl2, 24 hours for cytochrome - c, and 48 hours for caspase-3 and poly (adp - ribose) polymerase (parp). The protein activities were measured by using indirect- enzyme linked immunosorbent assay (elisa) and western blot analyses . Quantifications of developed proteins after the western blot analyses were done densitometrically by using image j software . Localization of caspase-3 was done by using an immunofluorescence study and was photographed under fluorescence microscope . The observers were blinded during observation as to whether they were observing the control and/or drug - treated materials . Data were analyzed, and the signicance of differences between the mean values was determined by using a one - way analysis of variance (anova) with fisher s least significant difference (lsd) post hoc tests by using spss 14-software (spss inc, chicago, il, usa). Ic50 values for 48 hours treatment with condurango 6c (3.57l/100l) and condurango 30c (2.43l/100l) against h460 were selected for the entire study (fig 1) the cell viability of both placebo - treated cells at maximum dose (5l/100l) was found to be very close to that of the untreated (control) ones . Cells were exposed to condurango 6c and 30c for 24 hours and 48 hours (0.5l/100l-5l/100l dose) with respective placebos (5l/100l). Results are expressed as mean percent of cell viability standard deviation (sd, n = 6). Signicance levels are presented as,p <0.05 vs. untreated cells andp <0.05 vs. both placebo - treated cells . The light microscopy (fig . 3) studies revealed that cell morphologies remained unaltered with intact cell membrane and cellular extensions in both untreated and placebo - treated cells . However, condurango 6c - treated cells showed gradual deformation with cellular shrinkage; condurango 30c - treated cells became gradually smaller and rounded with distorted nuclei and damaged cellular extensions, depicting the great apoptosis - inducing potential of condurango 30c . The fluorescence intensity in optical density (od) was too low in untreated and placebo - treated cells at different hour intervals . A significant increase in the fluorescence intensity was observed at 18 hours in both condurango 6c and 30c - treated cells (fig . Fluorescence microscopy showed a greater h2 dcfda - intensity in drug - treated cells at 18 h (fig . Signicance levels are presented as p <0.05 and * * * p <0.001 between untreated and condurango 6c and 30c - treated cells and between untreated and placebo - treated cells . Results showed up - regulation and downregulation of bax and bcl2, respectively, at 18 hours in response to both drug treatments as compared to drug - untreated cells (fig . Condurango 30c - treated sample showed significantly increased bax and decreased bcl2-expressions than condurango 6c and placebo - treated samples . Signicance * p <0.05 untreated (ut) vs condurango 6c and 30c, p <0.05 placebo (6c) vs condurango 6c andp <0.05 placebo (30c) vs condurango 30c . Figure 6a shows bright green fluorescence in drug - un - treated and cells at different time - points while condurango 6c and 30c - treated cells showed a gradual decrease in the fluorescence intensity with increasing time, but a marked decrease occurred at 24 hours, suggesting that mmp depolarizes the maximum at 24h that trigger cytochrome- c release from mitochondria . An increased expression of cytochrome - c was found in response to drugs at 24 hour of treatment (fig . Condurango 30c - treated cells showed more significant upregulation of cytochrome - c than condurango 6c - treated ones . Signicance levels are presented as * p <0.05 untreated (ut) vs condurango 6c and 30c, p <0.05 placebo (6c) vs condurango 6c andp <0.05 placebo (30c) vs condurango 30c . A gradual increase in the dapi - fluorescence intensity was observed in condurango 6c and 30c - treated cells at 48 hours of treatment (fig . Further, ao / eb- staining showed both a change in the fluorescence pattern from green (normal - cellular dna) to orange (nicked - cellular dna) and an increase in the fluorescence of eb in drug - treated cells (fig . Figure8c shows the formation of dna - laddering in both drug - treated samples, especially in condurango 30c - treated samples, compared to the dna in the drug - untreated groups . A gradual increase in the number of dutp - nicks was noted in drug - treated cells compared to drug - untreated cells (fig . This strongly suggests the abilities of both drugs to generate dna - nicks that induce apoptosis . Quantitative evaluation revealed the percent of tunel - positive nuclei was greater in condurango 30c - treated cells . The sub - diploid cell population at subg0/g1 was more highly increased in the condurango 6c and 30c - treated cells than in the placebo - treated and untreated cells (fig . Rt - pcr and elisa data showed upregulation of caspase-3 after condurango 6c and 30c treatment as compared to untreated samples (fig . The western blot analysis revealed that condurango 30c had more capacity to increase caspase-3 expression significantly by forming cleaved fragments (20kda and 12kda). P <0.05 untreated (ut) vs condurango 6c and 30c, p <0.05 placebo (6c) vs condurango 6c andp <0.05 placebo (30c) vs condurango 30c . The immunofluorescence study demonstrated a gradual increase in the localization of caspase-3 within cell - cytosol in drug - treated cells, especially in condurango 30c - treated cells (fig . Parp activation, the end process of the caspase-3-mediated pathway is designated by the formation of two cleaved fragments of 116kda (inactive) and 89kda (active). The western blot analysis showed the formation of two fragments in condurango 6c and 30c - treated cells at 48 h of treatment, which was nearly absent in drug - untreated samples (fig . Band - intensities confirmed gradual down - regulation of the 116kda fragment and significant up - regulation of the 89kda fragment, especially in the condurango 30c - treated cells . Present findings would demonstrate that exposure of nsclc- h460 cells to ic50 doses of both condurango 6c and 30c for 48 hours resulted in apoptotic cell - death . Further, the effects of condurango 30c, which actually was highly- diluted, caused relatively more palpable alterations in all parameters of this study than did condurango 6c . This phenomenon did not apparently follow the general pharmacological rule that the effect of a drug increases linearly with its concentration, but was in line with the claim of the higher the dilution, the stronger the effect as per homeopathic doctrine . Incidentally, apparent evidence for discernible effects produced by two dilutions, one below and one above avogadro s limit an enigma to many rationalists who believe in the accepted laws of physical sciences, as well as pharmacological sciences . However, claims are accumulating that homeopathic potencies beyond avogadro s limit show demonstrable beneficial/ curative effects against different diseases including cancer [20 - 22]. In fact, our earlier in vivo findings on benzo[a] pyrene - induced lung cancer in rats also convincingly demonstrated the ameliorative effect of condurango 30c, for which we tried to verify that the potentized remedy showed anti - cancer potential in nsclc cells . Apoptosis induction in cancerous cells is often targeted as one of the key events of cancer chemotherapy . Thus, one approach was to look at the generation of ros and to ascertain if it had a specific role in the induction of apoptosis . The time - course studies showed elevation of ros, mmp - depolarization and cytochrome - c release at 18 hours through 24 hours of treatment, indicating that these events were earlier than apoptotic execution at 48 hours . Our result for cytochrome - c release at 24 hours of treatment further suggested that these events occurred due to an alteration in the mitochondrial structure that could trigger apoptosis . Along with mitochondrial dysfunctions, ros generation may be initiated at early hours of drug exposure via bax- bcl2 modulation . In this study, we found increased and decreased expression of bax and bcl2, respectively, at 18 hours in the drug - treated series . However, surprisingly bax - bcl2 expressions were more significant in the condurango 30c - treated samples, which is a good indication of the higher efficacy of a more highly diluted homeopathic drug . Another indication of apoptosis is internucleosomal dna breakdown . Results of dapi and ao / eb - staining would suggest the formation of dna nicks, as further confirmed by dna - laddering in drug - treated cells . Tunel - positivity in both drug - treated cells as compared to placebo- treated cells would further strengthen ros - dependent dna - breakage - mediated apoptotic events . Interestingly quantitatively more dna damage was found in condurango 30c - treated cells, than was found in condurango 6c . Increased ratio of bax - bcl2 is well known to stimulate the release of cytochrome - c from mitochondria to promote activation of caspase-9 that binds to apaf-1 to lead to caspase-3 and parp - activation . Results provide clear indication of caspase-3 activation in drug - treated cells . Increased expression and formation of cleavage of caspase-3 and parp after drug exposure confirmed apoptosis induction via caspase-3-mediated- intrinsic pathway . However, noticeably, condurango 30c - treated cell showed more significant modulation of caspase-3 and parp cleavage than did condurango 6c - treated cells . These strongly support again the idea that more potentized condurango 30c had greater ability to induce apoptosis in nsclc - h460 . If the similar results are taken into consideration, one possible conclusion is that homeopathic drugs may act by interacting with certain high affinity receptors that regulate expressions of specific genes, but more work is necessary to identify some missing links . The present study has gain significance because it attempts to determine if potentized forms of condurango, below (6c) and above (30c) avogadro s limit, which are occasionally used to treat digestive problems and stomach cancer [22, 24], had abilities to inhibit lung cancer progression, in vitro . The various changes noted in this study can only be brought about by the activities of certain genes and by epigenetic modifications, which would support the hypothesis [3, 23] that ultra - highly - diluted drugs might somehow manage to correct expressions of relevant genes, the regulation of which had failed in cancer cells . Interestingly, many factors, including the presence of nanoparticles [5, 25], interaction between containers (e.g., silica from glass or a polymer) and drug molecules, etc ., are now under scrutiny and are implicated in modifications of the structural orientation, the size, and the physico - chemical properties of resultant homeopathic drugs even in absence of any original molecule [6, 26]. The overall results of this study suggest that condurango 30c has more apoptosis - inducing ability than condurango 6c, which is consistent with the claim made in the homeopathic doctrine.
Urinary tract infection (uti) is the most common infection contracted by renal allograft recipients . In patients of autosomal dominant polycystic kidney disease (adpkd), cyst infection presents a complex diagnostic and therapeutic challenge, especially in the post transplant period . Consequently, early and reliable detection of infected renal cyst is crucial for optimal patient management, especially when initial antibiotic therapy has failed . It has been infrequently reported in the post transplant scenario or in the adpkd patients . We report a case of post transplant xpn in the native adpkd kidney presenting as relapsing uti . 18-fluorodeoxyglucose (fdg)-positron emission computerized tomography (pet / ct) scan allowed the exact localization of the infection in the renal parenchyma and guided the therapeutic procedure with subsequent resolution of uti . A 53-year - old male of adpkd - ckd on maintenance hemodialysis since one and half years received a deceased donor renal transplant at our institute . His postoperative recovery was uneventful and he was discharged on tenth post transplant day with a serum creatinine of 1.2 mg / dl . He presented two weeks later with complains of fever, malaise and dysuria . On clinical examination, he was hemo - dynamically stable but febrile . Laboratory parameters included hemoglobin 10.2 gm / dl, leukocytes 8,600/cmm, platelets 2.9 lacs / cmm, glucose 186 mg / dl, urea 18 mg / dl, serum creatinine 1.7 mg / dl, sodium 138 meq / l, potassium 4.3 meq / l . Urine analysis showed, albumin 1 +, 40 - 50 leukocytes /high power field (hpf), red cells 4 - 5/hpf, no casts, bacteria, acid fast bacilli or fungus were found . Chest x - ray was normal, abdominal ultrasonography showed multiple hepatic and renal cysts in native kidneys, but was not suggestive of any infection, graft kidney was unremarkable . He was treated with antibiotics and responded well to the same and later discharged . Over the next three months patient presented twice with symptoms of uti and the urine cultures on both occasions grew escherichia coli . He was evaluated with ultrasonography and unenhanced computerized tomography (ct), which revealed a normal transplant kidney and multiple cysts without evidence of any infection, hemorrhage or calculi in native kidneys . Subsequently, patient underwent cystoureteroscopy with bilateral selective urine sampling from native kidneys, however culture revealed no growth . In an attempt to localize the source of infection it showed hyper - metabolic lesion arising in postero - inferior part of right native kidney [figure 1]. Pet / ct guided aspiration reveled purulent fluid, culture of the same yielded heavy growth of e. coli . On the basis of these findings, patient was subjected to right native kidney nephrectomy . The cut section of the gross specimen showed xanthomatous area [figure 2] and the histopathology was suggestive of xpn [figure 3]. Patient made a good recovery with s. creatinine returning to 1.3 mg / dl and urine culture being sterile on subsequent follow - up of three months . Pet / ct scan showing strong fdg uptake into postero inferior part of right native kidney (arrow) gross section of right kidney showing xanthogranulomatous area (arrow) light micrograph showing an interstitial infiltrate composed of neutrophils, mononuclear cells, and, most characteristically, lipid - laden macrophages (arrow). Febrile illness owing to complicated uti's secondary to renal or hepatic cyst infection is a common cause of graft dysfunction . If not localized early and redressed they can be a source of significant morbidity especially in an immuno - compromised host . It is characterized by replacement of renal parenchyma with diffuse or segmental cellular infiltrate of lipid laden macrophages called foam cells . Here, we report a case of rare occurrence of xpn in native polycystic kidney of a renal allograft recipient . In the literature, xpn in adpkd either in transplant scenario or otherwise has been infrequently reported. [46] in our case, conventional ct scan was non - contributory, however fdg pet / ct scan was helpful in localizing the site of infection . As the patient was not responding to conservative management it was decided to proceed with nephrectomy of infected kidney . Xpn was subsequently established on evaluation of gross specimen [figure 2] and histopathology [figure 3]. Fdg pet / ct scan in the recent times has emerged as a valuable tool for the transplant physician in localizing of infections especially in adpkd patients . Pet / ct can overcome interpretive challenges in identifying tissue infection, based on the high metabolic activity and increased uptake of the glucose - analogue fdg by inflammatory cells . Post transplant, uti especially in the adpkd patients is challenging to the transplant physician . This case highlights the rare occurrence of xpn as a cause of post transplant uti, especially in the absence of obstruction or stone disease . Fdg pet / ct imaging is a valuable tool in both localization and subsequent planning of therapeutic interventions for complicated utis associated with adpkd transplant patients.
Gestational diabetes mellitus (gdm) is defined as abnormal glucose tolerance that is first identified or diagnosed during pregnancy . It is estimated that approximately 2% to 5% of all pregnancies in korean women are complicated by gdm . The clinical significance of gdm is that it increases the risk of adverse pregnancy outcomes . In a recent large - scale multinational prospective study, increased maternal glucose concentration during pregnancy was significantly associated with increased neonatal birth weight, primary cesarean delivery, neonatal hypoglycemia, and increased placental c - peptide levels . After parturition, about 15% of gdm women had persistent diabetes at an early postpartum period of 2 months . Women with a previous history of gdm are at increased risk of future development of type 2 diabetes mellitus (t2 dm). The risk of t2 dm is 3.5 times greater in women with a history of gdm compared to the general population in koreans . In addition, offspring of gdm women are also at risk of developing obesity and t2 dm . The incidence of gdm is expected to rise, as it will parallel the increasing rate of obesity and t2 dm . Furthermore, the recent recommendation from the international association of diabetes and pregnancy study group has lowered the diagnostic threshold of gdm and is expected to increase the incidence of gdm . Gdm women have an increased positive family history of t2 dm . Compared to pregnant women with normal glucose tolerance, gdm women have a significantly greater parental history of t2 dm (13.2% vs. 30.1%, p <0.001). In addition, both gdm women and their offspring are at increased risk of future development of t2 dm . The heritability estimate of t2 dm was reported to be quite high (h = 0.69) in a recent study performed in europeans . These findings are indirect evidence that gdm has familial tendency . However, there is no study that has specifically evaluated the heritability of gdm using familial clustering or twins . It would be important to estimate the heritability of gdm and compare it with that of t2 dm in women . During normal pregnancy, women experience increased adiposity and weight gain, which begin near mid - pregnancy and progress throughout the third trimester . In this period, insulin resistance ensues as a consequence of multiple factors, including increased production of placental growth hormone, estrogen, and tumor necrosis factor [14, 15]. Pregnant women with normal glucose tolerance can increase their -cell insulin secretion in response to this increased insulin resistance during pregnancy . Although the mechanism of increased -cell insulin secretion during pregnancy is not fully understood, it is reported that prolactin, which increases during pregnancy, can repress islet menin levels and stimulate -cell proliferation in mice . In addition, recent reports suggest that -cell serotonin signaling is also a major determinant of -cell mass during pregnancy . It has been suggested from several clinical studies that gdm women have limited insulin secretion capacity that cannot compensate for the increased insulin resistance . Similar to gdm, t2 dm is also characterized by relative deficiency in insulin secretion in the face of increased insulin resistance . Various factors, including increased age, obesity, high - fat diet, and sedentary lifestyle, can induce insulin resistance, and those who do not have sufficient -cell insulin secretory capacity are more likely to develop t2 dm . Therefore, it is said that a large proportion of gdm women are experiencing future t2 dm in advance . Based on the findings that gdm women are at high risk of t2 dm and both gdm and t2 dm share similar pathophysiologies, it is reasonable to assume that they might also share similar genetic risk factors . Soon after the initial reports of t2 dm genome - wide association (gwa) studies [18 - 21], several studies investigated whether genetic variants that were identified through gwa studies of t2 dm were also associated with the risk of gdm [22, 23]. Genotyped variants in or near cdkal1, cdkn2a/2b, fto, hhex, igf2bp2, slc30a8, tcf7l2, kcnj11, and pparg in 869 gdm women and 632 carefully selected nondiabetic control subjects . They found that genetic variants in cdkal1 and cdkn2a/2b were highly associated with the risk of gdm (p <1 10). In addition, variants in hhex, igf2bp2, slc30a8, and tcf7l2 were all nominally associated with gdm (p <0.05). Among a total of 18 genetic variants studied, 9 reached a nominal significance level (p <0.05). In fig . 1, the risk allele frequency as well as the odds ratios of known t2 dm variants are compared among a control group (n = 632, men: women = 287: 345), t2 dm group (n = 761, men: women = 354: 407), and gdm group (n = 869) in koreans [22, 24]. For most of the variants, there was an increasing trend of risk allele frequencies from control to t2 dm and from t2 dm to gdm . . Also found that variants in tcf7l2, cdkal1, and tcf2 were significantly associated with the risk of gdm in europeans, consisting of 283 gdm women and 2,446 glucose - tolerant control women . Variants in kcnq1, which were first identified in an east asian t2 dm gwa study, were also significantly associated with the risk of gdm in koreans (p <0.05). Recently, it was reported that tph1 and htr2b play a crucial role in regulating pancreatic -cell mass during pregnancy in a mouse model and that their genetic alterations could result in gdm . A total 6 genetic variants in htr2b and 11 variants in tph1 were identified and genotyped in korean gdm women and control subjects . Although there were no significant associations of these variants with the risk of gdm, they were associated with measures of obesity and weight gain during pregnancy . Following these candidate approach studies was a two - staged gwa study that was performed in korean gdm women . A total of 468 gdm women and 1,242 nondiabetic control women were genotyped using affymetrix genome - wide human snp array 5.0 . Variants that passed the prespecified p - value threshold in the stage 1 genome scan were further genotyped in 931 gdm women and 783 nondiabetic control women . Two variants, one located in an intron of cdkal1 (rs7754840) and one near mtnr1b (rs10830962), were associated with a risk of gdm at a genome - wide significance level (p = 6.65 10 and p = 2.49 10, respectively). Although variants in mtnr1b have been previously reported to be associated with increased fasting glucose levels, the study was the first to report that mtnr1b variants are associated with gdm at a genome - wide significance level . One of the limitations was that it was not sufficiently powered to find truly novel genetic variants that were only specific in gdm . It should be noted that current gwa and gwa meta - analysis performed in t2 dm recruits more than 100,000 cases and controls . One of the interesting findings was that genetic variants of t2 dm were enriched in gdm subjects . Among the 34 confirmed t2 dm genetic variants, 8 were associated with a risk of gdm . In addition, when the -coefficients (or odds ratio) of the variants derived from the logistic regression were compared between gdm and t2 dm, there was a significant positive correlation of -coefficients between the two . These findings suggest that gdm and t2 dm might share similar genetic backgrounds, at least in part . Gwa studies have opened a new era in diabetes research . Our knowledge on the genetic predisposition of gdm as well as t2 dm is expected to increase even faster as next - generation sequencing technology is applied to this field . There should be even larger gwa studies on gdm, and gwa meta - analyses should be available . In this way, we could find variants that have smaller effect sizes but are more specific to gdm than t2 dm . In order to understand the genetic determinants of glucose and insulin concentration during pregnancy, a fast way is to see whether genetic variants that are known to affect glucose or insulin concentration in the normal population also affect glucose or insulin concentration during pregnancy . In addition, a gwa study on glucose or insulin concentration during pregnancy should also be helpful . In this way, we would be able to better understand the pathophysiology of gdm . By definition, gdm encompasses women with pre - exiting t2 dm, maturity onset diabetes of the young (mody), or even type 1 diabetes patients that were not diagnosed previously . In particular, about 15% of gdm patients remain diabetic at early postpartum periods, and a significant portion of these subjects might fall into the category of mody . The contribution of genetic variants known to cause mody, such as gck and hnf1a, in gdm has been reviewed in recent literature . However, whole - exome sequencing will provide us with better bird's eye view on the contribution of mody genes in gdm . In addition, it might be able to find novel mody genes in those who have persistent diabetes after gdm pregnancy and also have a strong family history of diabetes . One of the first steps in translating the genetic information into clinical practice would be to predict the future development of t2 dm in gdm women . Gdm women are at particularly high risk of developing t2 dm and require preventive measures and early screening of t2 dm . Genetic information might improve our prediction of t2 dm in women with a history of gdm . This is an area of active research [31, 32], and we are looking forward to studies that use genotype risk scores in predicting t2 dm in gdm women . A similar approach could also be applied in predicting gdm, as more genetic variants associated with gdm are expected to be revealed . The functional consequences of the current common genetic variants identified through gwa studies of gdm are not well understood yet . It is not known whether they are markers in linkage disequilibrium with nearby causal variants or whether they have unknown but relevant functional roles . Next - generation sequencing might give answers to these questions, but a huge number of samples and much effort will be required . Gdm women are at increased risk of developing t2 dm and have familial clustering of t2 dm . A common pathophysiology that is shared by gdm and t2 dm is impaired compensatory increase in insulin secretion to overcome the increased insulin resistance . The gwa study of gdm enabled us to investigate the common genetic risk factors of gdm, and it revealed that gdm and t2 dm share similar genetic backgrounds, at least in part (fig . Although this information has significantly improved our insight in the pathogenesis of gdm, there are more unanswered questions remaining that should be explicitly expressed . Using the technology of next - generation sequencing in addition, we hope that personalized genomic medicine could be available using the advances in the genetics of gdm and t2 dm.
Parkinson's disease (pd) is a common, progressive neurodegenerative disorder, the incidence of which rises with age, with the life - time risk of developing the disease standing at 1.5% (lees et al ., 2009). The two classical hallmarks are a progressive loss of dopaminergic neurons from the substantia nigra pars compacta (snpc), and the presence of aggregates of -synuclein, called lewy bodies, that are present in many regions of the central and peripheral nervous systems (lees et al ., 2009). The progressive loss of dopaminergic neurons in the snpc leads to the motor features of the disorder which are characterised by bradykinesia, akinesia and a resting tremor . There are also many non - motor symptoms such as cognitive dysfunction, orthostatic hypotension and gastrointestinal disturbances (lees et al ., 2009). Though administration of the dopamine precursor drug, levodopa, is a successful symptomatic treatment, its effectiveness wears off over time and levodopa - induced dyskinesias develop with prolonged use . There is therefore a critical need to develop new drugs and drugs targets to protect dopaminergic neurons and their axons from degeneration in pd . It has become increasingly recognized that epigenetic disturbances are found in patients with pd, and that these may play a role in parkinsonian pathology . One of the most intensively studied modes of epigenetic regulation is dna methylation (figure 1). This refers to the covalent methylation of residues in cpg dinucleotides within the dna sequence, by enzymes called dna methyltranferases (dnmts), and most commonly results in gene repression by blocking access to dna by transcription factors (labbe et al ., 2016). Evidence implicating global changes in the methylome is supported by observations of genome - wide changes in dna methylation in brain and blood samples from pd patients (masliah et al ., 2013). A distinctive pattern of methylation (both increased (hyper) and decreased (hypo)) was observed in both brain and peripheral blood leukocytes from these patients, involving many genes previously associated with pd (masliah et al ., such findings highlight the fact that methylation patterns have the potential to be employed as epigenetic biomarkers for pd . In support of these findings, a recent study examining methylation at the cellular level using induced pluripotent stem cell (ipsc)-derived dopaminergic neurons from patients with genetic (monogenic lrrk2-associated pd) and sporadic forms of pd showed extensive differences in dna methylation, and subsequent gene expression, in ipsc - derived dopaminergic neurons from these individuals compared to controls (fernandez - santiago et al ., intriguingly, these differences were not seen in parental skin cells, undifferentiated ipscs or ipscs not enriched in dopaminergic neurons (fernandez - santiago et al ., 2015), suggesting that there may be something unique about the dopaminergic methylome in pd . For example, administration of methionine (which increases global levels of dna methylation) was found to decrease levodopa - induced dyskinesias, whereas administration of rg-108 (which reduces global levels of dna methylation) exacerbated levodopa - induced dyskinesias (figge et al ., 2016). This highlights the importance of the dna methylome in pd - associated motor function, and raises the potential for pharmacological manipulation of the methylome as a viable therapeutic strategy for pd . Histone acetylation occurs when enzymes called histone acetyltransferases acetylate lysine residues in the n - terminal of histone proteins . This neutralizes the positive charge on the histone tail, decreasing their interaction with negatively - charged dna . This results in loosening of the dna around the histones, rendering the dna more accessible to transcription factor binding and enhanced gene transcription . These densely - methylated islands can directly prevent transcription factor binding and therefore reduce gene expression . Ac: acetyl group; cpg: cytosine - phosphoric acid - guanine motif; dnmt: dna methyltranferase; hat: histone acetyltransferase(s); hdac: histone deacetylase(s); mbp: myelin basic protein . A second intensively - studied mechanism of epigenetic regulation is post - translational modification of the n - terminal tails of histone proteins, around which dna is normally coiled (figure 1). Although there are many types of post - translational histone modifications, including methylation, acetylation, phosphorylation, ubiquitination and sumoylation, histone acetylation at lysine residues is particularly important (labbe et al ., 2016). Histone acetylation is regulated by a balance between histone acetyltransferases (hats) and histone deacetylases (hdacs). Hats add acetyl groups to histones, resulting in a less condensed chromatin structure, thereby facilitating transcriptional activation, whereas hdacs remove acetyl groups from histones, exerting the opposite effect (labbe et al ., 2016). As dysregulation in histone acetylation has been implicated in the pathogenesis of pd, drugs which alter levels of histone acetylation may have therapeutic potential (harrison and dexter, 2013). A recent study has shown that the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (mpp) increases the levels of acetylated histones in experimental models of pd, and that there are increased levels of acetylated histones in the brains of pd patients (park et al ., 2016). This suggests that changes in the levels of histone acetylation may either 1) play a causative role in the neurodegenerative process in pd, or 2) contribute to an endogenous compensatory response in an attempt to counteract the neurodegeneration . A number of studies have shown that pan- and class - specific hdac inhibitors have neuroprotective effects in cellular models (harrison and dexter, 2013). However, it is important to note that not all hdac inhibitors have been shown to be neuroprotective, with a recent study showing that a hdac1/2 inhibitor exacerbates mpp - induced toxicity in a sh - sy5y cell model of pd (park et al ., 2016). Conversely, another study showed that an alternative hdac1/2 inhibitor was neuroprotective against mpp - induced dopaminergic toxicity both in vitro and in vivo (choong et al ., 2016). Moreover, it has also been recently shown that other hdac inhibitors can protect other neuronal cell types affected by pd, as it was shown that hdac inhibition protected both dopaminergic and sympathetic neurons from mpp - induced cytotoxicity (collins et al ., 2015). These contrasting findings are not easy to reconcile, and may reflect subtle compositional and functional differences in the hdac inhibitor molecules . Furthermore, the precise phenotypic outcomes of hdac inhibitors may also be concentration dependent, as highlighted by our recent work on the p300/cbp hat described below . The potential of hdac inhibitors for clinical translation is highlighted by an on - going phase i clinical trial of the fda - approved drug glycerol phenylbutyrate (an hdac inhibitor), which is exploring the potential of this drug to increase the removal of -synuclein from the brain (nct02046434). In addition to hdac inhibition, an alternative approach to increase histone acetylation is the induction of hat activity using hat activators . However, there has been limited research into the potential of hat activators as potential drug therapies for pd . To begin to address this potential, we employed a selective and potent small molecular activator of p300/cbp known as ctpb (n-(4-chloro-3-trifluoromethyl - phenyl)-2-ethoxy-6-pentadecyl - benzamide) (balasubramanyam et al ., 2003). Ctpb is a benzamide that activates p300/cbp hat activity and induces p300/cbp hat - dependent transcriptional activation, but has no effect on p300/cbp - associated factor (pcaf) or histone deacetylase activity (balasubramanyam et al ., 2003). To investigate the neurotrophic potential of ctpb in pd, we examined the survival- and growth - promoting effects of ctpb in the sh - sy5y neuronal cell line, a widely used model of human dopaminergic and sympathetic neurons (hegarty et al ., 2016). We found that ctpb - induced p300/cbp hat activation dose - dependently promoted the survival and neurite growth of sh - sy5y cells, and that ctpb significantly increased histone acetylation in these cells, most likely through induction of p300/cbp hat activity . Moreover, this study found that ctpb was capable of protecting sh - sy5y cells from the cell death induced by the dopaminergic neurotoxin 6-hydroxydopamine (6-ohda) (hegarty et al ., 2016). Collectively these data suggest that increasing the levels of histone acetylation either through hdac inhibition or hat activation may be neuroprotective . However in contrast to this, another recent study found that garcinol - mediated inhibition of p300/cbp and pcaf hats protected sh - sy5y cells against mpp+-induced cell death (park et al ., 2016). Again, it is difficult to rationalize why both activation and inhibition of p300/cbp hats could be neuroprotective in these cellular models of pd, but it could reflect intrinsic differences between ctbp and garcinol, with garcinol (but not ctpb) targeting pcaf hat activity . Moreover, unpublished observations from our laboratory have shown that garcinol induces cell death in sh - sy5y cells, and that it exacerbates the toxic effects of 6-ohda (figure 2). Further research is required to determine the basis of these contrasting findings, but again such discrepancies may reflect differences between the concentrations of garcinol used in these studies . A key challenge for future research is how to optimize the delivery of hat - targetting molecules to the brain . Interestingly, carbon nanosphere - conjugated ctpb has the ability to cross the blood - brain barrier, localize to specific nuclei in the brain and induce hyperacetlyation in vivo (selvi et al ., 2008). Taken together, these studies demonstrate that small molecule - mediated p300/cbp hat activation may be an avenue to explore for neurotrophic effects in pd . Garcinol dose - dependently induces cell death in sh - sy5y cells, and significantly exacerbates the neurotoxic effects of 6-ohda . (a) standardised thiazolyl blue tetrazolium bromide (mtt) assay of sh - sy5y cells treated daily for 4div with either control or garcinol (0.5100 m), as indicated (* * p <0.01, * * * p <0.001, vs. control; anova with post - hoc tukey's test . Standardized mtt assay (b) and lactate dehydrogenase (ldh) assay (c) on sh - sy5y cells treated with 15 m 6-ohda in the presence or absence of 1 m garcinol for 24 hours (* * p <0.01, * * * p <0.001, vs. control; + p <0.05, + + + p <0.001, vs. 6-ohda alone; one - way anova with post - hoc tukey's test; n = 6). In summary, small molecule epigenetic modulators, such as those targeting dnmts, hdacs and hats, hold much promise as pharmacological modifiers of the epigenetic status of the cns, especially considering their ability to cross the blood - brain barrier . These drugs therefore have the ability to act both centrally and peripherally in the nervous system, and have the potential to protect all neurons affected by pd . However, further research is required to elucidate the precise mechanisms leading to the chronic epigenetic dysregulation observed in neurodegenerative diseases, such as pd . In addition to this, much more work is needed in translational animal models of pd for rationalizing the use of small molecule epigenetic modulators as a potential neuroprotective therapy for this disorder, including exploring strategies to deliver these drugs to the brain . As shown for ctpb, carbon nanosphere - conjugation offers a method to deliver such molecules to the brain . Although the beneficial effects of epigenetic modulators, such as hdac inhibitors, are yet to be reported in clinical trials for pd, there is much evidence to support the continued study of molecules that target the epigenome as novel neuroprotective therapies for pd.
Androgens control sexual function in the male and are central to the anabolic processes that underlie the development of male sexual and physiological characteristics [mooradian et al ., serum androgen levels are low prior to puberty and climb exponentially during adolescence in the male, leading to the enhanced muscle growth and lean body mass typical of the gender . In the mature adult they range from 600 to 700 ng / dl . As men age, androgen levels decline during the so - called andropause, reaching 450 to 500 ng / dl in their seventies and eighties [flynn and hellstrom, 2001]. Concomitantly, lean body mass decreases and older men are frailer, with a greater tendency to falls and bone fractures . Testosterone (t), acting via its more potent natural metabolite, dihydrotestosterone (dht), stimulates maturation of the prostate during sexual development . Dht is produced from t by 5--reduction, and it binds to the androgen receptor with about five times greater affinity than t. the enzyme 5--reductase is expressed tissue - specifically, most prominently in prostate and in hair follicles . This has been taken advantage of pharmacologically, with the generation of specific inhibitors of the enzyme for the treatment of androgen - dependent disorders . For example, finasteride is a 5--reductase inhibitor that prevents local synthesis of dht and is used to treat alopecia . T also has important physiological effects that are independent of dht, including anabolic effects on muscle, maintenance of testicular function, and inhibition of pituitary gonadotropin secretion . Prostate maturation results in the production of prostatic secretions that form a component of semen, and thus it is essential for normal fertility . Removal of circulating androgens by testicular or medical castration in humans and rodents results in apoptosis of prostatic cells and shrinkage of the prostate . The stimulatory effect of androgens on the prostate throughout adulthood is often thought to be primarily responsible for the increased risk of prostate cancer after the age of 40, although there is no direct proof of this . Nevertheless, localized and early stage metastatic prostate cancers are readily treatable by surgery and combined androgen blockade (cab). Cab typically consists of treatment with a gonadotropin - releasing hormone (gnrh) agonist (such as leuprolide), to shut down physiological sex steroid synthesis (surgical castration, or orchidectomy, has the same result); and an androgen antagonist (such as flutamide or bicalutamide), to shut down androgen - responsive pathways . Androgen antagonists have been approved in the us only for cab of advanced prostate cancer . Cab is an expensive therapy and men experience hot flushes and bone loss due to the depletion of anabolic steroids . Recently, monotherapy with androgen antagonists has shown to be as effective as cab in preventing disease recurrence [see et al ., 2002], and some european regulatory authorities have approved the use of bicalutamide for this purpose . However, both currently used androgen antagonists, flutamide (eulexin) and bicalutamide (casodex), cause breast tenderness and gynecomastia, which limit their use for monotherapy . These side effects are due to the propensity of the compounds to raise serum estrogen levels, which in turn is due to antagonism of normal feedback inhibition by testicular androgens at the pituitary . With feedback inhibition blunted, t synthesis continues, serum t levels rise, and the excess is aromatized into estrogen [mcleod and iversen, 2000]. Benign prostatic hyperplasia (bph) is a common condition in middle - aged and older men . The stromal compartment of the prostate grows larger and obstructs urethral flow, resulting in difficulty in urination . The etiology of bph is poorly understood, but the condition can be treated with inhibitors of dht formation (finasteride) or with adrenergic antagonists . Androgen antagonists are also effective [stone and clejan, 1991], but they are not used due to their side effects on the male breast . Testosterone and dht are also active in the skin, where they appear to maintain normal secretory activity and hair growth . Facial hair (hirsutism) is a common symptom of hyperandrogenism in women . In the scalp, where androgens have a different mechanism of action, excessive androgen levels cause male - pattern baldness (alopecia). Testosterone and dht act via the androgen receptor (ar), which is a member of the nuclear receptor superfamily . Binding of an agonist ligand to the receptor induces conformational changes that result in binding of the receptor - ligand complex to dna elements adjacent to target genes in the genome, followed by changes in gene expression . These gene expression changes lead to cellular effects including increases in proliferation and metabolic activity . Androgen antagonists bind to the receptor, preventing binding of the natural steroid, but do not produce the correct receptor conformational change . Thus, the classic view of receptor - antagonist complexes is that they fail to elicit normal changes in gene expression, disrupting androgen - signaling pathways ., 2000; sack et al ., 2001], and it has the typical helical sandwich structure found in other nuclear receptors . In the past several years, the concept of tissue - selective nuclear receptor ligands has emerged . This concept has come to fruition with estrogens, with the successful marketing of drugs such as raloxifene . Raloxifene is an estrogen antagonist in some estrogen - responsive tissues (breast and uterus), and an agonist in others (bone). While the molecular mechanism of action of these selective estrogen receptor modulators (serms) is not understood, a commonly accepted hypothesis is that tissue - specific coregulators interact with the steroid receptor - ligand complexes to modulate their activities in different ways [heinlein and chang, 2002]. The discovery of serms has raised the possibility of generating selective compounds for other pathways, including androgens (that is, selective androgen receptor modulators, or sarms) [zhi and martinborough, 2001]. For example, a compound that is an antagonist at the prostate, but an agonist or weak antagonist at the pituitary, would have an improved side effect profile over currently marketed androgen antagonists . Similarly, a compound that is a strong agonist on muscle, but an antagonist or weak agonist on prostate could be used to treat muscle - wasting conditions and age - related frailty, with a reduced concern for the potential to stimulate nascent or undetected prostate cancer . Three are non - steroidal and the fourth (cyproterone) is a steroid with mixed progestational and androgen antagonist activities . Of the non - steroids, bicalutamide is currently the biggest seller, probably due to its long half - life relative to flutamide, which facilitates once - per - day dosing . However, flutamide has recently come on the market as a generic in the united states, which is likely to significantly impact the future sales of bicalutamide here . Bicalutamide itself will come off patent in 2008 . Nilutamide s patent has also expired, but it was always a poor third to its competitors in terms of side effects and clinical use, and has not been developed as a generic compound . There is a large body of literature supporting their efficacy in animal models of alopecia [pan et al ., 1998; 2000] and in clinical trials treating alopecia [diamanti - kandarakis, 1999], hirsutism [muderris et al ., 2002; venturoli et al ., 1999; venturoli et al ., a new androgen antagonist, ru 58841, is in phase ii clinical trials in europe for the topical treatment of acne and alopecia . As mentioned above, androgen antagonists also have clinical efficacy in bph [stone and clejan, 1991]. To our knowledge, there are no non - steroidal androgens other than ru 58841 in clinical development . A number of companies, including bristol myers squibb, ligand pharmaceuticals, gtx technologies and karo bio have internal sarm research programs . The next section will review new classes of non - steroidal androgen receptor ligands that are in the public domain . Some progress has been made in identifying new structural classes of non - steroidal sarms since the launch of flutamide (figure 1, structure 1), nilutamide (figure 1, structure 2) and bicalutamide (figure 1, structure 3). Among many structures explored, the following three series have received the most attention: 2-quinolone and coumarins (figure 2, series 4), phthalimide analogs (figure 3, series 5) and bicalutamide derivatives (figure 4, series 6). As frequently seen in modulators of other steroid receptors such as the progesterone receptor, small structural changes can lead to the reversal of agonistic and antagonistic activity . Both 4a and 4b bind to ar in the nanomolar range, with kis of 26 nm and 21 nm, respectively . Compound 4a seems more potent and more efficacious in a cv-1 cell functional assay (4a: 74% efficacy, 27 nm antagonist potency; 4b: 39% efficacy, 34 nm antagonist potency). In addition, 4b showed some agonist activity in the same cells (39% efficacy, 125 nm potency). However, in castrated immature male rats, 4b demonstrated better efficacy as an antagonist . It inhibited testosterone - induced increases in prostate weight and seminal vesicle weight (4a: ed50 [prostate] = 18 mg / kg, ed50 [seminal vesicle] = 19 mg / kg; 4b: ed50 [prostate] = 3.1 mg / kg, ed50 [seminal vesicle] = 7.5 mg / kg), and was orally active [edwards et al ., 2000]. Compound 4c binds to ar with similar affinity (ki = 17 nm), but showed very good efficacy as an agonist in cv-1 cell functional assays (ec50 = 4 nm). In intact mature male rats, compound 4a did not raise serum levels of luteinizing hormone or testosterone, at 20 mg / kg or 40 mg / kg by the oral route . This was in contrast to flutamide and bicalutamide, which raised the levels of these hormones two- to eight - fold at these doses [hamann et al . Thus, compound 4a may have an advantage over current androgen antagonists because of its reduced potential for side effects in prostate cancer patients . Phthalamides (figure 3) such as 5a were identified as androgens antagonists with more potency than flutamide [miyachi et al . For example, compound 5b was as potent as bicalutamide in a cwr22 human prostate cancer xenograft model [salvati et al ., one of the compounds from this series, gtx-007, showed an agonist effect in increasing the weight of the levator ani muscles of castrated male rats . In addition, the compound did not suppress luteinizing hormone and follicle stimulating hormone levels [dalton et al . The structures of compounds 4a to 4c represent the 2-quinolones (figure 2). Compounds as frequently seen in modulators of other steroid receptors such as the progesterone receptor, small structural changes can lead to the reversal of agonistic and antagonistic activity . Both 4a and 4b bind to ar in the nanomolar range, with kis of 26 nm and 21 nm, respectively . Compound 4a seems more potent and more efficacious in a cv-1 cell functional assay (4a: 74% efficacy, 27 nm antagonist potency; 4b: 39% efficacy, 34 nm antagonist potency). In addition, 4b showed some agonist activity in the same cells (39% efficacy, 125 nm potency). However, in castrated immature male rats, 4b demonstrated better efficacy as an antagonist . It inhibited testosterone - induced increases in prostate weight and seminal vesicle weight (4a: ed50 [prostate] = 18 mg / kg, ed50 [seminal vesicle] = 19 mg / kg; 4b: ed50 [prostate] = 3.1 mg / kg, ed50 [seminal vesicle] = 7.5 mg / kg), and was orally active [edwards et al ., 2000]. Compound 4c binds to ar with similar affinity (ki = 17 nm), but showed very good efficacy as an agonist in cv-1 cell functional assays (ec50 = 4 nm). In intact mature male rats, compound 4a did not raise serum levels of luteinizing hormone or testosterone, at 20 mg / kg or 40 mg / kg by the oral route . This was in contrast to flutamide and bicalutamide, which raised the levels of these hormones two- to eight - fold at these doses [hamann et al ., 1999]. Thus, compound 4a may have an advantage over current androgen antagonists because of its reduced potential for side effects in prostate cancer patients . Phthalamides (figure 3) such as 5a were identified as androgens antagonists with more potency than flutamide [miyachi et al ., 1997]. Recently, for example, compound 5b was as potent as bicalutamide in a cwr22 human prostate cancer xenograft model [salvati et al ., 2002]. One of the compounds from this series, gtx-007, showed an agonist effect in increasing the weight of the levator ani muscles of castrated male rats . In addition, the compound did not suppress luteinizing hormone and follicle stimulating hormone levels [dalton et al . Androgen receptor antagonists have found therapeutic use in the treatment of androgen - responsive prostate cancer for over two decades . In particular, androgen antagonist monotherapy, rather than cab or antagonists in combination with orchidectomy, will likely see increased use, due to its relatively fewer negative effects on quality of life . Androgen antagonists will be approved for new clinical uses, including the treatment of hirsutism in women, alopecia in men and acne in both sexes . New sarm antagonists with improved tissue specificity, such as with reduced effects on the hypogonadal axis, will emerge for prostate cancer monotherapy, as well as for skin indications . It is possible that sarm antagonists will find use in bph, provided that they have sufficient efficacy . New laboratory tools, such as the availability of crystal structures of the receptor for rational drug design, will facilitate the discovery of new sarms . A promising area of research is on the development of sarm agonists for increasing lean body mass and muscle strength in hypogonadal men and in cancer and immunodeficient patients . Ultimately, sarm agonists may be used to counteract the frailty associated with aging . As well as being extremely safe overall, such drugs will have to stimulate muscle strength without increasing the risk of prostate cancer . Nevertheless, it is possible to see a day when tissue - specific androgens of many flavors will be of great medical benefit . 1, flutamide; 2, nilutamide; 3, bicalutamide see text for more details . See text for more details see text for more details
Small molecule natural products provide opportunities for scientific advances in chemistry and biology through their study . An excellent example of this line of investigation is the discovery and study of cell - specific apoptosis - inducing natural products . In the 1990s seto and hayakawa initiated a cell - based screening program aimed at the identification of new genotype - selective cytotoxic agents from microbial extracts. (1) in 1993 the seto group reported the isolation of apoptolidin, a 20-membered macrolide found to induce apoptosis in rat glia cells transformed with the adenovirus oncogene and produced by nocardiopsis sp . 200 mg/2 l of fermentation). (2) subsequently several minor apoptolidin congeners were isolated from the same microorganism by wender s group and given the names apoptolidin bd with apoptolidin adopting the name apoptolidin a (figure 1). (3) apoptolidins ad were reported to inhibit growth of h292 cancer cells (lung carcinoma) in the submicromolar range, with apoptolidin b showing the greatest activity (gi50 = 7 4 nm). Not surprisingly, the reported cell - selective induction of apoptosis by apoptolidin a stimulated investigations on several research fronts in order to better understand the reported cell - selective cytotoxicity . Khosla and co - workers determined that apoptolidin a and structurally related cytotoxic macrolides ossamycin and cytovaricin inhibit mitochondrial f0f1-atpase, identifying this enzyme as a promising target for the development of cell - selective anticancer agents. (4) the apoptolidins have proven to be rather labile compounds easily subjected to a base - induced acyl migration from the c19 to c20 hydroxyl group to produce isoapoptolidins (figure 1),(5) compounds possessing diminished activity against mitochondrial f0f1-atpase . The molecular complexity and instability of apoptolidin a has led wender and co - workers to search for semisynthetic analogs possessing superior stability and/or pharmacokinetic properties. (6) comparison of the enzyme inhibition of mitochondrial f0f1-atpase to antiproliferative activity in e1a - transformed rat fibroblasts of apoptolidin a derived analogs suggested either the existence of a secondary biological target or a more complex mode of action . The complex molecular architecture and novel cytotoxic profile of apoptolidin a has stimulated considerable interest from the synthetic community. (7) total syntheses of the fully glycosylated ensemble, apoptolidin a, have been reported by the groups of nicolaou and koert. (8) several groups including our own have described the synthesis of apoptolidinone (a). (9) we describe herein the total synthesis of apoptolidinones a and d and evaluation of their antiproliferative properties . An unsaturated 20-membered macrolactone conjugated to a stereochemically rich pyran ring constitutes the gross structural features of apoptolidinone a / d, reminiscent of polyketides such as the bafilomycin macrolides. (10) we anticipated macrolide i (scheme 1) would serve as a logical synthetic precursor to apoptolidinone . However, because of the tendency of apoptolidin to ring expand to iso - apoptolidin under basic conditions, proper choice of protecting groups was considered of paramount importance . As we gathered information from published studies and work from our own group, we ultimately identified triethylsily (p = tes) groups as the protecting groups of choice . Disconnection of the macrolide at c5c6 which relied on introduction of a vinyl boronate by a cross - metathesis reaction allowed, in principle, synthetic access to apoptolidinones a (r = me) and d (r = h). Disconnection at c11c12 and application of two stereoselective aldol retrons leads to the identification of fragments iivi as key building blocks for the assembly of apoptolidinone a / d . The c23c28 fragment (scheme 1, iv) was derived by a crimmins syn aldol reaction between the chlorotitanium enolate derived from oxazolidinethione 9 and aldehyde 4 (scheme 2). (11) the latter was easily prepared from (r)-glycidyl methyl ether (1) starting with epoxide ring opening with the carbanion derived from 1,3-dithiane followed by alcohol protection and dithiane hydrolysis . Reduction of the tes protected aldol 6 with lithium borohydride led to alcohol 7, which on oxidation under swern conditions gave aldehyde 8 . Reagents and conditions: (a) n - buli, 1,3-dithiane, thf, 40 c, 95%; (b) tbscl, imh, dmap, dmf, 0 c, 95%; (c) mei (xs), k2co3, mecn / water (10:1), 40 c, 90%; (d) 9, ticl4, 0 c then ()-sparteine, 78 to 0 c, ch2cl2, 95%; (e) tescl, imh, dmf, 0 c, 95%; (f) libh4, meoh, 0 c, 90%; (g) (cocl)2, dmso, et3n, 78 c, 88% . Our synthetic strategy (scheme 1) projected the equivalent of a c20c22 fragment (vi) to conjoin aldehydes iv and v through two regio- and stereoselective aldol reactions by one of two coupling orders (vi + iv + v and vi + v + iv). To this end we developed reaction conditions that would allow access to either isomeric silyl enol ethers 12 or 13 starting from tes protected 1-hydroxy-2-butanone 11 (scheme 3). (12) deprotonation of 11 under kinetic conditions (lda, thf, 78 c) followed by the addition of tmscl afforded a 15:73:12 mixture of isomers (z)-12, (e)-12, and (z)-13 (scheme 3). Thermodynamic conditions (tmsotf, et3n, thf, 0 c) afforded (z)-13 as the exclusive isomer as determined by gc analysis . Reagents and conditions: (a) tescl, imh, ch2cl2, 0 c, 77%; (b) lda, thf, 78 c then tmscl, 80%; (c) tmsotf, et3n, thf, 0 c, 92% . The c6c11 fragment that incorporates two stereocenters (c8 and c9) was to be introduced in the form a vinyl boronate (ii, scheme 1). To this end, we examined the asymmetric crotylation of 3-iodoacrolein (14)(15) and 3-borylacrolein (15)(16) employing either roush s (18)(17) or brown s (19) syn selective crotylating agent (scheme 4). (18) as reported earlier, addition of the (z)-crotylboronate reagent 18 to the pinacol ester of 3-boronoacrolein afforded, following in situ silylation, syn homoallylic ether 17 with modest enantioselectivity (80% ee) and yield (4043%). (7 g) the addition of 18 to 3-iodoacrolein proceeded with poor asymmetric induction (26% ee), whereas addition of the brown reagent (19) to 14 gave 16 in high enantiomeric excess (90% ee) and good yield (67%). Vinyl iodide 16 was readily converted to vinyl boronate 17 by lithiumhalogen exchange followed by a boronate quench . Construction of the c12c19 fragment (v, scheme 1) started from lactone 20, readily derived from (s)-malic acid(19) and converted to 3-methoxy--butyrolactone (21) (scheme 5). (20) reduction of lactone 21 with dibal - h afforded lactol 22, which on condensation with 1,3-propanedithiol afforded dithiane 23 . A five - carbon unit was introduced to aldehyde 24 by chelation - controlled addition of the grignard reagent derived from bromide 25, prepared from dihydrofuran according to kocienski s procedure as described by koert and co - workers in their reported synthesis of apoptolidinone . A dichloromethane solution of secondary alcohol 26 was treated sequentially with iodine followed by imidazole and triethylchlorosilane in one pot to provide vinyl iodide 28 in 8996% yield . Dithiane hydrolysis of 28 was efficiently accomplished using the fetizonjurion procedure to provide aldehyde 29 in 6474% yield. (22) with fragments iivi available, we turned our attention to their coupling to complete the synthesis of apoptolidinone a (scheme 6). Reagents and conditions: (a) ag2o, mei (solvent) 86%; (b) dibal - h, thf, 78 c; (c) 1,3-propanedithiol, bf3oet2, ch2cl2, 28 c, 86% from lactone 21; (d) (cocl)2, dmso, i - pr2net, 78 c, 98%; (e) 25, mg, 1,2-dibromoethane, et2o, 78 c, 85%; (f) tescl, imh, ch2cl2, 28 c; (g) i2, ch2cl2, 0 c, 90% from 26; (h) mei (xs), k2co3, mecn / ph 7 buffer (4:1), 28 c, 92% . Reagents and conditions: (a) 17, pd(ph3p)4, tl(oet), thf / h2o (3:1), 28 c, 86%; (b) 12, bf3oet2, cah2, ch2cl2, 94 c, 72%; (c) 2,4,6-trichlorobenzoyl chloride, et3n, dmap, phme, 78 c, 71%; (d) lhmds, thf, hmpa, 78 c, 2 h then 8, thf, 48%; (e) tesotf, 2,6-lutidine, ch2cl2, 0 c, 97%; (f) 34, isopropenyl pinacol boronic ester (5 equiv), phme, 50 c, 46 h, 22%; (g) pd(ph3p)4, tl(oet), thf / h2o (3:1), 28 c, 30 min, 84%; (h) hfpyridine, thf, 10 to 10 c, 48h, 94% . Initially, suzukimiyaura cross - coupling of aldehyde 29 and vinyl boronate 17 was accompanied by elimination of the -methoxy group of aldehyde 30 to afford the corresponding,-unsaturated aldehyde. (23) however optimization of coupling conditions [tl(oet), pd(pph3)4, thf(aq)] and reaction time (15 min) eventually led to an observed 70% yield of 30 without any observed elimination. (24) we next turned our attention to investigating two key stereocontrolled aldol reactions; leading to the formation of the c(19)c(20) and c(22)c(23) carboncarbon bonds . Mukaiyama aldol reaction between aldehyde 30 and an 8:1 mixture of silyl enol ether 12 and 13 afforded anti, syn-31a as the major isomer (31ad: 12:3.8:1.6:1) (figure 2). (25) the assigned c(19)c(20) syn relative stereochemistry rested on the observed coupling constant of the aldol product (j19,20 = 3.5 hz) and the proclivity for the formation of syn aldol products in the mukaiyama aldol reaction of silyl enol ethers (regardless of double bond geometry) with chiral aldehydes. (26) the assigned c(17)c(19) anti configuration rested on the well - established 1,3-asymmetric induction model proposed by evans and co - workers in the mid-1990s. (27) the second major isomer was assigned the syn, syn (31b) relative stereochemistry (j19,20 = 3.9 hz) (figure 2). The minor aldol products were assigned the structures of anti, anti-31c and syn, anti-31d (j19,20 = 4.7 and 4.2 hz), although the assignment of the c(17)c(19) relative configuration remained ambiguous . The carbonyl resonances in the c nmr of 31c and 31d were not observed due to the trace amount of material isolated . Yamaguchi esterification of anti, syn-31 with carboxylic acid 32(28) led to isolation of dienoate 33 in 83% yield. (29) we anticipated the matched double stereodifferentiating aldol reaction between a metal enolate derived from ketone 33 and aldehyde 8 to proceed with high stereoselectivity. (30) in the event kinetic deprotonation (lhmds, hmpa, thf, 78 c) of 33 followed by aldol condensation with aldehyde 8 afforded syn aldol product 35 as a single isomer in 48% yield . Following silylation of 35 a dichloromethane solution of alkene 36, isopropenyl boronate, and grubbs second - generation catalyst (34) held at reflux for 5 h afforded a 30% yield of vinylboronate 37 that was judged by h nmr spectroscopy analysis to be a single geometric isomer . However, analysis by hplc revealed a significant quantity of an isomeric byproduct as determined by further h nmr analysis. (31) employing the same catalyst in toluene heated to 50 c resulted in a 22% yield of 37 and significant reduction of the isomeric byproduct. (32) vinylboronate 37 derived by the latter procedure subjected to an intramolecular suzukimiyaura reaction delivered macrolactone 38 in a consistent 84% yield . Finally, desilylation of 38 provided apoptolidinone a in 94% yield . A synthesis of apoptolidinone d required only a change in the cross - metathesis partner of alkene 36 from isopropenyl boronic ester to 1-propenyl boronic ester . However, we decided to probe the assembly of alkene 36 by an alternate order of aldol coupling (cf ., we examined the addition of silyl enol ether (z)-13 to syn substituted aldehyde 8 (scheme 7). As pointed out by evans, syn substituted aldols are stereochemically nonreinforcing substrates where the c24 substitutent of 8 favors a felkin while the c25 a non - felkin addition . Literature precedent suggested the stereoselectivity of the addition of (z)-13 to 8 could not be predicted with confidence . In the event, we were surprised to observe treatment of a solution of 8 and (z)-13 with borontrifluoride etherate led not to a mukaiyama aldol reaction but instead silyl enol ether 39 as a single stereoisomer, the product of a heteroene reaction. (34) stereochemical assignment of c22 and c23 was based on nmr analysis of pyran 40, derived from 39 by exhaustive desilylation (p - tsa, meoh, 28 c, 69%). The noesy spectrum of 40 revealed noes from h-23 to 22-me and 24-me and h-23 to h-27. (35) this observation combined with the large coupling constants (j2223 = 9.9 hz and j2324 = 9.9 hz) led to the assigned relative stereochemistry shown, indicating a felkin addition to 8, the undesired stereoselectivity . Reagents and conditions: (a) 34, 1-propenyl pinacol boronic ester (10 equiv), phme, 80 c, 6 h then 40 c, 24 h, 53%; (b) pd(ph3p)4, tl(oet), thf / h2o (3:1), 28 c, 15 min, 95%; (c) hfpyridine, thf, 10 to 10 c, 48 h, 87% . Apoptolidinone d was completed starting with a cross - metathesis between 36 and 1-propenyl pinacol boronic ester promoted by grubbs second generation catalyst to deliver vinyl boronate 41 in superior yield and stereoselectivity relative to the same reaction with isopropenyl boronate leading to apoptolidinone a. macrocyclization of 41 under the previously described suzuki conditions gave 42 in 95% yield . Wender s group has reported gi50s for apoptolidin a (32 nm), apoptolidin b (7 nm), apoptolidin c (24 nm), and apoptolidin d (110 nm) against h292 (human lung carcinoma) cells . The effect reported by wender s group was cytostasis observed over a 23 day (ca . We observed that treatment of h292 lung carcinoma cells with apoptolidin a results in growth arrest . In addition, however, extended maintenance of cells in the presence of apoptolidin a results in a delayed toxicity not previously reported . The effect is drastic, resulting in> 95% cell death after 7 days in culture with apoptolidin a concentrations as low as 30 nm (figure 3). In contrast, the synthetic aglycones apoptolidonone a and apoptolidinone d were inactive in the assay, inhibiting neither cell growth nor viability at the concentrations tested . (a) cells were plated at a density of 500/well in 96-well plates and treated for 7 days with apoptolidin a, apoptolidinone a, and apoptolidinone d at concentrations from 3 nm to 10 m . Viability was measured by loading cells with 2 m calcein - am and reading plates using a spectramax (molecular dynamics) plate reader; abs = 494, em = 517 . (b) effective concentration 50 (ec50) values for apoptolidin a, apoptolidinone a, and apoptolidinone d. (c) bright field photomicrographs of h292 cells cultured for 7 days in the presence of either 10 or 30 nm apoptolidin a. in conclusion, we have developed a convergent synthesis of apoptolidinones a and d. evaluation of the cytotoxicity of these aglycones using human lung cancer cells (h292) confirms the importance of the sugars of apoptolidin in expressing overall cytotoxicity as reported by koert and co - workers. (8e) current efforts are aimed at the development of synthetic probes aimed at interrogating the cellular target(s) of the apoptolidins . To a solution of diene 30 (0.40 g, 0.76 mmol) and silyl enol ethers (7:1, 12/13; 0.83 g, 3.03 mmol, 4 equiv) in dichloromethane (32 ml) at 0 c was added cah2 (64 mg, 1.52 mmol, 2 equiv). After 15 min, the reaction was cooled to 94 c and bf3oet2 (105 l, 1.52 mmol, 2 equiv) was added . The reaction mixture was stirred for 45 min at 94 c and quenched with saturated nahco3 (25 ml), and the aqueous layer was extracted with dichloromethane (3 50 ml). The combined organic layers were dried (mgso4), filtered, and concentrated in vacuo . The residue was purified by flash chromatography (hexanes / etoac, 20:115:1) to afford 0.39 g (72%) of alcohol 31ad as a 12:3.8:1.6:1 mixture of diastereoisomers (by hplc, 21.4 mm 25 cm column, 49 min gradient, 010% ethyl acetate in hexanes). For synthetic purposes the mixture can be advanced to the next step without further purification, as the resulting mixture of yamaguchi esterification products can be easily separated to afford the desired ester 33 . Tr = 34.25 min; []d 9.1 (c 2.1, chcl3); ir (neat) 3505, 2953, 2873, 1724, 1462, 1418, 1244, 1091, 1018 cm; h nmr (500 mhz c6d6) 6.25 (d, j = 16.0 hz, 1h), 6.055.98 (m, 1h), 5.63 (dd, j = 16.0, 7.5 hz, 1h), 5.55 (t, j = 7.0 hz, 1h), 5.085.03 (m, 2h), 4.244.18 (m, 1h), 4.07 (t, j = 7.5 hz, 1h), 4.04 (d, j = 4.0 hz, 1h). J = 10.0, 4.0, 2.0 hz, 1h), 3.30 (s, 3h), 2.61 (d, j = 9.0 hz, 1h), 2.532.38 (m, 4h), 2.252.16 (m, 1h), 1.951.89 (m, 1h), 1.881.82 (m, 1h), 1.76 (s, 3h), 1.661.60 (m, 1h), 1.601.52 (m, 1h), 1.11 (d, j = 6.5 hz, 3h), 1.050.99 (m, 21h), 0.91 (t, j = 8.0 hz, 9h), 0.680.62 (m, 12 h), 0.52 (q, j = 8.0 hz, 6h); c nmr (125 mhz, c6d6) 213.3, 141.3, 135.7, 133.7, 132.5, 128.6, 114.2, 81.7, 80.8, 78.1, 71.6, 70.6, 58.2, 45.2, 33.5, 32.2, 31.7, 25.3, 15.0, 12.6, 7.3, 7.1, 7.1, 6.9, 5.4, 5.4, 5.0; hrms (maldi) m / z 749.5078 [(m + na) calcd for c39h78o6si3na, 749.5004]. Tr = 31.75 min; []d 2.6 (c 0.16, chcl3); ir (neat) 3498, 2955, 2877, 1716, 1459, 1414, 1239, 1083, 1006, 966, 845, 677 cm-1; 1h nmr (500 mhz c6d6) 6.27 (dd, j = 15.5, 4.5 hz, 1h), 6.075.98 (m, 1h), 5.65 (dd, j = 15.5, 7 hz, 1h), 5.565.50 (m, 1h), 5.105.04 (m, 2h), 4.254.19 (m, 1h), 4.17 (d, j = 4 hz, 1h), 4.09 (t, j = 6.2 hz, 1h), 3.933.88 (m, 1h), 3.443.39 (m, 1h), 3.30 (d, j = 4.5 hz, 1h), 3.09 (s, 3h), 2.73258 (m, 2h), 2.452.32 (m, 2h), 2.242.14 (m, 1h), 2.04 (d, j = 15 hz, 1h), 1.831.73 (m, 2h), 1.76 (s, 3h), 1.591.52 (m, 1h), 1.151.09 (m, 6h), 1.070.94 (m, 27h), 0.690.56 (m, 18h); c nmr (125 mhz, c6d6) 211.9, 141.4, 135.7, 133.8, 132.4, 128.8, 114.3, 84.5, 81.9, 78.1, 73.8, 71.6, 57.1, 45.2, 32.9, 31.9, 31.8, 25.2, 15.1, 12.7, 7.4, 7.2, 7.0, 5.5, 5.4, 5.2; hrms (maldi) m / z 749.5020 [(m + na) calcd for c39h78o6si3na, 749.5004]. Tr = 32.80 min; []d 9.3 (c 0.08, chcl3); ir (neat) 3470, 3400, 2955, 2913, 2878, 2361, 2337, 1714, 1459, 1414, 1377, 1238, 1096, 1008, 968, 912, 844, 741, 671, 558, 522 cm; h nmr (500 mhz c6d6) 6.26 (d, j = 19.5 hz, 1h), 6.085.97 (m, 1h), 5.65 (dd, j = 19.5, 9.2 hz, 1h), 5.585.52 (m, 1h), 5.105.04 (m, 2h), 4.254.20 (m, 1h), 4.14 (d, j = 5.8 hz, 1h), 4.09 (t, j = 7.9 hz, 1h), 4.003.95 (m, 1h), 3.603.57 (m, 1h), 3.27 (s, 3h), 3.01 (d, j = 5 hz, 1h), 2.622.47 (m, 2h), 2.452.32 (m, 2h), 2.272.15 (m, 1h), 2.001.92 (m, 1h), 1.77 (s, 3h), 1.651.55 (m, 1h), 1.13 (d, j = 8.6 hz, 3h), 1.081.00 (m, 18h), 0.95 (t, j = 10.0 hz, 9h), 0.66 (q, j = 9.8 hz, 12h), 0.58 (q, j = 9.9 hz, 6h);c nmr (150 mhz, c6d6) 141.4, 135.8, 133.8, 132.5, 128.7, 114.2, 84.5, 82.4, 81.9, 78.1, 73.9, 72.4, 71.6, 71.1, 58.0, 57.1, 45.3, 32.9, 32.5, 32.0, 31.9, 25.2, 15.1, 12.7, 7.4, 7.2, 7.0, 6.9, 5.6, 5.5, 5.4, 5.2, 5.1; hrms (maldi) m / z 749.5038 [(m + na) calcd for c39h78o6si3na, 749.5004]. Tr = 36.80 min; []d 50.0 (c 0.01, chcl3); ir (neat) 2955, 2878, 1713, 1459, 1414, 1379, 1239, 1105, 1007, 968, 912, 845, 741, 583, 553, 525 cm; h nmr (500 mhz c6d6) 6.30 (d, j = 19.6 hz, 1h), 6.085.98 (m, 1h), 5.67 (dd, j = 19.5, 9.1 hz, 1h), 5.565.50 (m, 1h), 5.115.04 (m, 2h), 4.304.17 (m, 1h), 4.11 (t, j = 7.9 hz, 1h), 4.07 (d, j = 5.2 hz, 1h), 3.943.89 (m, 1h), 3.48 (d, j = 12.6 hz, 1h), 3.29 (s, 3h), 2.552.32 (m, 4h), 2.222.12 (m, 1h), 1.911.85 (m, 1h), 1.78 (s, 3h), 1.711.59 (m, 2h), 1.511.42 (m, 1h), 1.13 (d, j = 8.5 hz, 3h), 1.101.00 (m, 18h), 0.94 (t, j = 9.9 hz, 9h), 0.750.62 (m, 12h), 0.56 (q, j = 9.9 hz, 6h); c nmr (150 mhz, c6d6) 141.4, 135.7, 133.8, 132.2, 128.9, 114.3, 82.1, 81.5, 78.1, 72.9, 70.7, 57.9, 45.3, 34.2, 34.0, 32.1, 25.3, 15.1, 12.7, 7.3, 7.2, 7.1, 7.0, 5.6, 5.5, 5.0; hrms (maldi) m / z 749.5030 [(m + na) calcd for c39h78o6si3na, 749.5004]. To a solution of vinyl boronate 37 (5 mg, 3.1 mol) in thf / h2o (4 ml, 3:1, degassed) was added pd(ph3p)4 (0.7 mg, 0.62 mol, 0.2 equiv). The resulting yellow solution was stirred for 5 min before tloet (0.3 l, 4.6 mol, 1.5 equiv) was added . The solution was stirred for 15 min (color turned from yellow to gray). The reaction was quenched with saturated nahco3 (5 ml), and the aqueous layer was extracted with dichloromethane (3 10 ml). The combined organic layers were dried (mgso4), filtered, and concentrated in vacuo . The residue was purified by flash chromatography (hexanes / etoac, 25:1) to afford 3.5 mg (84%) of lactone 38 as a colorless oil: []d + 17.5 (c 0.32, chcl3); ir (neat) 3497, 3382, 2954, 2915, 2877, 1700, 1459, 1241, 1111, 1081, 968, 835, 781, 741; h nmr (500 mhz, c6d6) 7.49 (s, 1h), 6.26 (s, 1h), 6.06 (d, j = 16.0 hz, 1h), 5.715.67 (m, 1h), 5.57 (t, j = 8.0 hz, 1h), 5.38 (dd, j = 16.0, 9.0 hz, 1h), 5.16 (d, j = 10.0 hz, 1h), 5.00 (d, j = 4.0 hz, 1h), 4.314.28 (m, 1h), 4.24 (dd, j = 9.5, 1.0 hz, 1h), 3.943.89 (m, 1h), 3.82 (t, j = 8.5 hz, 1h), 3.60 (dt, j = 8.5, 2.5 hz, 1h), 3.523.44 (m, 1h), 2.48 (s, 3h), 3.393.36 (m, 2h), 3.16 (s, 3h), 3.02 (t, j = 8.5 hz, 1h), 2.612.56 (m, 1h), 2.532.46 (m, 1h), 2.161.90 (m, 4h), 2.07 (s, 3h), 1.82 (s, 3h), 1.801.74 (m, 1h), 1.63 (d, j = 1.0 hz, 3h), 1.59 (s, 3h), 1.41 (d, j = 7.0 hz, 3h), 1.361.26 (m, 6h), 1.21 (d, j = 7.0 hz, 3h), 1.171.13 (m, 21h), 1.10 (d, j = 7.0 hz, 3h), 1.081.02 (m, 18 h), 1.00 (s, 9h), 0.970.80 (m, 18 h), 0.750.64 (m, 15 h), 0.18 (s, 3h), 0.16 (s, 3h); c nmr (125 mhz, c6d6) 209.8, 168.4, 146.2, 145.5, 141.5, 136.4, 133.3, 132.3, 132.2, 131.8, 129.3, 123.4, 82.1, 81.1, 77.1, 76.2, 73.9, 73.1, 70.4, 70.0, 61.0, 58.7, 47.3, 43.6, 41.0, 40.8, 36.2, 34.9, 30.1, 26.1, 24.8, 18.3, 17.9, 17.4, 16.3, 14.0, 11.9, 11.1, 10.3, 7.5, 7.4, 7.3, 7.2, 7.1, 6.6, 5.9, 5.8, 5.6, 5.4, 4.0, 4.5; hrms (esi) m / z 1371.9600 [(m + li) calcd for c73h144o11si6li, 1371.9484]. To solution of 6.4 mg (4.7 mol) macrolactone 38 in 2 ml of thf at 10 c was added hf - py (0.1 ml) dropwise . After 1 h the temperature after 36 h the reaction mixture was diluted with et2o, washed with water, and concentrated in vacuo, leaving ca (it was noticed that drying over mgso4 significantly decreased the amount of product in the solution .) The residue was purified by flash chromatography (ch2cl2/meoh, 15:1) to afford 3 mg (94%) of apoptolidinone a as a white solid: []d + 68(c 0.15, chcl3); ir (neat) 3395, 2925, 1664, 1596, 1390, 1258, 1095, 1023, 968, 754, 710 cm; h nmr (500 mhz, cd3od) 7.37 (s, 1h), 6.19 (s, 1h), 6.10 (d, j = 16.0 hz, 1h), 5.64 (dd, j = 9.0, 7.0 hz, 1h), 5.33 (dd, j = 15.5, 8.5 hz, 1h), 5.325.29 (m, 1h), 5.22 (d, j = 10.5 hz, 1h), 4.09 (ddd, j = 9.0, 3.5, 2.5 hz, 1h), 3.77 (app t, j = 9.5 hz, 1h), 3.74 (dd, j = 11.0, 5.0 hz, 1h), 3.533.58 (m, 2h), 3.453.42 (m, 1h), 3.36 (s, 3h), 3.30 (s, 3h), 3.233.15 (m, 2h), 2.73 (dd, j = 10.0, 4.5 hz, 1h), 2.522.41 (m, 2h), 2.19 (s, 3h), 2.182.13 (m, 1h), 2.11 (s, 3h), 2.092.02 (m, 2h), 1.92 (d, j = 1.0 hz, 3h), 1.781.72 (m, 2h), 1.67 (s, 3h), 1.601.51 (m, 2h), 1.441.37 (m, 1h), 1.321.26 (m, 1h), 1.13 (d, j = 6.5 hz, 3h), 1.02 (d, j = 6.5 hz, 3h), 0.88 (d, j = 7.0 hz, 3h); c nmr (100 mhz, cd3od) 172.7, 149.1, 147.3, 143.7, 137.7, 134.9, 133.1, 133.0, 132.6, 129.6, 123.8, 101.3, 83.8, 80.6, 78.6, 75.5, 74.6, 73.7, 72.3, 69.2, 68.1, 61.4, 59.4, 41.0, 40.8, 38.6, 38.4, 36.6, 36.4, 24.5, 17.8, 17.8, 16.4, 14.0, 12.2, 12.1, 5.3; hrms (esi) m / z 687.4298 [(m + li) calculated for c37h60lio11, 687.4296]. To solution of 19.9 mg (14.7 mol) macrolactone 42 in 4 ml of thf at 10 c was added hf - py (0.2 ml) dropwise . After 1 h the temperature was raised to 0 c and after an additional 12 h to 10 c . After 36 h the reaction mixture was diluted with et2o, washed with water, and concentrated in vacuo leaving ca . (it was noticed that drying over mgso4 significantly decreased the amount of product in the solution .) The residue was purified by flash chromatography (ch2cl2/meoh, 15:1) to afford 8.5 mg (87%) of apoptolidinone d as a off - white solid: []d + 67.5; ir (neat) 3382, 2927, 2362, 1666, 1599, 1457, 1390, 1255, 1100, 1024, 965, 754 cm; h nmr (600 mhz, cd3od) 7.39 (s, 1h), 6.276.22 (m, 2h), 5.99 (d, j = 15.6 hz, 1h), 5.57 (t, j = 8.1 hz, 1h), 5.485.44 (m, 1h), 5.325.28 (m, 2h), 4.08 (dt, j = 8.4, 2.4 hz, 1h), 3.783.73 (m, 2h), 3.553.50 (m, 1h), 3.53 (d, j = 1.2 hz, 1h), 3.423.35 (m, 2h), 3.37 (s, 3h), 3.26 (s, 3h), 3183.12 (m, 2h), 2.66 (dd, j = 9.6, 5.4 hz, 1h), 2.492.42 (m, 1h), 2.302.23 (m, 1h), 2.17195 (m, 5h), 2.13 (s, 3h), 2.08 (s, 3h), 1.791.72 (m, 2h), 1.721.53 (m, 3h), 1.64 (s, 3h), 1.431.22 (m, 4h), 1.18 (d, j = 6.6 hz, 3h), 1.02 (d, j = 6.6 hz, 3h), 0.87 (d, j = 6.6 hz, 3h); c nmr (150 mhz, cd3od) 172.6, 147.5, 146.0, 143.0, 137.4, 135.0, 133.5, 132.2, 130.9, 126.6, 124.2, 101.3, 84.0, 80.0, 78.5, 75.4, 74.9, 73.8, 72.5, 69.2, 68.1, 61.4, 59.4, 46.7, 40.9, 38.5, 38.3, 36.3, 36.1, 24.8, 18.6, 15.6, 13.9, 12.2, 12.1, 5.3; hrms (maldi) m / z 689.3881 [(m + na) calcd for c36h58nao11, 689.3877].
Violence can be defined as overt behavior that involves the intent to inflict noxious stimulation or to behave destructively toward another organism . Violent behavior in human society is a complex output of biological, behavioral, and social factors . Epilepsy and violent behavior have long been regarded as similar because of their episodic or impulsive natures . Violent behavior among patients with epilepsy can be categorized into peri - ictal violence (preictal, ictal, and postictal), which occurs around the time of a seizure attack, and interictal violence, which has less of a temporal relationship with a seizure attack . There has been a belief that people with epilepsy are more prone to committing violent acts than the general population; however, there has been no convincing evidence to indicate that epilepsy is associated with violent behavior . The prevalence of violence among patients with epilepsy can vary according to the definition of violent behavior, epilepsy subtypes, and the origin of the study population . For example, temporal lobe epilepsy had been reported to be related to a high rate of about 7% of violent acts . Some studies have indicated that epileptic prisoners do not commit more violent crimes than nonepileptic prisoners . Another report stated that the rate of epilepsy in prison populations was similar to those found in most economically disadvantaged communities, suggesting that socioeconomic conditions are an important factor leading patients with epilepsy to commit criminal behavior . Combined medical diseases, such as alcohol dependence, brain injury, and psychosis, and frustrated social achievement may contribute to violent behavior . It is conceivable that only a minority of patients with epilepsy commit a violent crime . If we can elucidate potential violence - aggravating factors among patients with epilepsy, we may be able to understand the pathomechanism of aggression and possibly prevent further violent behavior . In this study, we set out to evaluate the clinical and criminal characteristics of epileptic patients who committed violent crimes . The national forensic hospital in gongju is a hospital - based correctional institution that was founded in 1987 by the ministry of justice of the republic of korea . It is a unique forensic hospital in the republic of korea, and those criminals who have committed crimes as a manifestation of their psychiatric or neurological diseases are incarcerated in the institution . When patients are admitted to the hospital, we perform routine blood tests, electroencephalography (eeg), and the korean version of the wechsler adult intelligence scale, which measures the intelligent quotient (iq). Brain magnetic resonance imaging (mri) is considered when outside data is not available . Between october 2007 and september 2008, patients with epilepsy who were incarcerated in the forensic hospital due to violent crime were enrolled in the study . Epilepsy was diagnosed when a patient had two or more unprovoked seizure attacks or when antiepileptic drugs had been prescribed for a diagnosis of epilepsy . All of the patients had been diagnosed with epilepsy before their crimes, and one neurologist confirmed the diagnosis after admission to the forensic hospital . The violent crimes included homicide, attempted murder, rape, assault, arson, and robbery . The seizure - control state was determined as good if a patient was in a state of remission for longer than a year . The study was reviewed and approved by the institutional review board of the national forensic hospital in gongju . A spearman test was performed to test for any correlation between the age of the patient at their first crime and values including epilepsy onset age, iq, and educational achievement with spss for windows (version 12.0; spss, inc ., chicago, il, usa). The national forensic hospital in gongju is a hospital - based correctional institution that was founded in 1987 by the ministry of justice of the republic of korea . It is a unique forensic hospital in the republic of korea, and those criminals who have committed crimes as a manifestation of their psychiatric or neurological diseases are incarcerated in the institution . When patients are admitted to the hospital, we perform routine blood tests, electroencephalography (eeg), and the korean version of the wechsler adult intelligence scale, which measures the intelligent quotient (iq). Brain magnetic resonance imaging (mri) is considered when outside data is not available . Between october 2007 and september 2008, patients with epilepsy who were incarcerated in the forensic hospital due to violent crime were enrolled in the study . Epilepsy was diagnosed when a patient had two or more unprovoked seizure attacks or when antiepileptic drugs had been prescribed for a diagnosis of epilepsy . All of the patients had been diagnosed with epilepsy before their crimes, and one neurologist confirmed the diagnosis after admission to the forensic hospital . The violent crimes included homicide, attempted murder, rape, assault, arson, and robbery . The seizure - control state was determined as good if a patient was in a state of remission for longer than a year . The study was reviewed and approved by the institutional review board of the national forensic hospital in gongju . A spearman test was performed to test for any correlation between the age of the patient at their first crime and values including epilepsy onset age, iq, and educational achievement with spss for windows (version 12.0; spss, inc ., chicago, il, usa). There were 17 epilepsy patients who had committed a violent crime, and these patients constituted 2.2% of the total patient population (761) in the forensic hospital . The mean age of the patients at epilepsy onset and at the time of their first crime was 15.5 14.5 years and 33.9 12.3 years, respectively . All of the patients were diagnosed with localization - related epilepsy, which involved either frontal lobe epilepsy or temporal lobe epilepsy (table 1). Possible etiologies included head trauma in three patients, cerebral infection in two patients, and stroke in another . Brain mri data, which was available for seven patients, revealed various structural lesions, including cerebral infarction, polymicrogyria, meningioma, surgical excision, and encephalomalatic change after head injury . Fifteen out of the 17 patients were not taking regular antiepileptic medication prior to their crimes, partly because of economic problems or adverse effects of the drugs, but mostly because they did not recognize the importance of drug maintenance . Ten patients were in a remission state with monotherapy, and five were being administered two antiepileptic medications . Other combined medical conditions included mental retardation in seven patients, alcohol abuse in four, schizophrenia in three, and dementia in one . The types of crimes among the patients included murder in six patients, attempted murder in three, rape in three, assault in two, robbery in two, and arson in one . Most of the patients did not experience overt seizure attacks before and after the crime, and they did not lose their consciousness or memory during the crime . However, two patients stated that they did not remember their criminal behavior, suggesting the possibility of postictal confusion . Although the two patients experienced overt seizure attacks after admission, they had not shown any alteration of consciousness with violent behavior . Ten patients (58.8%) had previous criminal records, which were due to similar types of crimes as those discussed in this paper . Seven patients were in a heavily drunken state when they committed their crimes (table 1). Eight patients experienced psychosis, and two patients committed their crimes in response to auditory or visual hallucinations . In eight patients, a positive correlation existed between the age of onset of their first crimes and the patients iq scores, as illustrated in table 2 and figure 1 (r = 0.533, p = 0.033). Educational achievement was defined as the number of years that were spent in a school (table 1). Seven out of 17 patients had finished middle school (i.e., they spent nine years in the school system), which is the final step of compulsory education in korea . Occupational experience was mostly confined to temporary and daily work in seven patients, while two patients had regular occupations . Four patients were divorced, and one patient was widowed because she had murdered her husband . Regular contact by family members of at least one visit per year was maintained in five patients . All of the patients were diagnosed with localization - related epilepsy, which involved either frontal lobe epilepsy or temporal lobe epilepsy (table 1). Possible etiologies included head trauma in three patients, cerebral infection in two patients, and stroke in another . Brain mri data, which was available for seven patients, revealed various structural lesions, including cerebral infarction, polymicrogyria, meningioma, surgical excision, and encephalomalatic change after head injury . Fifteen out of the 17 patients were not taking regular antiepileptic medication prior to their crimes, partly because of economic problems or adverse effects of the drugs, but mostly because they did not recognize the importance of drug maintenance . Ten patients were in a remission state with monotherapy, and five were being administered two antiepileptic medications . Other combined medical conditions included mental retardation in seven patients, alcohol abuse in four, schizophrenia in three, and dementia in one . The types of crimes among the patients included murder in six patients, attempted murder in three, rape in three, assault in two, robbery in two, and arson in one . Most of the patients did not experience overt seizure attacks before and after the crime, and they did not lose their consciousness or memory during the crime . However, two patients stated that they did not remember their criminal behavior, suggesting the possibility of postictal confusion . Although the two patients experienced overt seizure attacks after admission, they had not shown any alteration of consciousness with violent behavior . Ten patients (58.8%) had previous criminal records, which were due to similar types of crimes as those discussed in this paper . Seven patients were in a heavily drunken state when they committed their crimes (table 1). Eight patients experienced psychosis, and two patients committed their crimes in response to auditory or visual hallucinations . In eight patients, a positive correlation existed between the age of onset of their first crimes and the patients iq scores, as illustrated in table 2 and figure 1 (r = 0.533, p = 0.033). Educational achievement was defined as the number of years that were spent in a school (table 1). Seven out of 17 patients had finished middle school (i.e., they spent nine years in the school system), which is the final step of compulsory education in korea . Occupational experience was mostly confined to temporary and daily work in seven patients, while two patients had regular occupations . Four patients were divorced, and one patient was widowed because she had murdered her husband . Regular contact by family members of at least one visit per year was maintained in five patients . In this national center - based retrospective study, the most common epilepsy subtype in patients with violent crimes was localization - related epilepsy, and these patients showed favorable responses to medical treatment . It was noted that violent crimes were rarely observed during ictal or postictal periods, suggesting that neuropsychological and social factors might interplay and lead to violence rather than the violence being caused by just the epileptic seizure itself . Our study suggested that a low level of intelligence was related to an earlier onset of crimes . Educational achievement was markedly limited among the epileptic criminals, as were occupational experience and family support . The temporal relationship between seizure attacks and violent crimes was very subtle in most cases, suggesting that interictal violent behavior was more commonly associated with crimes than that of ictal or postictal periods . Although there have been several reports about peri - ictal violence, those seemed to be associated with purposeless movement or resistive activity against restriction . There was a recent report illustrating violent crimes that were probably due to epileptic automatism . However, it is very challenging to prove violent behaviors as an ictal manifestation, given that video - eeg monitoring is required to determine whether there were ictal discharges during the behavior . Two of our patients reported decreased levels of consciousness and amnesia after committing rape, but seizure attacks cannot be guaranteed at that time because such a complex behavior would be difficult to perform during a seizure attack . In addition, a high level of emotional excitability could also result in transient memory dysfunction . It is generally accepted that violent behavior among epilepsy patients is most commonly observed in the interictal period . Low intelligence has been regarded as one of the major risk factors of violence among epilepsy patients . Our results revealed a positive correlation between the age at the first crime and iq score, suggesting that epilepsy patients with low intelligence might be prone to violent behavior at a younger age . Although it is hard to ascertain that low intelligence is a direct cause of violent crime among epilepsy patients, low intelligence may force them to remain in a low socioeconomic state, which may possibly increase criminal behaviors . Another possible explanation is that structural brain abnormalities may result in epilepsy, intellectual disability, and impulse control dysfunction, and each of these can contribute to an overall disability . However, it is also possible that early criminal behavior might have caused social discrimination, including a lack of educational opportunities, which would have resulted in a low iq measured upon admission . Two of our patients committed a violent crime in response to a psychotic manifestation in the form of auditory hallucinations and persecutory delusions . Those crimes might have been prevented if the patients had been adequately treated with antipsychotics . Considering that seven out of 17 patients were drunk before the crime, inebriation seems to be another violence - provoking factor among epileptic patients, justifying educational programs for the responsible use of alcohol and/or abstinence programs . Although korea has had a mandatory educational system since 1954, and given that the majority of the population has completed middle school after 1993, our study revealed that ten patients (58.8%) could not graduate from middle school . The undereducated proportion of patients in our study was much larger compared to the percentage of general criminals who did not finish middle school (6.5%). The level of educational achievement among epileptic criminals seemed to be stationary compared to a previous study from the same institution, which revealed that 45.5% (10 out of 22 patients) of patients had not graduated from middle school . Poor employment conditions among epilepsy criminals was shown in another study, which reported a 92.3% (12 out of 13 patients) unemployment rate . Family support and marital relationships were lacking in the majority of the patients, which was reflected by a high divorce rate and lack of family contact during incarceration . Disrupted educational achievement, occupational opportunities, and family background may prohibit social integration after discharge and trigger future crime . Although this is a single national center - based study, our study population likely included the entire population of violent criminals with epilepsy specific to the selected time period because there is only one forensic hospital in south korea . We chose those patients with violent crime in order to homogenize the study population; however, selection bias may have taken place since the court ultimately decides the referral of epileptic criminals to the forensic hospital, even though medical advice also plays a substantial role . In addition, a small percentage of the study population may have produced biased results . Further studies comparing epilepsy patients with and without violent behavior may increase our understanding of the relationship between epilepsy and aggression . Low intelligence, alcohol abuse, and psychosis appear to be associated with criminal activity among epilepsy patients . Medical interventions to treat psychosis and alcohol abuse and social reinforcement may be helpful to prevent future violent behavior.
The online version of this article (doi:10.1186/s13321 - 015 - 0089-z) contains supplementary material, which is available to authorized users . Recent work has demonstrated the power of network - based approaches in drug discovery [13]. We have shown previously that a large semantic network of drug target interactions provides a powerful framework for predicting new associations and that an algorithm that predict drug - target associations by using this network performs surprisingly well, even without training datasets or incorporating target preference . In this work, we apply a random walk - based link prediction algorithm based on chen et al . To a more extensive drug we combine three networks drug drug, target target, and drug target to construct a heterogeneous network of drugs and targets . The links between drugs are obtained by quantifying molecular similarity with chemical fingerprints and examining the shared targets . The links between targets are obtained by calculating sequence similarity between proteins and again examining the shared drugs . Random walk is a useful mathematical framework that provides a systematic way to measure importance of nodes in a network . Pagerank, developed for ranking web pages, measures page clicks of hypothetical web surfers who randomly click hyperlinks in the network of webpages . Since it is possible for the surfer to be trapped in a dead - end webpage that does not have any outgoing link, at each time step the surfer may jump to a random webpage with a probability c. interestingly, this formulation also provides a simple way to define a random walk - based distance from a node a (or a set of nodes) to every other node, namely by allowing the random walkers to jump only to the source node a (or the source set of nodes) and restart from there . As a result, it is more likely to find the random walker at the vicinity of the source node than at a distant part of the network, and thus we are able to estimate the relevance (closeness) of each node with respect to the source node . The prediction method applies this idea to identify drugs and targets that are relevant to a set given set of drugs and targets . Consider an undirected, unweighted network g = (v, e), where v is the set of nodes and e is the set of links . For each pair of nodes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$a, b \in v $$\end{document}a, bv we can assign a proximity score by executing the following procedure: (1) we start a random walker from a. (2) at each time step, with the probability 1 c, the walker walks to one of the neighbors, b, according to the transition probability matrix wab = sab / ka, where sab is the adjacency matrix of the network and (sab equals 1 if node a and b are connected, 0 otherwise) ka denotes the degree of a. (3) with the probability c, the walker goes back to a. (4) after many time steps the probability of finding the random walker at node x converges to the steady - state probability, which is our proximity score sax . This approach allows us to generate candidate targets for a given drug even if the drug does not have any known targets . If there is a missing interaction between drug d and target t, we expect that d is more likely to interact with other targets that are similar to t, and drugs that are similar to d are more likely to interact with t. therefore, we expect to see many indirect paths that connect d and t. these indirect paths are likely to be captured by the random walkers . We apply the rwr algorithm to a drug target network and use an external dataset extracted from chembl 15 (544 drugs and 467 proteins) at bioactivity cutoff points of 10 and 1 m to quantitatively evaluate the performance and robustness of the approach . We compile a set of approved drugs from drugbank database (version 3.0), consisting of 727 compounds and 3519 protein targets (additional file 1). To construct the network between drugs, we incorporate two types of similarity measures: chemical (structural) similarity and target similarity . We calculate chemical similarity between drugs by using the jaccard index (tanimoto coefficient) between their chemical fingerprints . The jaccard index is defined as the size of the intersection of two sets divided by the size of the union of the sets, ranging between 0 and 1 . For binary vectors like chemical fingerprints, it is defined as c/(a + b c) where c is the number of bits in common, a is the number of bits in one of the fingerprints, and b is the number of bits in the other fingerprint . We use four types of chemical features namely, mdl maccs166 keys (fragmental descriptors), ecfp6 fingerprints (extended connectivity fingerprint path 6), 2d pharmacophore fingerprints (phfp4) and rocs program which uses tanimoto combo similarity which combines shape and color measures of a compound, we calculate them with rocs program . Ecfp (extended connectivity fingerprint) encodes information on atom - centered fragments that is derived from the variant of the morgan algorithm . Ecfps are generated using the neighborhood of each non - hydrogen atom into multiple circular layers up to a given diameter . These atom - centric substructural features are then mapped into integer codes using a hashing procedure, which constitute the extended - connectivity fingerprint . Ecfp can, for instance, represent a very large number of features (over 4 billion), do not rely on predefined dictionary of features, can represent stereochemical information, and can be interpreted as the presence of particular substructures . Pharmacophore fingerprints consist of pairs, triplets, or quartets of molecular features and the corresponding bond distances among them . We use phfp_4 (quartets which includes number of bonds in the shortest path between the features) fingerprints for the calculation . The feature vectors of quartets involve four pharmacophoric features, six euclidean distances separating those features, and an indication of chirality . For 3d alignment and similarity we used rocs 3.2, which is a shape - similarity method based on the tanimoto - like overlap of volumes . The alignment was developed using the combo score, which combines the tanimoto shape score with the color score that added the score for the appropriate overlap of groups with similar properties (donor, acceptor, hydrophobe, cation, anion, and ring) [http://docs.eyesopen.com/rocs/shape_theory.html] defined by smarts . Conformers for the data set is created using omega, about 250 conformers with rmsd threshold of 0.6 is generated . Rocs score performed in color optimization mode where it optimizes the molecular overlay to maximize both the shape overlap and the color overlap obtained by aligning groups with the same properties that are contained in the color force field file . This overlay is then subsequently scored using the sum of shape tanimoto for the overlay and the color score called tanimoto combo score . We use cs to refer the n by n chemical compounds similarity matrix . For the 727 drugs we used different chemical descriptors to calculate the tanimoto similarity distribution to create a view of 1 shows that for four fingerprints (166 maccs keys, phfp4, 3d rocs, and ecfp6), 0.56% had a similarity above 0.7 for the maccs keys, 0.31% had similarity above 0.4 for phfp4, 0.88% had similarity above 1.2 tanimoto combo score for rocs, 0.24% had similarity above 0.3 for ecfp6 . The mean similarity is 0.346, 0.019, 0.742, and 0.063 for maccs, phfp4, rocs, ecfp6 fingerprints, respectively . This indicates how diverse chemical structures are in the drug dataset (additional file 2).fig . 1plots showing the compounds similarity distributions according to the four types of chemical fingerprints . A rocs similarity, b phfp4 similarity, c maccs similarity, d ecfp similarity . Plots showing the compounds similarity distributions according to the four types of chemical fingerprints . A rocs similarity, we extracted 3,519 target proteins across all available species and their sequences from the drugbank database . As proteins in other species may provide useful information in our network - based approach, we keep all the proteins regardless of species . Note we calculate the sequence similarity matrix ts by using the r biostrings package and the normalization procedure proposed by bleakley and yamanishi: 1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$t_{s} = \frac{{sw\left ({g, g^ {'}} \right)}}{{\sqrt {sw\left ({g, g} \right)} \sqrt {sw\left ({g^ {'}, g^ {'}} \right)}}}, $$\end{document}ts = swg, gswg, gswg,g,where sw (,) means the original smith waterman similarity score . We construct a drug - target relationship matrix a whose element a(i, j) is 1 if drug i interacts with target j, otherwise 0 . The matrix is sparse; the total number of connections among the drugs and targets is only 2,557, with 687 drugs having at least one known target and with 628 proteins having at least one drug . 2a) that affect nervous systems mostly psychoanaleptics and psycholeptics have the largest number of interactions . As most drugs are metabolized by cytochrome p450, which serves as an important protein target and enzyme for the drugs, the interaction between important enzymes cyp3a4, cyp2d6 and cyp3a5 are not considered on the drug target interaction matrix except for the drug paliperidone, which has interactions to all the three cytochromes targets mentioned above.fig . 2 a drugs with the most target associations and b targets with the most drug associations . A drugs with the most target associations and b targets with the most drug associations . The top frequent targets are muscarinic receptor (acm1), adrenoreceptor alpha 1a (ada1a), histamine receptors (5ht2a), and dopamine receptors (drd2). In addition to the drug drug similarity matrix cs (based on chemical similarity) and target target similarity matrix ts (based on sequence similarity), we introduce additional measure of drug drug and target target similarities based on the network structure . \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{s}^{n} $$\end{document}csn is a drug drug similarity matrix based on the number of shared targets between drugs; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$t_{s}^{n} $$\end{document}tsn is a target target similarity matrix based on the shared drugs . The similarity between two drugs di and dj is quantified by jaccard coefficient, which is defined by:2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{s}^{n} (d_{i}, d_{j}) = \frac{{m_{l} (i, j)}}{{m_{l} \left ({i, i} \right) + m_{l} \left ({j, j} \right) - m_{l} (i, j)}}, $$\end{document}csn(di, dj)=ml(i, j)mli, i+mlj, j - ml(i, j),where ml is the inner product of the drug target interaction matrix . We define the final drug drug similarity matrix sd by taking a linear combination of the chemical similarity matrix (cs) and target sharing similarity matrix (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{s}^{n} $$\end{document}csn). Similarly, the final target target similarity matrix st is calculated using the sequence similarity matrix (ts) and drug sharing similarity matrix \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(t_{s}^{n}) $$\end{document}(tsn).3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${s_{d} = \, w_{d} c_{s} + \, \left ({1 - w_{d}} \right)c_{s}^{n}} $$\end{document}sd = wdcs+1-wdcsn4\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$s_{t} = \, w_{t} t_{s} + \, \left ({1 - w_{t}} \right)t_{s}^{n} $$\end{document}st = wtts+1-wttsn we combined drug drug, drug target, and target target networks into a undirected heterogeneous network . Many nodes have connections to both drugs and targets and we call them bridge nodes . At a bridge node, a random walker may jump to a node with the other type or to a node with the same type . For instance, if a random walker is at a drug node, it can jump to one of the connected target nodes with the probability, or jump to connected drug nodes with the probability 1 . We call the parameter the jumping probability . If is 0, a random walker will explore only one type of networks . Most importantly, the probability p(i) is the probability of finding the random walker at node i in the steady state . It gives a measure of probability of source and target node (proximity) between node i and the source nodes where the random walks restarts . The transition matrix is represented by,\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$w = \left [{\begin{array}{{20}c} {w_{tt}} & {w_{td}} \\ {w_{td}} & {w_{dd}} \\ \end{array}} \right] $$\end{document}w = wttwtdwtdwddhere wtt is the target to target transition matrix, wdd is the drug to drug transition matrix, wdt is drug to target transition matrix and wtd is target to drug transition matrix . The calculation of each of the transition matrix in discussed in chen et al . . (5) given below5\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p_{t + 1} = \, \left ({1 - c} \right)w^{t} p_{t} + \, cp_{0} $$\end{document}pt+1=1-cwtpt+cp0pt is a vector in which ith elements holds the probability of finding the random walker at node i at time step t. initial probability vector p0 controls the restart probability c.6\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p_{0} = \left [{\begin{array}{{20}c} {(1 - \eta) u_{0}} \\ {\eta v_{0}} \\ \end{array}} \right]\left ({\text{initial probability matrix}} \right) $$\end{document}p0=(1-)u0v0initial probability matrixu0 and v0 be the initial probability vectors for target network and drug network, respectively . Parameter controls the importance of two kinds of seed nodes, i.e. Drug node and target node . We tested the importance parameter \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} for different values ranging from 0 to 1 . After a number of iteration steps, the pt converges to a steady - state probability vector p, where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p_{\infty} = \left [{\begin{array}{{20}c} {u_{\infty}} \\ {v_{\infty}} \\ \end{array}} \right] $$\end{document}p=uv. In practice, we consider pt = p if the change between pt and pt + 1 (measure by the frobenius norm) is less than 10 . For finding novel targets for a given drug, we set the drug and the targets that are directly connected to the drug as our seed nodes . We set t2, t3, and d3 as the source nodes, namely u0 = and v0 = the stationary probability p represents the expected relevance of each drugs and targets regarding the source node set t2, t3 and d3 . For instance, if the value for t1 is the largest among t1, t4 and t5, then we expect that t1 is most likely to interact with d3 . We compile a set of approved drugs from drugbank database (version 3.0), consisting of 727 compounds and 3519 protein targets (additional file 1). To construct the network between drugs, we incorporate two types of similarity measures: chemical (structural) similarity and target similarity . We calculate chemical similarity between drugs by using the jaccard index (tanimoto coefficient) between their chemical fingerprints . The jaccard index is defined as the size of the intersection of two sets divided by the size of the union of the sets, ranging between 0 and 1 . For binary vectors like chemical fingerprints, it is defined as c/(a + b c) where c is the number of bits in common, a is the number of bits in one of the fingerprints, and b is the number of bits in the other fingerprint . We use four types of chemical features namely, mdl maccs166 keys (fragmental descriptors), ecfp6 fingerprints (extended connectivity fingerprint path 6), 2d pharmacophore fingerprints (phfp4) and rocs program which uses tanimoto combo similarity which combines shape and color measures of a compound, we calculate them with rocs program . Ecfp (extended connectivity fingerprint) encodes information on atom - centered fragments that is derived from the variant of the morgan algorithm . Ecfps are generated using the neighborhood of each non - hydrogen atom into multiple circular layers up to a given diameter . These atom - centric substructural features are then mapped into integer codes using a hashing procedure, which constitute the extended - connectivity fingerprint . Ecfp can, for instance, represent a very large number of features (over 4 billion), do not rely on predefined dictionary of features, can represent stereochemical information, and can be interpreted as the presence of particular substructures . Pharmacophore fingerprints consist of pairs, triplets, or quartets of molecular features and the corresponding bond distances among them . We use phfp_4 (quartets which includes number of bonds in the shortest path between the features) fingerprints for the calculation . The feature vectors of quartets involve four pharmacophoric features, six euclidean distances separating those features, and an indication of chirality . For 3d alignment and similarity we used rocs 3.2, which is a shape - similarity method based on the tanimoto - like overlap of volumes . The alignment was developed using the combo score, which combines the tanimoto shape score with the color score that added the score for the appropriate overlap of groups with similar properties (donor, acceptor, hydrophobe, cation, anion, and ring) [http://docs.eyesopen.com/rocs/shape_theory.html] defined by smarts . Conformers for the data set is created using omega, about 250 conformers with rmsd threshold of 0.6 is generated . Rocs score performed in color optimization mode where it optimizes the molecular overlay to maximize both the shape overlap and the color overlap obtained by aligning groups with the same properties that are contained in the color force field file . This overlay is then subsequently scored using the sum of shape tanimoto for the overlay and the color score called tanimoto combo score . We use cs to refer the n by n chemical compounds similarity matrix . For the 727 drugs we used different chemical descriptors to calculate the tanimoto similarity distribution to create a view of 1 shows that for four fingerprints (166 maccs keys, phfp4, 3d rocs, and ecfp6), 0.56% had a similarity above 0.7 for the maccs keys, 0.31% had similarity above 0.4 for phfp4, 0.88% had similarity above 1.2 tanimoto combo score for rocs, 0.24% had similarity above 0.3 for ecfp6 . The mean similarity is 0.346, 0.019, 0.742, and 0.063 for maccs, phfp4, rocs, ecfp6 fingerprints, respectively . This indicates how diverse chemical structures are in the drug dataset (additional file 2).fig . 1plots showing the compounds similarity distributions according to the four types of chemical fingerprints . A rocs similarity, b phfp4 similarity, c maccs similarity, d ecfp similarity . Plots showing the compounds similarity distributions according to the four types of chemical fingerprints . A rocs similarity, b phfp4 similarity, c maccs similarity, d ecfp similarity . We extracted 3,519 target proteins across all available species and their sequences from the drugbank database . As proteins in other species may provide useful information in our network - based approach, we keep all the proteins regardless of species . Note we calculate the sequence similarity matrix ts by using the r biostrings package and the normalization procedure proposed by bleakley and yamanishi: 1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$t_{s} = \frac{{sw\left ({g, g^ {'}} \right)}}{{\sqrt {sw\left ({g, g} \right)} \sqrt {sw\left ({g^ {'}, g^ {'}} \right)}}}, $$\end{document}ts = swg, gswg, gswg,g,where sw (,) means the original smith waterman similarity score . We construct a drug - target relationship matrix a whose element a(i, j) is 1 if drug i interacts with target j, otherwise 0 . The matrix is sparse; the total number of connections among the drugs and targets is only 2,557, with 687 drugs having at least one known target and with 628 proteins having at least one drug . 2a) that affect nervous systems mostly psychoanaleptics and psycholeptics have the largest number of interactions . As most drugs are metabolized by cytochrome p450, which serves as an important protein target and enzyme for the drugs, the interaction between important enzymes cyp3a4, cyp2d6 and cyp3a5 are not considered on the drug target interaction matrix except for the drug paliperidone, which has interactions to all the three cytochromes targets mentioned above.fig . 2 a drugs with the most target associations and b targets with the most drug associations . A drugs with the most target associations and b targets with the most drug associations . The top frequent targets are muscarinic receptor (acm1), adrenoreceptor alpha 1a (ada1a), histamine receptors (5ht2a), and dopamine receptors (drd2). In addition to the drug drug similarity matrix cs (based on chemical similarity) and target target similarity matrix ts (based on sequence similarity), we introduce additional measure of drug drug and target target similarities based on the network structure . \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{s}^{n} $$\end{document}csn is a drug drug similarity matrix based on the number of shared targets between drugs; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$t_{s}^{n} $$\end{document}tsn is a target target similarity matrix based on the shared drugs . The similarity between two drugs di and dj is quantified by jaccard coefficient, which is defined by:2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{s}^{n} (d_{i}, d_{j}) = \frac{{m_{l} (i, j)}}{{m_{l} \left ({i, i} \right) + m_{l} \left ({j, j} \right) - m_{l} (i, j)}}, $$\end{document}csn(di, dj)=ml(i, j)mli, i+mlj, j - ml(i, j),where ml is the inner product of the drug target interaction matrix . We define the final drug drug similarity matrix sd by taking a linear combination of the chemical similarity matrix (cs) and target sharing similarity matrix (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{s}^{n} $$\end{document}csn). Similarly, the final target target similarity matrix st is calculated using the sequence similarity matrix (ts) and drug sharing similarity matrix \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(t_{s}^{n}) $$\end{document}(tsn).3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${s_{d} = \, w_{d} c_{s} + \, \left ({1 - w_{d}} \right)c_{s}^{n}} $$\end{document}sd = wdcs+1-wdcsn4\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$s_{t} = \, w_{t} t_{s} + \, \left ({1 - w_{t}} \right)t_{s}^{n} $$\end{document}st = wtts+1-wttsn we compile a set of approved drugs from drugbank database (version 3.0), consisting of 727 compounds and 3519 protein targets (additional file 1). To construct the network between drugs, we incorporate two types of similarity measures: chemical (structural) similarity and target similarity . We calculate chemical similarity between drugs by using the jaccard index (tanimoto coefficient) between their chemical fingerprints . The jaccard index is defined as the size of the intersection of two sets divided by the size of the union of the sets, ranging between 0 and 1 . For binary vectors like chemical fingerprints, it is defined as c/(a + b c) where c is the number of bits in common, a is the number of bits in one of the fingerprints, and b is the number of bits in the other fingerprint . We use four types of chemical features namely, mdl maccs166 keys (fragmental descriptors), ecfp6 fingerprints (extended connectivity fingerprint path 6), 2d pharmacophore fingerprints (phfp4) and rocs program which uses tanimoto combo similarity which combines shape and color measures of a compound, we calculate them with rocs program . Ecfp (extended connectivity fingerprint) encodes information on atom - centered fragments that is derived from the variant of the morgan algorithm . Ecfps are generated using the neighborhood of each non - hydrogen atom into multiple circular layers up to a given diameter . These atom - centric substructural features are then mapped into integer codes using a hashing procedure, which constitute the extended - connectivity fingerprint . Ecfp can, for instance, represent a very large number of features (over 4 billion), do not rely on predefined dictionary of features, can represent stereochemical information, and can be interpreted as the presence of particular substructures . Pharmacophore fingerprints consist of pairs, triplets, or quartets of molecular features and the corresponding bond distances among them . We use phfp_4 (quartets which includes number of bonds in the shortest path between the features) fingerprints for the calculation . The feature vectors of quartets involve four pharmacophoric features, six euclidean distances separating those features, and an indication of chirality . For 3d alignment and similarity we used rocs 3.2, which is a shape - similarity method based on the tanimoto - like overlap of volumes . The alignment was developed using the combo score, which combines the tanimoto shape score with the color score that added the score for the appropriate overlap of groups with similar properties (donor, acceptor, hydrophobe, cation, anion, and ring) [http://docs.eyesopen.com/rocs/shape_theory.html] defined by smarts . Conformers for the data set is created using omega, about 250 conformers with rmsd threshold of 0.6 is generated . Rocs score performed in color optimization mode where it optimizes the molecular overlay to maximize both the shape overlap and the color overlap obtained by aligning groups with the same properties that are contained in the color force field file . This overlay is then subsequently scored using the sum of shape tanimoto for the overlay and the color score called tanimoto combo score . We use cs to refer the n by n chemical compounds similarity matrix . For the 727 drugs we used different chemical descriptors to calculate the tanimoto similarity distribution to create a view of 1 shows that for four fingerprints (166 maccs keys, phfp4, 3d rocs, and ecfp6), 0.56% had a similarity above 0.7 for the maccs keys, 0.31% had similarity above 0.4 for phfp4, 0.88% had similarity above 1.2 tanimoto combo score for rocs, 0.24% had similarity above 0.3 for ecfp6 . The mean similarity is 0.346, 0.019, 0.742, and 0.063 for maccs, phfp4, rocs, ecfp6 fingerprints, respectively . This indicates how diverse chemical structures are in the drug dataset (additional file 2).fig . 1plots showing the compounds similarity distributions according to the four types of chemical fingerprints . A rocs similarity, b phfp4 similarity, c maccs similarity, d ecfp similarity . Plots showing the compounds similarity distributions according to the four types of chemical fingerprints . A rocs similarity, b phfp4 similarity, c maccs similarity, d ecfp similarity . We extracted 3,519 target proteins across all available species and their sequences from the drugbank database . As proteins in other species may provide useful information in our network - based approach, we keep all the proteins regardless of species . We calculate the sequence similarity matrix ts by using the r biostrings package and the normalization procedure proposed by bleakley and yamanishi: 1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$t_{s} = \frac{{sw\left ({g, g^ {'}} \right)}}{{\sqrt {sw\left ({g, g} \right)} \sqrt {sw\left ({g^ {'}, g^ {'}} \right)}}}, $$\end{document}ts = swg, gswg, gswg,g,where sw (,) means the original smith waterman similarity score . We construct a drug - target relationship matrix a whose element a(i, j) is 1 if drug i interacts with target j, otherwise 0 . The matrix is sparse; the total number of connections among the drugs and targets is only 2,557, with 687 drugs having at least one known target and with 628 proteins having at least one drug . 2a) that affect nervous systems mostly psychoanaleptics and psycholeptics have the largest number of interactions . As most drugs are metabolized by cytochrome p450, which serves as an important protein target and enzyme for the drugs, the interaction between important enzymes cyp3a4, cyp2d6 and cyp3a5 are not considered on the drug target interaction matrix except for the drug paliperidone, which has interactions to all the three cytochromes targets mentioned above.fig . 2 a drugs with the most target associations and b targets with the most drug associations . A drugs with the most target associations and b targets with the most drug associations . The top frequent targets are muscarinic receptor (acm1), adrenoreceptor alpha 1a (ada1a), histamine receptors (5ht2a), and dopamine receptors (drd2). In addition to the drug drug similarity matrix cs (based on chemical similarity) and target target similarity matrix ts (based on sequence similarity), we introduce additional measure of drug drug and target target similarities based on the network structure . \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{s}^{n} $$\end{document}csn is a drug drug similarity matrix based on the number of shared targets between drugs; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$t_{s}^{n} $$\end{document}tsn is a target target similarity matrix based on the shared drugs . The similarity between two drugs di and dj is quantified by jaccard coefficient, which is defined by:2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{s}^{n} (d_{i}, d_{j}) = \frac{{m_{l} (i, j)}}{{m_{l} \left ({i, i} \right) + m_{l} \left ({j, j} \right) - m_{l} (i, j)}}, $$\end{document}csn(di, dj)=ml(i, j)mli, i+mlj, j - ml(i, j),where ml is the inner product of the drug target interaction matrix . We define the final drug drug similarity matrix sd by taking a linear combination of the chemical similarity matrix (cs) and target sharing similarity matrix (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{s}^{n} $$\end{document}csn). Similarly, the final target target similarity matrix st is calculated using the sequence similarity matrix (ts) and drug sharing similarity matrix \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(t_{s}^{n}) $$\end{document}(tsn).3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${s_{d} = \, w_{d} c_{s} + \, \left ({1 - w_{d}} \right)c_{s}^{n}} $$\end{document}sd = wdcs+1-wdcsn4\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$s_{t} = \, w_{t} t_{s} + \, \left ({1 - w_{t}} \right)t_{s}^{n} $$\end{document}st = wtts+1-wttsn many nodes have connections to both drugs and targets and we call them bridge nodes . At a bridge node, a random walker may jump to a node with the other type or to a node with the same type . For instance, if a random walker is at a drug node, it can jump to one of the connected target nodes with the probability, or jump to connected drug nodes with the probability 1 . We call the parameter the jumping probability . If is 0, a random walker will explore only one type of networks . Most importantly, the probability p(i) is the probability of finding the random walker at node i in the steady state . It gives a measure of probability of source and target node (proximity) between node i and the source nodes where the random walks restarts . The transition matrix is represented by,\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$w = \left [{\begin{array}{{20}c} {w_{tt}} & {w_{td}} \\ {w_{td}} & {w_{dd}} \\ \end{array}} \right] $$\end{document}w = wttwtdwtdwddhere wtt is the target to target transition matrix, wdd is the drug to drug transition matrix, wdt is drug to target transition matrix and wtd is target to drug transition matrix . The calculation of each of the transition matrix in discussed in chen et al . . (5) given below5\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p_{t + 1} = \, \left ({1 - c} \right)w^{t} p_{t} + \, cp_{0} $$\end{document}pt+1=1-cwtpt+cp0pt is a vector in which ith elements holds the probability of finding the random walker at node i at time step t. initial probability vector p0 controls the restart probability c.6\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p_{0} = \left [{\begin{array}{{20}c} {(1 - \eta) u_{0}} \\ {\eta v_{0}} \\ \end{array}} \right]\left ({\text{initial probability matrix}} \right) $$\end{document}p0=(1-)u0v0initial probability matrixu0 and v0 be the initial probability vectors for target network and drug network, respectively . Parameter controls the importance of two kinds of seed nodes, i.e. Drug node and target node . We tested the importance parameter \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} for different values ranging from 0 to 1 . After a number of iteration steps, the pt converges to a steady - state probability vector p, where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p_{\infty} = \left [{\begin{array}{{20}c} {u_{\infty}} \\ {v_{\infty}} \\ \end{array}} \right] $$\end{document}p=uv. In practice, we consider pt = p if the change between pt and pt + 1 (measure by the frobenius norm) is less than 10 . For finding novel targets for a given drug, we set the drug and the targets that are directly connected to the drug as our seed nodes . We set t2, t3, and d3 as the source nodes, namely u0 = and v0 = the stationary probability p represents the expected relevance of each drugs and targets regarding the source node set t2, t3 and d3 . For instance, if the value for t1 is the largest among t1, t4 and t5, then we expect that t1 is most likely to interact with d3 . We evaluated our approach using a perturbed network where we have removed some links to measure how well our approach re - identifies those removed links . There are five parameter to explore: the restart probability c, the jumping probability, the relative importance, which controls the relative importance between two types of seeds, wd and wt that weigh the drug and target similarity matrices and network based similarity measure of the drugs and proteins, respectively . Among these five parameters, we have tested because, to our knowledge, the restart probability c, jumping probability, and wd and wt are not likely to affect the results in a significant way . First, it is known that in most cases the choice of restart probability c does not affect performance of pagerank algorithm and other pagerank - based algorithms . For instance, the results of pagerank are highly insensitive to the choice of restart probability [14, 15]. It has been shown that the prediction results from rwr are also robust [7, 1416]. Because of these evidences, we here simply adopt the previously used value of 0.3 . Second, the robustness of (jumping probability) has already been discussed [1517]. It has been shown that the weight parameters wd and wt are robust among the prediction results . In our drug target network 684 (94%) drugs have at least one target . We prepare a test network of 684 drugs where we remove one links from 684 drugs with a total of 684 drug the links include drugs which has only one target in order to see if the method able to predict single known interaction . We divide the number of actual targets that are in the top n lists by the number of tests (684) and call the fraction as recoveredfraction. We also used a random set to calculate the statistics with same parameters and found that the results are way better than random set . We tested our results with different values of wd and wt ranging from 0 to 1 and found that at extreme point like 0 and 1 the performances drops radically but the performance gets best on values of wd and wt of 0.5 given in additional file 3: sheet 3 . We test different values of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} for the four different chemical fingerprints to identify the optimal value of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} and the right of chemical features . We observed that the prediction performance becomes optimal when \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} is small but not 0 . We found optimal performance at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta = 0.0 1 $$\end{document}=0.01 . For all the other values of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta \left ({0 . 9} \right) $$\end{document}0.1 - 0.9 the prediction rate for all fingerprints is equal . We find nearly 28% of the true interactions out of 684 can be retrieved at the top 10 rank positions and more than 38% of the interactions can be retrieved at the top 50 rank positions . We also prepare 10 test networks of drugs that have more than two targets links, where we randomly remove 1001,000 links . Using the 10 test networks we predicted the removed links . We repeat this process, from preparing a test network to calculating the recovered fraction, 50 times to obtain the average recovered fraction. From table 1 we can see that if we remove 100 links it gave us the best prediction rates and as we increase the number of removed links to 1,000 the prediction rates falls . From table 2 shows the recovered fraction rates for top 10, 25, 50, 100, 200, 500, 1,000 retrieved targets we also find almost 32% of the true interactions can be retrieved at the top 10 rank positions for each of the test networks and more than 75% of the true interactions can be retrieved at the top 50 rank positions . This indicates that the method performs well if we remove links from drugs which are having at least two or more known interactions, since it uses the given interaction information in the network . We also measured the area under accumulation curve, area under roc curve auc (top 10%), bedroc and enrichment factor given in table 1 . The area under the receiver operating characteristic (roc) curve (auc) is widely used to evaluate the performance of the ranking method . The advantage of using auc is, the value ranges from 0 to 1 with 0.5 corresponding to randomness . Another key criterion for measuring the success of ranking prediction is the enrichment of annotated associations among top ranking associations . The higher the percentage of annotated associations among the top ranking associations, the better the performance of the prediction . The enrichment criterion is evaluated by enrichment factor (ef) [16, 17]. Ef reflects the capability of a screening application to detect true links (true positives) compared to random selection . Thus, its value should always be greater than 1 and the higher it is, the better the enrichment performance . When we are predicting links it should rank true links in the top - ranking list . Metric likes roc not sensitive to early recognition for example considering cases like where (1) true links are retrieved at beginning of a rank ordered list, (2) where true links are randomly distributed and (3) where true links, which are retrieved in the middle of the rank, ordered list . In all of the above cases roc is 0.5 but in terms of early recognition we see that case (1) is better than (2) and (3). To overcome these limitations methods such as rie and bedroc, one can test whether the method is able to rank true links early or not.table 1shows the recovered fraction values for top 10, 25, 50, 100, 200, 500 and 1,000 ranks with the number of links removednumber of links removedauacaucbedrocefauc (top 10%)1000.9470.9910.8339.230.8672000.9380.9950.8279.1000.8573000.9300.9950.8188.950.8454000.9200.9910.8058.790.8305000.9160.9970.8018.710.8246000.9080.9950.7898.560.8127000.8990.9810.7808.420.8028000.8850.9970.7618.200.7839000.8690.9550.7417.910.7651,0000.8540.9560.7157.620.741table 2shows the recovered fraction values for top 10, 25, 50, 100, 200, 500 and 1,000 ranks with the number of links removed #of links removedtop 10 (%) top 25 (%) top 50 (%) top 100 (%) top 200 (%) top 500 (%) top 1,000 (%) 10032.2478.2487.7690.7491.9293.2293.8820031.9277.9587.2689.8691.1592.3793.1230032.1478.3186.8289.4890.6891.8092.6340032.0477.485.3488.0789.2490.3391.4550032.6277.3985.0487.5688.7089.9591.160032.5376.2183.6886.2387.5488.8690.1670032.575.6482.6985.1886.5787.8989.3380033.0674.1380.8883.4584.8686.3587.9790033.5872.1478.4981.0482.7784.5786.381,00033.7169.8176.00878.3180.2282.1284.42 shows the recovered fraction values for top 10, 25, 50, 100, 200, 500 and 1,000 ranks with the number of links removed shows the recovered fraction values for top 10, 25, 50, 100, 200, 500 and 1,000 ranks with the number of links removed we found that the performance of the algorithm for ranking the targets by different chemical features is approximately same which indicates using this approach a user can identify protein targets with any one set of chemical features . We used public 166 maccs keys, ecfp6, phfp4 and 3d rocs to perform the analysis and it is surprising that the commercial programs feature performance is same as the 166 public maccs keys . As a baseline, we randomized the interactions and similarity matrices and performed rwr and found the random set prediction rate was way below our original prediction rate as given in additional file 3: sheet 1 . In addition to the internal evaluation using link perturbation approach, we evaluate the performance of our method using an external dataset, namely chembl version 15 database . From chembl 15 data we extract all the drugs and targets that have activity values not more than 1 m (additional file 3: sheet 4) and 10 m (additional file 3: sheet 4) with units ic50, ki, kd, ec50, ac50, lc50, and gi50 . Our training model is based on drugbank and uniprot database so we mapped the drugs and targets chembl ids with the drugbank ids and uniprot ids . We used pubchem mapping tool (http://pubchem.ncbi.nlm.nih.gov/idexchange/) to map chembl ids to drugbank ids and the uniprot mapping tool (http://www.uniprot.org/?tab=mapping) to map target chembl ids to uniprot ids . It gives us 544 drugs and 467 protein targets, with 3,463 and 564 drug target interactions those are below 10 and 1 m, respectively . Naturally, there are lots of interactions that are present in both drugbank and chembl . We tested performance of parameter \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} at different values on chembl 1 m set and 10 m having which have more than 0, 1 and 2 target relations . Figures 3 and 4 shows the recovered fractions against the rank with different \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} (eta) values for chembl datat at 1 and 10 m cutoff with different fingerprints respectively.fig . 3showing the recovered fractions against the rank with different \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} (eta) values for chembl datat at 1 m cutoff . The recovered fraction is calculated by the number of targets retrieved at different rank positions over total number true interactions . A maccs, b phfp4, c rocs, d ecfp4.fig . 4showing the recovered fractions against the rank with different \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} the recovered fraction is calculated by the number of targets retrieved at different rank positions over total number true interactions . Showing the recovered fractions against the rank with different \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} (eta) values for chembl datat at 1 m cutoff . The recovered fraction is calculated by the number of targets retrieved at different rank positions over total number true interactions . Showing the recovered fractions against the rank with different \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} (eta) values for chembl data at 10 m cutoff . The recovered fraction is calculated by the number of targets retrieved at different rank positions over total number true interactions . A maccs, b phfp4, c rocs, d ecfp4 . From tables 3 and 4 we observe that rwr performance is better for 1 m target than 10 m because at 10 m we have lots off targets from different classes and as a result of that the prediction rate falls . For chembl 1 m dataset, drugs having more than 0, 1 and 2 targets we achieve bedroc score of 0.433, 0.553 and 0.611, respectively, which is much better than a random set of interactions . To test whether random walk performs better than just a simple sequence similarity search we took the approved drugs and it s known targets from the chembl 10 m dataset and performed sequence similarity based sech against 3,519 targets and ranked them . We found rwr performance is way better in ranking targets than performing simple sequence based search . This is the first time that the random walk - based method is evaluated using a binding assay dataset (cf . [3, 5]).table 3shows the types of data we used the drug target interaction having more than 1 and 2 drug interactions, area under the accumulated curve (auac), area under the roc curve (auc), bedroc and, enrichment factor (ef) and auc top 10%data typesnumber of targetsauac (%) auc (%) bedrocefauc (top 10%)chembl 1 m (rwr)>00.7090.9950.4335.0580.455chembl 1 m (seq)>00.6700.6700.3964.480.414chembl 1 m (random rwr)>00.4940.4930.0751.0900.079chembl 10 m (rwr)>00.5960.8370.3233.8650.351chembl 10 m (seq)>00.5180.5180.2372.6410.2555chembl 10 m (random rwr)>00.3940.3640.0360.9540.029chembl 1 m (rwr)>10.7840.7840.5536.2860.569chembl 1 m (seq)>10.6520.6510.3904.5070.412chembl 1 m (random rwr)>10.4830.4830.0811.2900.083chembl 10 m (rwr)>10.6130.610.3534.0910.378chembl 10 m (seq)>10.5510.5520.2793.0840.300chembl 10 m (random rwr)>10.5140.5140.0751.2440.088chembl 1 m (rwr)>20.8230.8240.6116.8660.631chembl 1 m (seq)>20.7010.7050.5135.1090.469chembl 1 m>20.5330.5330.06711.4650.065chembl 10 m (rwr)>20.6320.6330.3994.5690.422chembl 10 m (seq)>20.5690.5690.2983.030.315chembl 10 m (random rwr)>20.5210.5210.2621.950.125table 4shows the types of data we used the drug target interaction having more than 1 and 2 drug interactions and sensitivity (hit rate) at top 10, 25, 50, 100 and 200 predicted targetsdata typesnumber of targetstop 10 (%) top 25 (%) top 50 (%) top 100 (%) top 200 (%) chembl 1 m (rwr)>00.1440.3420.4700.5320.607chembl 1 m (seq)>00.1640.3150.3940.4200.430chembl 1 m (random rwr)>00.0020.0130.0180.0360.021chembl 10 m (rwr)>00.110.2470.3240.3860.409chembl 10 m (seq)>00.1220.1830.2340.2490.254chembl 10 m (random rwr)>00.0140.0230.0350.0480.079chembl 1 m (rwr)>10.2740.4770.5500.5800.614chembl 1 m (seq)>10.1890.3500.4280.4720.513chembl 1 m (random rwr)>10.0070.0230.0380.0760.091chembl 10 m (rwr)>10.2200.2770.3480.4170.446chembl 10 m (seq)>10.130.2120.2760.2960.302chembl 10 m (random rwr)>10.0140.0230.0350.0480.079chembl 1 m (rwr)>20.2710.5180.5980.6340.677chembl 1 m (seq)>20.190.3930.530.560.598chembl 1 m>20.0060.0180.0340.0550.08chembl 10 m (rwr)>20.2330.2970.3530.42990.472chembl 10 m (seq)>20.130.220.2950.3160.324chembl 10 m (random rwr)>20.0120.0280.0400.0570.093 shows the types of data we used the drug target interaction having more than 1 and 2 drug interactions, area under the accumulated curve (auac), area under the roc curve (auc), bedroc and, enrichment factor (ef) and auc top 10% shows the types of data we used the drug target interaction having more than 1 and 2 drug interactions and sensitivity (hit rate) at top 10, 25, 50, 100 and 200 predicted targets here, as a case study we investigate the target profiles of the popular top selling drugs in 2012 . First, we consider \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u_{\infty}, $$\end{document}u, the steady - state probability vector for the targets in our framework, as target profile of a drug . We find that some targets are associated with many drugs (see table 5). For instance, adrenoceptor alpha 1a appears in 60% of drug s top 10 target association lists; serotonin receptor 5ht2a appear in 43%; and adrenoceptor alpha 1b in 35% . Most drugs shown on the table 5 mostly belong to the rhodopsin class of gpcr s . In additional file 4, 5 shows a bipartite network of 110 drugs with top 10 predicted targets for each drug.table 5the top 10 associated targets of 110 drugs with true percentage of associated target before prediction and predicted percentage of associationtargetspercentage of drugs associated with the targets based on drugbank and chembl (%) percentage of drug associations appearing in top-10 target list in our prediction (%) ada1a 7.2760 5ht2a 4.5443.63 ada1b 7.2735.45 5ht1a 4.5433.63 adrb1 5.4531.81 5ht1b 5.4530.90 5ht2c 3.6330 acm2 9.0926.36 5ht3a 4.5425.45 the size and label of the target nodes is proportional to the degree of the nodes . The top 10 associated targets of 110 drugs with true percentage of associated target before prediction and predicted percentage of association shows the network of the top 10 predicted targets of 110 drugs . The size and label of the target nodes is proportional to the degree of the nodes . We took some random drugs and tried to find known binding associations to protein targets . We searched three databases chembl, pdsp, and pubchem using the binding coefficients like ic50 and ki . Further investigation may have significant values on understanding side effects of existing drugs as well as repurposing them.table 6drug target interactions with association values from different databasesproteindrugsourceactivity tpeactivity m 5ht2a carvedilolpubchem aid 625192ic500.41 5ht2a desloratadinepubchem aid 625192ic500.033 kcnh2 lidocainechemblic50263.02 adrb1 salmetorolchemblic500.501 5ht1a amphetaminepdsp databaseki6.6 hdac2 atorvastatinchemblic5022.5 ada1a duloxentinepdspki10 acm1 montelukastpubchem aid 625153ic508.045 sc6a4 quetiapinepdspki10 drug target interactions with association values from different databases finally, let us summarize the contributions of this paper . First, we offer a general approach that takes the whole drug target network into account without separating protein categories, in contrast to the previous study . The number of interactions between drugs and targets is 2,557, which makes 684 drugs to have at least one known target and 457 drugs to have two or more interactions . The proteins in the dataset are grouped under 15 different categories according to chembl target classifications (https://www.ebi.ac.uk/chembl/target/browser). Out of 3,519 proteins, 1,386 proteins the number of drugs that have at least two interactions with proteins that are categorized is 412 . Among these 412 drugs, the number of drugs that have interactions with proteins from multiple groups is 169 . In other words, we estimate that about 40% of drugs have interactions across multiple groups according chembl dataset . Therefore, it is more reasonable to consider all proteins together, rather than running the prediction model separately for each category . Second, we further investigate the methodology by presenting a benchmark of a parameter in conjunction with the four chemical fingerprint types: maccs 166 keys, ecfp6 fingerprints, phfp4 fingerprints, and rocs . In the previous study, the parameter space of is not explored below 0.1, but we find that we can improve the performance by decreasing eta below 0.1 . We also find that the performance is robust under the choice of chemical fingerprinting method, particularly when is around the optimum (~0.01). Very small \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} eta means the walk in the target network is much more important than the walk on the drug drug network . In a sense we evaluated our approach using a perturbed network where we have removed some links to measure how well our approach re - identifies those removed links . There are five parameter to explore: the restart probability c, the jumping probability, the relative importance, which controls the relative importance between two types of seeds, wd and wt that weigh the drug and target similarity matrices and network based similarity measure of the drugs and proteins, respectively . Among these five parameters, we have tested because, to our knowledge, the restart probability c, jumping probability, and wd and wt are not likely to affect the results in a significant way . First, it is known that in most cases the choice of restart probability c does not affect performance of pagerank algorithm and other pagerank - based algorithms . For instance, the results of pagerank are highly insensitive to the choice of restart probability [14, 15]. It has been shown that the prediction results from rwr are also robust [7, 1416]. Because of these evidences, we here simply adopt the previously used value of 0.3 . Second, the robustness of (jumping probability) has already been discussed [1517]. It has been shown that the weight parameters wd and wt are robust among the prediction results . In our drug target network 684 (94%) drugs have at least one target . We prepare a test network of 684 drugs where we remove one links from 684 drugs with a total of 684 drug the links include drugs which has only one target in order to see if the method able to predict single known interaction . We divide the number of actual targets that are in the top n lists by the number of tests (684) and call the fraction as recoveredfraction. We also used a random set to calculate the statistics with same parameters and found that the results are way better than random set . We tested our results with different values of wd and wt ranging from 0 to 1 and found that at extreme point like 0 and 1 the performances drops radically but the performance gets best on values of wd and wt of 0.5 given in additional file 3: sheet 3 . We test different values of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} for the four different chemical fingerprints to identify the optimal value of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} and the right of chemical features . We observed that the prediction performance becomes optimal when \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} is small but not 0 . We found optimal performance at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta = 0.0 1 $$\end{document}=0.01 . For all the other values of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta \left ({0 . 9} \right) $$\end{document}0.1 - 0.9 the prediction rate for all fingerprints is equal . We find nearly 28% of the true interactions out of 684 can be retrieved at the top 10 rank positions and more than 38% of the interactions can be retrieved at the top 50 rank positions . We also prepare 10 test networks of drugs that have more than two targets links, where we randomly remove 1001,000 links . Using the 10 test networks we predicted the removed links . We repeat this process, from preparing a test network to calculating the recovered fraction, 50 times to obtain the average recovered fraction. From table 1 we can see that if we remove 100 links it gave us the best prediction rates and as we increase the number of removed links to 1,000 the prediction rates falls . From table 2 shows the recovered fraction rates for top 10, 25, 50, 100, 200, 500, 1,000 retrieved targets we also find almost 32% of the true interactions can be retrieved at the top 10 rank positions for each of the test networks and more than 75% of the true interactions can be retrieved at the top 50 rank positions . This indicates that the method performs well if we remove links from drugs which are having at least two or more known interactions, since it uses the given interaction information in the network . We also measured the area under accumulation curve, area under roc curve auc (top 10%), bedroc and enrichment factor given in table 1 . The area under the receiver operating characteristic (roc) curve (auc) is widely used to evaluate the performance of the ranking method . The advantage of using auc is, the value ranges from 0 to 1 with 0.5 corresponding to randomness . Another key criterion for measuring the success of ranking prediction is the enrichment of annotated associations among top ranking associations . The higher the percentage of annotated associations among the top ranking associations, the better the performance of the prediction . The enrichment criterion is evaluated by enrichment factor (ef) [16, 17]. Ef reflects the capability of a screening application to detect true links (true positives) compared to random selection . Thus, its value should always be greater than 1 and the higher it is, the better the enrichment performance . When we are predicting links it should rank true links in the top - ranking list . Metric likes roc not sensitive to early recognition for example considering cases like where (1) true links are retrieved at beginning of a rank ordered list, (2) where true links are randomly distributed and (3) where true links, which are retrieved in the middle of the rank, ordered list . In all of the above cases roc is 0.5 but in terms of early recognition we see that case (1) is better than (2) and (3). To overcome these limitations methods such as rie and bedroc, one can test whether the method is able to rank true links early or not.table 1shows the recovered fraction values for top 10, 25, 50, 100, 200, 500 and 1,000 ranks with the number of links removednumber of links removedauacaucbedrocefauc (top 10%)1000.9470.9910.8339.230.8672000.9380.9950.8279.1000.8573000.9300.9950.8188.950.8454000.9200.9910.8058.790.8305000.9160.9970.8018.710.8246000.9080.9950.7898.560.8127000.8990.9810.7808.420.8028000.8850.9970.7618.200.7839000.8690.9550.7417.910.7651,0000.8540.9560.7157.620.741table 2shows the recovered fraction values for top 10, 25, 50, 100, 200, 500 and 1,000 ranks with the number of links removed #of links removedtop 10 (%) top 25 (%) top 50 (%) top 100 (%) top 200 (%) top 500 (%) top 1,000 (%) 10032.2478.2487.7690.7491.9293.2293.8820031.9277.9587.2689.8691.1592.3793.1230032.1478.3186.8289.4890.6891.8092.6340032.0477.485.3488.0789.2490.3391.4550032.6277.3985.0487.5688.7089.9591.160032.5376.2183.6886.2387.5488.8690.1670032.575.6482.6985.1886.5787.8989.3380033.0674.1380.8883.4584.8686.3587.9790033.5872.1478.4981.0482.7784.5786.381,00033.7169.8176.00878.3180.2282.1284.42 shows the recovered fraction values for top 10, 25, 50, 100, 200, 500 and 1,000 ranks with the number of links removed shows the recovered fraction values for top 10, 25, 50, 100, 200, 500 and 1,000 ranks with the number of links removed we found that the performance of the algorithm for ranking the targets by different chemical features is approximately same which indicates using this approach a user can identify protein targets with any one set of chemical features . We used public 166 maccs keys, ecfp6, phfp4 and 3d rocs to perform the analysis and it is surprising that the commercial programs feature performance is same as the 166 public maccs keys . As a baseline, we randomized the interactions and similarity matrices and performed rwr and found the random set prediction rate was way below our original prediction rate as given in additional file 3: sheet 1 . In addition to the internal evaluation using link perturbation approach, we evaluate the performance of our method using an external dataset, namely chembl version 15 database . From chembl 15 data we extract all the drugs and targets that have activity values not more than 1 m (additional file 3: sheet 4) and 10 m (additional file 3: sheet 4) with units ic50, ki, kd, ec50, ac50, lc50, and gi50 . Our training model is based on drugbank and uniprot database so we mapped the drugs and targets chembl ids with the drugbank ids and uniprot ids . We used pubchem mapping tool (http://pubchem.ncbi.nlm.nih.gov/idexchange/) to map chembl ids to drugbank ids and the uniprot mapping tool (http://www.uniprot.org/?tab=mapping) to map target chembl ids to uniprot ids . It gives us 544 drugs and 467 protein targets, with 3,463 and 564 drug target interactions those are below 10 and 1 m, respectively . Naturally, there are lots of interactions that are present in both drugbank and chembl . We tested performance of parameter \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} at different values on chembl 1 m set and 10 m having which have more than 0, 1 and 2 target relations . Figures 3 and 4 shows the recovered fractions against the rank with different \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} (eta) values for chembl datat at 1 and 10 m cutoff with different fingerprints respectively.fig . 3showing the recovered fractions against the rank with different \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} (eta) values for chembl datat at 1 m cutoff . The recovered fraction is calculated by the number of targets retrieved at different rank positions over total number true interactions . A maccs, b phfp4, c rocs, d ecfp4.fig . 4showing the recovered fractions against the rank with different \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} (eta) values for chembl data at 10 m cutoff . The recovered fraction is calculated by the number of targets retrieved at different rank positions over total number true interactions . Showing the recovered fractions against the rank with different \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} (eta) values for chembl datat at 1 m cutoff . The recovered fraction is calculated by the number of targets retrieved at different rank positions over total number true interactions . Showing the recovered fractions against the rank with different \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} (eta) values for chembl data at 10 m cutoff . The recovered fraction is calculated by the number of targets retrieved at different rank positions over total number true interactions . A maccs, b phfp4, c rocs, d ecfp4 . From tables 3 and 4 we observe that rwr performance is better for 1 m target than 10 m because at 10 m we have lots off targets from different classes and as a result of that the prediction rate falls . For chembl 1 m dataset, drugs having more than 0, 1 and 2 targets we achieve bedroc score of 0.433, 0.553 and 0.611, respectively, which is much better than a random set of interactions . To test whether random walk performs better than just a simple sequence similarity search we took the approved drugs and it s known targets from the chembl 10 m dataset and performed sequence similarity based sech against 3,519 targets and ranked them . We found rwr performance is way better in ranking targets than performing simple sequence based search . This is the first time that the random walk - based method is evaluated using a binding assay dataset (cf . [3, 5]).table 3shows the types of data we used the drug target interaction having more than 1 and 2 drug interactions, area under the accumulated curve (auac), area under the roc curve (auc), bedroc and, enrichment factor (ef) and auc top 10%data typesnumber of targetsauac (%) auc (%) bedrocefauc (top 10%)chembl 1 m (rwr)>00.7090.9950.4335.0580.455chembl 1 m (seq)>00.6700.6700.3964.480.414chembl 1 m (random rwr)>00.4940.4930.0751.0900.079chembl 10 m (rwr)>00.5960.8370.3233.8650.351chembl 10 m (seq)>00.5180.5180.2372.6410.2555chembl 10 m (random rwr)>00.3940.3640.0360.9540.029chembl 1 m (rwr)>10.7840.7840.5536.2860.569chembl 1 m (seq)>10.6520.6510.3904.5070.412chembl 1 m (random rwr)>10.4830.4830.0811.2900.083chembl 10 m (rwr)>10.6130.610.3534.0910.378chembl 10 m (seq)>10.5510.5520.2793.0840.300chembl 10 m (random rwr)>10.5140.5140.0751.2440.088chembl 1 m (rwr)>20.8230.8240.6116.8660.631chembl 1 m (seq)>20.7010.7050.5135.1090.469chembl 1 m>20.5330.5330.06711.4650.065chembl 10 m (rwr)>20.6320.6330.3994.5690.422chembl 10 m (seq)>20.5690.5690.2983.030.315chembl 10 m (random rwr)>20.5210.5210.2621.950.125table 4shows the types of data we used the drug target interaction having more than 1 and 2 drug interactions and sensitivity (hit rate) at top 10, 25, 50, 100 and 200 predicted targetsdata typesnumber of targetstop 10 (%) top 25 (%) top 50 (%) top 100 (%) top 200 (%) chembl 1 m (rwr)>00.1440.3420.4700.5320.607chembl 1 m (seq)>00.1640.3150.3940.4200.430chembl 1 m (random rwr)>00.0020.0130.0180.0360.021chembl 10 m (rwr)>00.110.2470.3240.3860.409chembl 10 m (seq)>00.1220.1830.2340.2490.254chembl 10 m (random rwr)>00.0140.0230.0350.0480.079chembl 1 m (rwr)>10.2740.4770.5500.5800.614chembl 1 m (seq)>10.1890.3500.4280.4720.513chembl 1 m (random rwr)>10.0070.0230.0380.0760.091chembl 10 m (rwr)>10.2200.2770.3480.4170.446chembl 10 m (seq)>10.130.2120.2760.2960.302chembl 10 m (random rwr)>10.0140.0230.0350.0480.079chembl 1 m (rwr)>20.2710.5180.5980.6340.677chembl 1 m (seq)>20.190.3930.530.560.598chembl 1 m>20.0060.0180.0340.0550.08chembl 10 m (rwr)>20.2330.2970.3530.42990.472chembl 10 m (seq)>20.130.220.2950.3160.324chembl 10 m (random rwr)>20.0120.0280.0400.0570.093 shows the types of data we used the drug target interaction having more than 1 and 2 drug interactions, area under the accumulated curve (auac), area under the roc curve (auc), bedroc and, enrichment factor (ef) and auc top 10% shows the types of data we used the drug target interaction having more than 1 and 2 drug interactions and sensitivity (hit rate) at top 10, 25, 50, 100 and 200 predicted targets here, as a case study we investigate the target profiles of the popular top selling drugs in 2012 . First, we consider \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u_{\infty}, $$\end{document}u, the steady - state probability vector for the targets in our framework, as target profile of a drug . We find that some targets are associated with many drugs (see table 5). For instance, adrenoceptor alpha 1a appears in 60% of drug s top 10 target association lists; serotonin receptor 5ht2a appear in 43%; and adrenoceptor alpha 1b in 35% . Most drugs shown on the table 5 mostly belong to the rhodopsin class of gpcr s . In additional file 4, 5 shows a bipartite network of 110 drugs with top 10 predicted targets for each drug.table 5the top 10 associated targets of 110 drugs with true percentage of associated target before prediction and predicted percentage of associationtargetspercentage of drugs associated with the targets based on drugbank and chembl (%) percentage of drug associations appearing in top-10 target list in our prediction (%) ada1a 7.2760 5ht2a 4.5443.63 ada1b 7.2735.45 5ht1a 4.5433.63 adrb1 5.4531.81 5ht1b 5.4530.90 5ht2c 3.6330 acm2 9.0926.36 5ht3a 4.5425.45 5ht1d 5.4523.63 acm3 9.0921.81 5ht7r 4.5418.18fig . The size and label of the target nodes is proportional to the degree of the nodes . The top 10 associated targets of 110 drugs with true percentage of associated target before prediction and predicted percentage of association shows the network of the top 10 predicted targets of 110 drugs . The size and label of the target nodes is proportional to the degree of the nodes . We took some random drugs and tried to find known binding associations to protein targets . We searched three databases chembl, pdsp, and pubchem using the binding coefficients like ic50 and ki . Further investigation may have significant values on understanding side effects of existing drugs as well as repurposing them.table 6drug target interactions with association values from different databasesproteindrugsourceactivity tpeactivity m 5ht2a carvedilolpubchem aid 625192ic500.41 5ht2a desloratadinepubchem aid 625192ic500.033 kcnh2 lidocainechemblic50263.02 adrb1 salmetorolchemblic500.501 5ht1a amphetaminepdsp databaseki6.6 hdac2 atorvastatinchemblic5022.5 ada1a duloxentinepdspki10 acm1 montelukastpubchem aid 625153ic508.045 sc6a4 quetiapinepdspki10 drug target interactions with association values from different databases finally, let us summarize the contributions of this paper . First, we offer a general approach that takes the whole drug target network into account without separating protein categories, in contrast to the previous study . The number of interactions between drugs and targets is 2,557, which makes 684 drugs to have at least one known target and 457 drugs to have two or more interactions . The proteins in the dataset are grouped under 15 different categories according to chembl target classifications (https://www.ebi.ac.uk/chembl/target/browser). Out of 3,519 proteins, 1,386 proteins the number of drugs that have at least two interactions with proteins that are categorized is 412 . Among these 412 drugs, the number of drugs that have interactions with proteins from multiple groups is 169 . In other words, we estimate that about 40% of drugs have interactions across multiple groups according chembl dataset . Therefore, it is more reasonable to consider all proteins together, rather than running the prediction model separately for each category . Second, we further investigate the methodology by presenting a benchmark of a parameter in conjunction with the four chemical fingerprint types: maccs 166 keys, ecfp6 fingerprints, phfp4 fingerprints, and rocs . In the previous study, the parameter space of is not explored below 0.1, but we find that we can improve the performance by decreasing eta below 0.1 . We also find that the performance is robust under the choice of chemical fingerprinting method, particularly when is around the optimum (~0.01). Very small \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta $$\end{document} eta means the walk in the target network is much more important than the walk on the drug drug network . In a sense we have demonstrated that rwr approach provides a powerful way of predicting of drug target interactions . First, it provides a natural way to integrate multiple types of information such as drug drug similarity, target target similarity, and existing drug target interactions into a coherent framework . Second, in contrast to other approaches like short - path - based methods, the random walk framework incorporates the network structure around a single or multiple points of interests extensively, taking into account not only the closeness of targets, but also the multitude of the paths to the targets . These properties allow us to predict novel targets even for the drugs that have no known target, by connecting such drugs to the network through the drug drug similarity . Still, the performance of rwr could be further improved by incorporating more known drug target interactions . We have studied the performance of the method under the variations of parameter and the choice of fingerprints methods, showing that while training the model one can use any of the chemical features as similarity matrix with parameter \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta = 0.0 1 $$\end{document}=0.01 to obtain the predicted results, without significantly affecting the outcomes . Additional file 1:binary drug target matrix consisting of 727 drugs and 3,519 proteins.additional file 2:four drug drug similarity matrices.additional file 3:predictions results.additional file 4:110 drugs predicted results with 3,519 targets . We thank jeremy yang university of new mexico for sharing the code to do nn rocs and also helped in creating the chembl subset of compounds in the study . We thank jaehong shin, ying ding and other members of the cheminformatics group at indiana university for discussion and comments.
Claims data are generated primarily from administrative transactions and are essential for the accounting and reimbursement purposes associated with health care delivery . Increasingly, however, administrative claims data are repurposed for real - world observational studies in the united states and other developed countries . Typically maintained in regularly updated repositories, claims data provide convenient and easy access to researchable details on demographics, clinical characteristics, health care utilization frequency and type of prescription medication fills, inpatient hospitalization, and the use of outpatient, emergency, and physician offices and the cost of care.1,2 claims databases efficiently and comprehensively capture structured data such as diagnostic codes from the international classification of diseases version 9 (icd-9-cm) or generic product identifier medication codes . In general, these databases do not enable access to data on patients race or ethnicity, laboratory test values and other clinical measures or to unstructured data, including clinicians progress notes, all of which could be valuable in identifying disease and establishing patient status . It is essential, therefore, when assessing outcomes related to disease identified with claims data, to understand the degree of agreement between claims - based observations and actual physician diagnoses, and such validations are not available for several diseases . Pneumonia was associated with more than 1.1 million inpatient hospitalizations and 50,000 deaths in 2010,3,4 and is more common and more fatal in copd patients than in those without copd.5 in the first year after a copd diagnosis, individuals are at 16 times the risk for pneumonia compared to those without copd.6 in a recent study the incidence rate of community acquired pneumonia was 22.4 events per 1,000 person years in the 10 years following the diagnosis of copd, and more than 50% higher in those categorized as having severe copd.7 furthermore, the economic impact of pneumonia is greater for those with copd, illustrated by a doubling of direct medical costs following an inpatient hospitalization for pneumonia compared to those without copd in a study of older individuals.8 thus, pneumonia is especially of interest in individuals with copd, and administrative claims databases can be a useful tool for studying this disease if the data are determined to be sufficiently valid . In the past few years alone, pneumonia has been widely researched using claims data in patients with and without copd, with studies focusing on the economic and clinical impact of the disease, the development of algorithms to predict inpatient readmission, and measuring the safety and efficacy of treatments.822 although the accuracy of claims - based diagnoses is critical to the sensitivity and validity of observational research, this remains largely unexamined for pneumonia . To our knowledge, no study has validated claims - based pneumonia diagnoses across different points of service such as inpatient hospitalizations, emergency departments (eds), and physicians offices . The current study aimed to determine the validity of pneumonia diagnosis codes in a large us - based administrative claims database by comparing patients claims - based records and their medical records at the time of their observed pneumonia diagnosis . The objective was to confirm the accuracy of claims - based diagnoses of pneumonia by using the physicians diagnoses (medical record - based) as the reference standard . This retrospective cohort study utilized claims data for commercially insured individuals from the healthcore integrated research environment . The healthcore integrated research environment contains a diverse spectrum of longitudinal claims data for more than 32 million individuals, and has the capability of linking claims data to providers, which facilitates the selection of medical records . All study materials were handled in compliance with the health insurance portability and accountability act, and a limited dataset was used for all analyses, as defined by the privacy rule . A national, institutional review board (irb), the new england irb, reviewed the protocol and approved this study prior to the researchers obtaining patient medical records . Healthcore was granted a waiver of authorization to obtain the medical records without patient authorization of release after the irb determined the use or disclosure of protected health information in this research project involved no more than minimal risk to the individuals . This validation study was nested within a larger, retrospective cohort study (nct01921127)23,24 comparing the effectiveness of two common inhaled corticosteroid / long - acting 2-agonist combination (ics / laba) medications in copd patients (icd-9-cm code 491.xx, 492.xx, or 496.xx). Pneumonia, a complication in many copd patients, was analyzed as a secondary outcome, and the validation study was performed to assess the accuracy of the pneumonia diagnosis code (icd-9-cm code 480.xx486.xx) to reflect a documented diagnosis of pneumonia reported in the medical records . Patients initiating ics / laba between march 1, 2009 and march 31, 2012 were followed for 1 year following treatment initiation, during which patients diagnosed with pneumonia were identified . Only the first event was assessed for patients with more than one claim with a diagnosis of pneumonia . A professional medical record abstraction agency, employing trained and experienced medical data reviewers, obtained and abstracted the patient medical records using a study - specific instrument developed by the research team . The medical record abstractors had advanced education in nursing (registered nurse [rn], licensed practical nurse [lpn], licensed vocational nurse [lvn]), pharmacy (pharmd, registered pharmacist [rph]) and/or medicine (medical doctor, doctor of osteopathic medicine [do]). Prior to the start of the study, all data abstractors participated in a project - specific data collection training session utilizing the data collection manual and materials specifically developed for the study . The validity of the diagnostic claims was determined by the presence of a diagnosis of pneumonia documented in the medical records . The confirmation of a pneumonia diagnosis in the patient record was obtained via either a diagnosis code for pneumonia in the record or text from a physician note, assessment, or diagnostic summary stating that pneumonia was present . Additional information abstracted from the records included demographic characteristics (sex, race, ethnicity, and smoking status), symptoms pertaining to pneumonia (cough, fever, chest pain, chills, dyspnea, rales, rhonchi, wheezing, distant breath sounds, and temperature> 100f), diagnostic tests and the presence of abnormal results related to pneumonia (respiratory rate, heart rate, chest x - ray, chest computerized axial tomography [cat] scan, sputum gram stain, sputum culture, and blood culture), and the administration or prescribing of antibiotics . A target final sample size of 400 patients was chosen a priori to allow for the estimation of validity with a maximum margin of error <5% . A threshold of 5% margin of error was selected to achieve a confidence interval (ci) no more than 10% wide . Additionally, robust sample sizes, ideally of 100 records, were wanted for each of the subgroup analyses by place of service (inpatient / ed versus [vs] outpatient) and chart type (paper, electronic, or a combination of both paper and electronic records [hybrid]), with the assumption that no subgroup would account for less than one quarter of all records based on the claims results and prior experience . The primary outcome of the validation analysis was calculated using the positive predictive value (ppv) of the claims diagnosis for pneumonia relative to the medical record review, the reference standard . Ppv was calculated as the number of pneumonia cases with a documented pneumonia diagnosis in the medical records (diagnosed pneumonia) (true positives) divided by the total number of cases identified from the claims data (true positives plus false positives). Results were reported overall and stratified by place of service (inpatient or ed vs outpatient) and medical record type (paper, electronic, or hybrid). A 95% ci for the estimated ppv was constructed using clopper - pearson (exact) confidence limits . Additional descriptive statistics are reported for each of the demographic characteristics, symptoms, diagnostic tests, and antibiotic use . All analyses were performed using sas version 9.2 (sas institute inc ., cary, nc, usa). This retrospective cohort study utilized claims data for commercially insured individuals from the healthcore integrated research environment . The healthcore integrated research environment contains a diverse spectrum of longitudinal claims data for more than 32 million individuals, and has the capability of linking claims data to providers, which facilitates the selection of medical records . All study materials were handled in compliance with the health insurance portability and accountability act, and a limited dataset was used for all analyses, as defined by the privacy rule . A national, institutional review board (irb), the new england irb, reviewed the protocol and approved this study prior to the researchers obtaining patient medical records . Healthcore was granted a waiver of authorization to obtain the medical records without patient authorization of release after the irb determined the use or disclosure of protected health information in this research project involved no more than minimal risk to the individuals . This validation study was nested within a larger, retrospective cohort study (nct01921127)23,24 comparing the effectiveness of two common inhaled corticosteroid / long - acting 2-agonist combination (ics / laba) medications in copd patients (icd-9-cm code 491.xx, 492.xx, or 496.xx). Pneumonia, a complication in many copd patients, was analyzed as a secondary outcome, and the validation study was performed to assess the accuracy of the pneumonia diagnosis code (icd-9-cm code 480.xx486.xx) to reflect a documented diagnosis of pneumonia reported in the medical records . Patients initiating ics / laba between march 1, 2009 and march 31, 2012 were followed for 1 year following treatment initiation, during which patients diagnosed with pneumonia were identified . Only the first event was assessed for patients with more than one claim with a diagnosis of pneumonia . A professional medical record abstraction agency, employing trained and experienced medical data reviewers, obtained and abstracted the patient medical records using a study - specific instrument developed by the research team . The medical record abstractors had advanced education in nursing (registered nurse [rn], licensed practical nurse [lpn], licensed vocational nurse [lvn]), pharmacy (pharmd, registered pharmacist [rph]) and/or medicine (medical doctor, doctor of osteopathic medicine [do]). Prior to the start of the study, all data abstractors participated in a project - specific data collection training session utilizing the data collection manual and materials specifically developed for the study . The validity of the diagnostic claims was determined by the presence of a diagnosis of pneumonia documented in the medical records . The confirmation of a pneumonia diagnosis in the patient record was obtained via either a diagnosis code for pneumonia in the record or text from a physician note, assessment, or diagnostic summary stating that pneumonia was present . Additional information abstracted from the records included demographic characteristics (sex, race, ethnicity, and smoking status), symptoms pertaining to pneumonia (cough, fever, chest pain, chills, dyspnea, rales, rhonchi, wheezing, distant breath sounds, and temperature> 100f), diagnostic tests and the presence of abnormal results related to pneumonia (respiratory rate, heart rate, chest x - ray, chest computerized axial tomography [cat] scan, sputum gram stain, sputum culture, and blood culture), and the administration or prescribing of antibiotics . A target final sample size of 400 patients was chosen a priori to allow for the estimation of validity with a maximum margin of error <5% . A threshold of 5% margin of error was selected to achieve a confidence interval (ci) no more than 10% wide . Additionally, robust sample sizes, ideally of 100 records, were wanted for each of the subgroup analyses by place of service (inpatient / ed versus [vs] outpatient) and chart type (paper, electronic, or a combination of both paper and electronic records [hybrid]), with the assumption that no subgroup would account for less than one quarter of all records based on the claims results and prior experience . The primary outcome of the validation analysis was calculated using the positive predictive value (ppv) of the claims diagnosis for pneumonia relative to the medical record review, the reference standard . Ppv was calculated as the number of pneumonia cases with a documented pneumonia diagnosis in the medical records (diagnosed pneumonia) (true positives) divided by the total number of cases identified from the claims data (true positives plus false positives). Results were reported overall and stratified by place of service (inpatient or ed vs outpatient) and medical record type (paper, electronic, or hybrid). A 95% ci for the estimated ppv was constructed using clopper - pearson (exact) confidence limits . Additional descriptive statistics are reported for each of the demographic characteristics, symptoms, diagnostic tests, and antibiotic use . All analyses were performed using sas version 9.2 (sas institute inc ., cary, nc, usa). A total of 1,345 patients had 1 claim with a pneumonia diagnosis during an inpatient hospitalization, ed visit, or outpatient visit over the 12-month follow - up period; 1,022 of the diagnoses were from claims containing provider contact information, and were deemed eligible for medical record abstraction . A random subset of 800 patients was selected with the goal of abstracting information on 400 patients, allowing for the possibility that up to 50% of targeted records may be unobtainable . There were 730 record requests to providers because an initial review showed 70 patients with insufficient contact information . Medical records were obtained for 407 patients; however 19 were unusable due to missing information on birth dates or sex, or had unmatched names, or incorrect time periods, which resulted in 388 abstracted records (figure 1). The average age of patients with abstracted records was 67.8 (11.6) years old, and 53.9% were female (table 1). Of the 47.9% of patients with information on race noted in the record, 92.5% were white . Patients smoking status was reported as active (23.2%), former (36.3%), never (10.3%), and status unknown for the remaining 30.2% . Patients whose records were abstracted (n=388) and pneumonia patients identified with claims data (n=1,345) had similar mean age (67.8 and 67.4 years), sex (53.9% and 52.9% female), and health plan type (60.3% and 60.6%, enrolled in preferred provider organizations), respectively . A slightly greater proportion of patients were from the midwest in the record - reviewed vs the claims group (51.8% vs 47.1%) and a slightly smaller proportion from the northeast (12.1% vs 17.5%). A documented pneumonia diagnosis was found in 311 out of the total 388 available medical records (table 2), indicating that 80.2% (ppv 95% ci: 75.8% to 84.0%) of pneumonia positive patients identified with the claim code algorithm were diagnosed with pneumonia according to the medical records . Stratification by health care setting showed that claims originating from an inpatient or ed (n=185) had a higher ppv than those from outpatient settings (n=203) (87.6% vs 73.4%, respectively). Claims that were linked to paper charts had the same ppv as the overall study population, 80.2% (95% ci: 71.1%87.5%), while those linked to electronic medical records (emr) had a lower ppv, 73.3% (95% ci: 65.5%80.2%), and those with data from both paper charts and emr had a higher ppv, 87.6% (95% ci: 80.9%92.6%). Diagnosed pneumonia cases (n=311) vs those without a documented diagnosis in the medical records (n=77) included fewer females (51.4% vs 63.6%) and a higher proportion of smokers (26.0% active and 39.2% former smokers vs 11.7% and 24.7%, respectively). Within all examined medical records, coughing (61.3%), was the most prevalent symptom reported, followed by dyspnea (53.9%), wheezing (42.5%), distant breath sounds (31.2%), rhonchi (28.9%), fever (26.0%), and rales (25.5%) (table 3). There was a clear discrepancy in the frequency of reported symptoms between records with and without a pneumonia diagnosis; just 15.8% of patients with a pneumonia diagnosis documented in the medical records had none of the symptoms listed, while more than half (53.2%) of those without a diagnosis had no symptoms mentioned . Furthermore, individual symptoms were 25 times more prevalent in diagnosed cases compared with those without a diagnosis documented in the medical records . Diagnostic testing was more common in diagnosed pneumonia cases vs those without a documented diagnosis of pneumonia in the medical record (table 4). Respiratory rate testing was performed in 74.3% of diagnosed pneumonia cases compared with 42.9% of records that had no pneumonia diagnosis . Within those that had a test, abnormal respiratory rate results were reported for 55.0% of those with a documented diagnosis of pneumonia and 45.5% of those without . Similarly, chest x - ray and cat scans were performed more often in those with vs without a documented diagnosis (77.8% and 24.8% vs 51.9% and 11.7%, respectively), and abnormal results in those with diagnosed pneumonia were found for 89.6% of x - rays and 90.5% of cat scans compared to 61.5% and 77.8% of x - rays and cat scans within patients without a diagnosis documented in the charts . Medical record reviews indicated that antibiotics were administered or prescribed to 79.7% of the diagnosed pneumonia cases compared with 35.1% that had no documented diagnosis (table 5). Levofloxacin (25.8%), azithromycin (25.0%), and ceftriaxone (24.2%) were the three most commonly used antibiotics in this patient sample; all three are commonly used to treat pneumonia . The lack of evidence of antibiotic use in 20% of confirmed cases may suggest that many of those cases were suspected to be viral in origin . A total of 1,345 patients had 1 claim with a pneumonia diagnosis during an inpatient hospitalization, ed visit, or outpatient visit over the 12-month follow - up period; 1,022 of the diagnoses were from claims containing provider contact information, and were deemed eligible for medical record abstraction . A random subset of 800 patients was selected with the goal of abstracting information on 400 patients, allowing for the possibility that up to 50% of targeted records may be unobtainable . There were 730 record requests to providers because an initial review showed 70 patients with insufficient contact information . Medical records were obtained for 407 patients; however 19 were unusable due to missing information on birth dates or sex, or had unmatched names, or incorrect time periods, which resulted in 388 abstracted records (figure 1). The average age of patients with abstracted records was 67.8 (11.6) years old, and 53.9% were female (table 1). Of the 47.9% of patients with information on race noted in the record, 92.5% were white . Patients smoking status was reported as active (23.2%), former (36.3%), never (10.3%), and status unknown for the remaining 30.2% . Patients whose records were abstracted (n=388) and pneumonia patients identified with claims data (n=1,345) had similar mean age (67.8 and 67.4 years), sex (53.9% and 52.9% female), and health plan type (60.3% and 60.6%, enrolled in preferred provider organizations), respectively . A slightly greater proportion of patients were from the midwest in the record - reviewed vs the claims group (51.8% vs 47.1%) and a slightly smaller proportion from the northeast (12.1% vs 17.5%). A documented pneumonia diagnosis was found in 311 out of the total 388 available medical records (table 2), indicating that 80.2% (ppv 95% ci: 75.8% to 84.0%) of pneumonia positive patients identified with the claim code algorithm were diagnosed with pneumonia according to the medical records . Stratification by health care setting showed that claims originating from an inpatient or ed (n=185) had a higher ppv than those from outpatient settings (n=203) (87.6% vs 73.4%, respectively). Claims that were linked to paper charts had the same ppv as the overall study population, 80.2% (95% ci: 71.1%87.5%), while those linked to electronic medical records (emr) had a lower ppv, 73.3% (95% ci: 65.5%80.2%), and those with data from both paper charts and emr had a higher ppv, 87.6% (95% ci: 80.9%92.6%). Diagnosed pneumonia cases (n=311) vs those without a documented diagnosis in the medical records (n=77) included fewer females (51.4% vs 63.6%) and a higher proportion of smokers (26.0% active and 39.2% former smokers vs 11.7% and 24.7%, respectively). Within all examined medical records, coughing (61.3%), was the most prevalent symptom reported, followed by dyspnea (53.9%), wheezing (42.5%), distant breath sounds (31.2%), rhonchi (28.9%), fever (26.0%), and rales (25.5%) (table 3). There was a clear discrepancy in the frequency of reported symptoms between records with and without a pneumonia diagnosis; just 15.8% of patients with a pneumonia diagnosis documented in the medical records had none of the symptoms listed, while more than half (53.2%) of those without a diagnosis had no symptoms mentioned . Furthermore, individual symptoms were 25 times more prevalent in diagnosed cases compared with those without a diagnosis documented in the medical records . Diagnostic testing was more common in diagnosed pneumonia cases vs those without a documented diagnosis of pneumonia in the medical record (table 4). Respiratory rate testing was performed in 74.3% of diagnosed pneumonia cases compared with 42.9% of records that had no pneumonia diagnosis . Within those that had a test, abnormal respiratory rate results were reported for 55.0% of those with a documented diagnosis of pneumonia and 45.5% of those without . Similarly, chest x - ray and cat scans were performed more often in those with vs without a documented diagnosis (77.8% and 24.8% vs 51.9% and 11.7%, respectively), and abnormal results in those with diagnosed pneumonia were found for 89.6% of x - rays and 90.5% of cat scans compared to 61.5% and 77.8% of x - rays and cat scans within patients without a diagnosis documented in the charts . Medical record reviews indicated that antibiotics were administered or prescribed to 79.7% of the diagnosed pneumonia cases compared with 35.1% that had no documented diagnosis (table 5). Levofloxacin (25.8%), azithromycin (25.0%), and ceftriaxone (24.2%) were the three most commonly used antibiotics in this patient sample; all three are commonly used to treat pneumonia . The lack of evidence of antibiotic use in 20% of confirmed cases may suggest that many of those cases were suspected to be viral in origin . This study tested the validity of pneumonia diagnoses among copd patients identified in a large us administrative claims database using medical records as a reference standard . This study differs from prior investigations on this subject in a number of important ways . Prior studies have examined the validity of claims data in diagnosing pneumonia.2529 however, those studies were either limited to a single hospital or ed,25,26 to children in pediatric hospitals,28 or to only hospitalizations due to pneumonia.29 we used icd-9-cm codes to identify pneumonia diagnoses in copd patients from outpatient, ed, and inpatient settings using a claims database that covers more than 30 million managed care patients across the us . Although there are major differences in the design of our study compared to others, the ppv calculated for claims - based pneumonia diagnoses in the current study was similar to those reported by aronsky et al (79.4%80.8% ppv based on the algorithm that used different combinations of icd-9-cm codes)25 and whittle et al (89%).26 both of these ppvs were similar to the 87.6% ppv found in inpatient or ed cases in our study . Compared to outpatient claims, pneumonia diagnoses in inpatient or ed claims were found to be more accurate (87.6% vs 73.4% ppv). Furthermore, the presence of a chest x - ray or chest cat scan was more common in inpatient and ed cases, of which, all but one individual (99.4%) with documented pneumonia had evidence of either procedure; conversely, chest imaging was present in just 61.1% of cases originating in an outpatient setting with documented pneumonia . Further research is needed to investigate differences in validity by place of service since there is more detailed information in inpatient medical records compared to those in physicians offices . Our study suggests that differences in pneumonia outcomes may be more difficult to study in the outpatient setting; since neither medical records nor claims data strongly support the accuracy of the medical diagnosis of pneumonia as evidenced by the lack of imaging in significant numbers of individuals . One goal of this research was to confirm that a patient was diagnosed with pneumonia during the same health care encounter in which we observed a diagnosis of pneumonia in the claims data . The goal was not to determine whether the diagnosis given by the physician was accurate; however, there was evidence that pneumonia diagnoses that were simultaneously documented in the medical records were usually accurately diagnosed by the physician for example, patients with a pneumonia diagnosis documented in the medical records had abnormal diagnostic test results and symptoms of pneumonia at least twice as often compared with those who did not have a diagnosis in the medical records . Additionally, the higher smoking rates in those with a documented diagnosis is consistent with research that has shown smoking to be the strongest independent risk factor of pneumonia in immunocompetent non - elderly adults.30,31 conversely, although nearly 20% of patients in the claims data did not have a pneumonia diagnosis documented in the medical records, there was evidence that some of those patients may have had pneumonia . The prevalence of individual symptoms being less common in unconfirmed cases may be a function of the pneumonia being truly absent, but also may be a result of additional omissions in medical records (ie, physicians who omitted a note of pneumonia in the record may also be more likely to leave out notes of specific symptoms). Furthermore, more than 20% of patients with an unconfirmed diagnosis had three or more symptoms that are associated with pneumonia; of the 40 patients who had a chest x - ray, 61.5% had an abnormal result, and 35.1% unconfirmed cases were given antibiotics during their visit . Thus, although the medical record was considered the reference standard in this analysis, they might not be 100% accurate, and the results could be an underestimate of the true ppv of the claims diagnosis . This study was able to directly link patients records from an administrative claims database to their medical records for the exact time they had an insurance claim for a diagnosis of pneumonia in outpatient, inpatient, and eds . This allowed the confirmation that pneumonia diagnoses observed in the claims database were present in the medical records, where the medical record was considered the reference standard in the validation analysis . Along with capturing the presence of a pneumonia diagnosis, we were also able to obtain information on the presence of pneumonia symptoms, diagnostic lab testing, and administration of antibiotics during the visit of interest . The high rates of symptoms and abnormal test results in the medical record documented cases provided further evidence that most were true pneumonia patients . Medical records, though used as the reference standard here, are not 100% accurate . For example, it was possible for records to be at multiple locations for a single patient during the same time period, due to different providers having separate paper charts and/or emr systems; from which a record from one provider may have had a pneumonia diagnosis while another did not . Each event had a medical record pulled from a single location and, thus, it was possible to miss some records with a pneumonia diagnosis . It is possible, that a patient was diagnosed with pneumonia but it was not noted in the record, or that part of the medical record was missing, or that a diagnosis for pneumonia was in the record but missed by the abstractor . However, all medical record abstractors were trained and experienced in the health care field and participated in project - specific data collection training that utilized the data collection manual and materials specifically developed for this study . Less than 80% of patients with pneumonia documented in the medical record had a chest x - ray noted, a procedure that should be performed prior to diagnosing pneumonia.32 it may be that imaging was performed but not included in the chart, performed at an alternative medical site that was not captured, or that the provider felt there was enough evidence to make a diagnosis of pneumonia without an imaging test . While a clinical diagnosis of pneumonia without a chest x - ray may not be clinically sound, the goal of this study was not to validate whether or not the clinician made a correct diagnosis, instead it was to determine whether or not the administrative claims accurately reflected the diagnosis made by the clinician . Some of the comorbidities of copd that include congestive heart failure, pulmonary emboli, and mucus plugging during a copd exacerbation may be diagnosed as pneumonia with an abnormal radiograph and pulmonary symptoms, treated with antibiotics, and yet not be pneumonia at all . Biomarkers such as brain natriuretic peptide for congestive heart failure and d - dimer for thromboembolic disease should be captured in future prospective studies . However, medical records were considered the gold standard as they are the repository for the clinical information documented by the medical provider and other health care professionals and utilized for the care of the patient . Furthermore, medical records have been used as the gold standard in a number of other studies to determine the validity of pneumonia diagnoses.2729,33 because this study examined only patients with a pneumonia diagnosis observed in claims, the negative predictive value, sensitivity, and specificity of pneumonia identified via administrative claims could not be estimated from this study . Lastly, all patients in the larger study design were required to have 12 months of follow - up and all pneumonia diagnoses were captured during those 12 months . Thus, only pneumonia patients who survived until the end of the follow - up period were included, and so the most severe cases of pneumonia resulting in death were not represented in this validation study . The validity recorded in this study might have been higher if cases of pneumonia that resulted in death were included, since these cases in an inpatient setting would be expected to have higher validity compared to outpatient diagnoses . This study was able to directly link patients records from an administrative claims database to their medical records for the exact time they had an insurance claim for a diagnosis of pneumonia in outpatient, inpatient, and eds . This allowed the confirmation that pneumonia diagnoses observed in the claims database were present in the medical records, where the medical record was considered the reference standard in the validation analysis . Along with capturing the presence of a pneumonia diagnosis, we were also able to obtain information on the presence of pneumonia symptoms, diagnostic lab testing, and administration of antibiotics during the visit of interest . The high rates of symptoms and abnormal test results in the medical record documented cases provided further evidence that most were true pneumonia patients . Medical records, though used as the reference standard here, are not 100% accurate . For example, it was possible for records to be at multiple locations for a single patient during the same time period, due to different providers having separate paper charts and/or emr systems; from which a record from one provider may have had a pneumonia diagnosis while another did not . Each event had a medical record pulled from a single location and, thus, it was possible to miss some records with a pneumonia diagnosis . It is possible, that a patient was diagnosed with pneumonia but it was not noted in the record, or that part of the medical record was missing, or that a diagnosis for pneumonia was in the record but missed by the abstractor . However, all medical record abstractors were trained and experienced in the health care field and participated in project - specific data collection training that utilized the data collection manual and materials specifically developed for this study . Less than 80% of patients with pneumonia documented in the medical record had a chest x - ray noted, a procedure that should be performed prior to diagnosing pneumonia.32 it may be that imaging was performed but not included in the chart, performed at an alternative medical site that was not captured, or that the provider felt there was enough evidence to make a diagnosis of pneumonia without an imaging test . While a clinical diagnosis of pneumonia without a chest x - ray may not be clinically sound, the goal of this study was not to validate whether or not the clinician made a correct diagnosis, instead it was to determine whether or not the administrative claims accurately reflected the diagnosis made by the clinician . Some of the comorbidities of copd that include congestive heart failure, pulmonary emboli, and mucus plugging during a copd exacerbation may be diagnosed as pneumonia with an abnormal radiograph and pulmonary symptoms, treated with antibiotics, and yet not be pneumonia at all . Biomarkers such as brain natriuretic peptide for congestive heart failure and d - dimer for thromboembolic disease should be captured in future prospective studies . However, medical records were considered the gold standard as they are the repository for the clinical information documented by the medical provider and other health care professionals and utilized for the care of the patient . Furthermore, medical records have been used as the gold standard in a number of other studies to determine the validity of pneumonia diagnoses.2729,33 because this study examined only patients with a pneumonia diagnosis observed in claims, the negative predictive value, sensitivity, and specificity of pneumonia identified via administrative claims could not be estimated from this study . Lastly, all patients in the larger study design were required to have 12 months of follow - up and all pneumonia diagnoses were captured during those 12 months . Thus, only pneumonia patients who survived until the end of the follow - up period were included, and so the most severe cases of pneumonia resulting in death were not represented in this validation study . The validity recorded in this study might have been higher if cases of pneumonia that resulted in death were included, since these cases in an inpatient setting would be expected to have higher validity compared to outpatient diagnoses . This study demonstrated that administrative claims data provide an accurate basis for the diagnosis of pneumonia in copd patients across multiple service settings . Diagnostic accuracy varies by treatment setting and tends to be better in inpatient and eds vs outpatient settings . Additional research will help to validate whether the service setting has a bearing on the accuracy of pneumonia diagnosis and whether differences were a consequence of data type or the actual content of the data inherent in claims and medical records . With the increasing amount of research being performed utilizing administrative claims it is important to understand the validity of such data . Confirming the validity of pneumonia diagnoses in claims data allows researchers to confidently study copd associated pneumonia and investigate the safety of copd therapies utilizing longitudinal observational data.
A cornerstone of ligand optimization in drug discovery research is the comparison of activity and property data for a collection of molecules which are related by similar structural cores. (1) in order to rationalize the relationship between structure and activity, it is often beneficial to organize the structures in the form of a hierarchical tree . Structures with a common core fragment are arranged in branches, in which each parent fragment is a smaller, pared - down substructure that is common to each of the children . If the tree is well constructed, considerable insight can be gained regarding which core fragments and which peripheral substituents are responsible for the properties of interest, such as binding affinity against some number of protein targets, toxicity, and relevant physical properties . Given a collection of arbitrary molecular structures, there is typically no single unambiguous way to arrange them in a tree such that each parent node is a substructure of all its children . If the molecules happened to be synthesized in a particular sequence, such as by introducing a variety of substituents in a stepwise fashion to some number of similar core fragments, it may be sensible to produce a fragmentation tree which is based on the synthetic procedures . Or, if a set of common scaffolds is already known, it may be sensible to start with these scaffolds as the root fragments, and from these, construct the descendency hierarchy . If the collection of molecules has significant structural similarity, but no specific information about common substructures is available, then algorithms exist for estimating which parts of a structure are most scaffold - like . These can be used to generate a fragmentation sequence which can then be expressed as a tree. (2) while the fragmentation techniques have been well developed, one part of the process remains conspicuously undocumented . A fragmentation tree of molecules with similar common scaffolds is significantly less informative if the 2d coordinates of the molecular diagrams are not arranged such that common fragments are drawn with a common layout and uniform orientation . Molecular structures are often sketched by chemists showing a standard orientation when structures are added to a database, but this is not always the case . For a small group of structures, or for a rare showcase example, it is typically not a herculean task to manually ensure that all of the fragments are properly drawn and aligned . For larger collections, or if charged with the task of regularly regenerating this data, one would at least begin to feel like sisyphus . Unfortunately, even if a good 2d depiction algorithm is already available, the procedure of generating 2d layout coordinates that honor common fragment ancestry, with group layout decisions made in the context of the global optimum, is not trivial . In this work, a method will be described for obtaining 2d coordinates suitable for structure diagrams, which are chosen for both independent aesthetic appeal and for clearly showing the common substructure patterns by means of layout and orientation . Also discussed are higher level presentation methods for making use of the structureactivity information, which is contained in the fragmentation hierarchy . In the following section, methods will be described for generating a fragment tree, mapping sibling fragments onto each other in an optimal way, then using this mapping to guide the 2d depiction process . The results section will describe methods of presenting this information in the context of examining structureactivity relationships . For each molecule in the data set, it is necessary to propose a scheme whereby the molecule is peeled away in some number of steps, such that the last remaining fragments are the most scaffold - like . The definition of scaffolds, and the fragmentation sequences which relate them to the whole molecules, may be adapted to suit the data . The method published by schuffenhauer and ertl et al . Has been found to consistently produce agreeable results for pharmacologically relevant molecules. (3) these decomposition rules are such that the last remaining fragments are typically those that have been used as scaffolds and tend to be common substructures for a particular drug discovery campaign . One modification is applied, which is to collapse sequential breaking of fused aromatic ring systems into a single fragmentation step . This fragmentation method is used exclusively in this work, but the layout methods described are general and apply equally well to any scheme which is appropriate to the data . The fragments generated for each molecule need to be organized in the form of a tree . This can be accomplished by first producing canonical string representations for each fragment (e.g., smiles). (4) the canonical strings are arranged from smallest to largest for each molecule, and sorted by alphabetical order . In this way, for a given row and column, the fragments above or below are considered to be part of the same tree node if all the fragments up to and including that column are equal, which is illustrated in figure 1 . (a) shows the fragmentation sequence for four molecules which share a common root, which are grouped according to common fragments, while (b) shows the corresponding tree representation, which subsumes adjacent, analogous fragments into individual nodes . In the following section, it will be necessary to know the correspondence between each fragment and its parent fragment . This can be accomplished by assigning an arbitrary label to each atom in the original molecule, and retaining these labels as the fragmentation proceeds, for example for any node position in the tree which has more than one child molecule, the objective is ultimately to obtain an ideal 2d depiction layout and orientation such that analogous atoms in related molecules are placed in the same location, in order to make visual perception of the common features as easy as possible . Before this can be accomplished, it is first necessary to find an atom - to - atom mapping scheme that relates each of the equivalent substructure fragments to each other . When all of the fragments are nonsymmetrical and nondegenerate, this process is not especially complicated, since there will only be one correct mapping between any pair of fragments . When the potential mappings are degenerate, it is desirable to consider an ensemble of possible solutions, and find a combination that leads to the most compatible topological overlap of whole molecules . Consider the example shown in figure 1, where four molecules share a common root fragment, the pyridine core . Pyridine itself has permutational symmetry, which means that when two such fragments are mapped onto each other, it is necessary to choose a mapping from multiple pairwise combinations . An arbitrary numbering scheme is chosen for the pyridine root fragment of the first molecule . As shown for the first option, the descendent fragment also has a benzyl substituent at atom number 6, for the second option, the benzyl fragment is attached to atom number 2 . For the pyridine root fragment, there are multiple ways to map the branches to each other, which are not of equal quality . For this simple example, it is clear that a suboptimal atom - to - atom mapping scheme would eventually lead to molecules being superposed on top of each other in a manner that misleads rather than elucidates the common structural features . Unfortunately, the number of possible solutions grows combinatorially, which means that it is not viable to rely on an algorithm which examines every combination to find the best topological overlap . While adequate results could be achieved by using a greedy algorithm, that is, assign the possible graph automorphisms for each fragment sequentially and pick the case that best matches the previous assignments, the method described here uses the unary quadratic optimization (uqo) method,(5) which has previously been applied to the problem of common scaffold detection. (6) each fragment can have one possible assigned permutation, and the quality of the ensemble can be reasonably expressed as the sum of compatibility scores between each pair of assignments . The interaction energy between each pair of states not belonging to the same fragment is expressed in terms of the compatibility between the substituents which are not part of the shared substructure . Figure 3 shows two molecules that share a commonly assigned benzene fragment, with the tentative mapping numbers shown . The similarity of the substitution patterns is obtained by a breadth first search of the molecular graph, starting from each direct substitution point . For the first shell, the molecule in 3a is described as [2-n, 6-c], for a singly bonded nitrogen atom adjacent to position 2 and a singly bonded carbon atom at position 6 . The molecule in 3b is described as [2-o, 6-c]. For the second shell, the patterns are [2-c, 2-c, 6=o, 6-c] and [2-c, 6-o, 6-c], while the third shell is [2-c, 2-c] and [6-c, 6-c]. Substitution points are assigned as being the topologically closest mapped atom, while the highest bond order is taken if there is a choice of pathways . Similarity between two fragments is determined by examining one shell at a time, and matching up pairs of atoms that are assigned to the same mapped atom . The score is computed as follows: 1 point for each pair, 1 point for both being carbon or both being heteroatoms, and 1 point for having the same bond order . When there are multiple mapping permutations, the best matched set of pairs is selected . The score for each shell is divided by n, which is the shell number, which starts at 1 and is incremented with each step in the breadth first search . This method of comparing substituents is simple and fast, and emphasizes the value of sharing any substition at analogous positions, with those having similar topology and heterosubstitution being further favored . The assignment of a common numbering system is done recursively . For each unique root fragment, the uqo equation is formulated and solved, which yields a unique solution for each member of the group . Once the atom equivalency is established for the group of substructures which share a tree node, these assignments are stored in the fragments themselves, and also percolated through down the tree . The descendent nodes therefore share the common mapping within their partial substructures . The process is continued for each child fragment . Figure 4 illustrates the mapping procedure in a stepwise fashion . In figure 4a, the assignment has been completed for the pyridine root fragment, which has been mapped for all four molecules . As can be seen, the mapping is such that the substitution is always at the atom labeled as number 6, which is an optimal solution . In figure 4b and c, the process has been recursively applied to each of the distinct children of the root fragments, and a large proportion of the structures have been mapped . These two steps are carried out separately, so the numbering systems used for the benzyl and the methylfuran substituents are not related . Figure 4d shows the completed mapping, where tree singletons are assigned arbitrary numbers for the remaining atoms . Stepwise assignment of a common numbering system, for a fragmentation tree consisting of 4 molecules . The method for 2d layout of atom coordinates which we use in this work normally operates by searching for a globally optimum aesthetic ideal, which is the appropriate goal for depiction of individual molecules. (7) when examining a set of molecules, such as the fragmentation tree we have described thus far, it is important to be able to represent common fragments with a common depiction motif . While this is often the case for unconstrained depiction, it cannot be relied upon, since arbitrary substituents may influence overall layout decisions in conflicting ways . Consider the following simple fragment, which has a prominent degree of freedom in the form of a methylene linker: two different derivatives of this fragment clearly indicate that a free - for - all depiction is not the most suitable way to produce a common layout, since the common substructure fragments which correspond to the overall aesthetic ideal are no longer superimposable: to solve this problem, we make use of the tree structure and the mapping numbering scheme, to build up the fragment depictions sequentially . While the results must honor the requirement of common layout for common fragments, degrees of freedom within the constituent fragments are dealt with in a way that emphasizes overall layout aesthetics . As a first step, we submit each of the input molecules to the depiction layout algorithm, without constraints, and store the new coordinates within the molecule datastructure . While many of the structures will have their coordinates further modified by subsequent depiction layout procedures, the coordinates obtained by unconstrained layout are used during the following procedure for clustering purposes . The tree is analyzed recursively, starting with each root fragment . For each sibling node, the 2d coordinates of the fragments comprising the node are extracted, and clustered in a greedy fashion . Starting with the first set of coordinates as the first cluster, each subsequent set of coordinates is added to the same cluster as any fragment for which its coordinates can be superposed with an rmsd of less than 0.1 or to a new cluster if none . The common atom mapping scheme derived in the previous step if there is more than one cluster, which can occur when fragments have layout degrees of freedom such as rotational symmetry or aliphatic chains, the cluster with the largest number of members is selected . The first set of coordinates in this cluster is used as the definitive reference . For fragments that were found in the same cluster, this transformation is applied simultaneously to all fragments belonging to a particular molecule, not just the fragment under consideration . Fragments which are not in the reference cluster require more sophisticated treatment . In each case, the whole molecule for the fragment is resubmitted to the depiction layout algorithm, with a special constraint: the coordinates for the fragment are submitted as a preblock, which forces the layout algorithm to construct the rest of the 2d molecule around the predefined fragment, whose coordinates are taken from the reference fragment . This process has been previously described for drawing molecules with common scaffolds, and is used to obtain coordinates which are optimal for the collection of molecules, if not necessarily each individual molecule. (6) the coordinates from all of the fragments corresponding to the whole molecule are updated accordingly . The fragmentation patterns for 3 molecules are shown, each of which has been subjected to unconstrained depiction layout . Consider the fragments in column (b), which form a single node in the resulting tree, and are directly descended from the common root fragment shown in (a). The fragments (1,b) and (2,b) can be superposed onto each other, and so they are part of the same cluster of size 2 . Fragment (3,b) cannot be superposed, and so it is assigned to its own cluster of size 1 . The coordinates of (1,b) are used as the reference point . For the fragment (2,b), the coordinates are superposed onto the reference . For fragment (3,b), the coordinates are obtained by redepiction, using the reference coordinates as the preblock . Same fragmentation tree as shown in figure 5, after the colligative depiction layout procedure has been applied . At the completion of the previous step, each of the root branches has been arranged and oriented to show the common features within the branch . There is as yet no frame of reference for comparing the structures within different root branches, because there is no common ancestor and hence not even a partial common mapping system . There is, however, quite a high likelihood that the root branches are structurally related, and so it is useful to devise a scheme to orient them in a common way by means of translation / rotation / inversion . To do this, we take advantage of the fact that the constituent fragments of the root branches are depicted in a very constrained way . Their 2d shape now encodes a significant amount of information, which is generally not the case for unconstrained depiction layout . Therefore, it is quite viable to search for a single transformation for each whole branch which maximizes the overall shape overlap of the 2d structures . Since the orientation is a relatively imprecise step, it is sufficient to use a greedy algorithm, rather than a more rigorous clustering method . One begins by first defining the reference set to be the root branch with the largest number of constituent molecules . The subject set is the root branch with the second highest molecule count . For the subject set, an orientation is selected such that its combined 2d shape is most similar to that of the reference set . The orientation is applied to the subject set, and then it is merged into the reference set . A new subject set is selected, and the algorithm proceeds until all of the root branches have been processed . To compare the shapes of two sets, each of the molecules in each set is first translated so that the center of the root fragment is at the origin . A grid is defined, which is large enough to capture the bounds of each set as it is rotated around the origin. (8) for each set, grid values are defined by addition of a gaussian function, for each atom in each molecule: where r is the distance from the grid point to the center of the corresponding atom and n is the number of molecules in the set . The two grids are now directly comparable, and their similarity can be computed: where i and j iterate over each of the grid points, r is the grid for the reference set and s is the grid for the subject set . Lower values indicate more similar grids . To find the most similar orientation, the subject set is varied by rotating about the origin in increments of 5; inverting along one of the axes; and translating by [dx, dy] {2, 1, 0, 1, 2}, which makes for a total of 3600 evaluations . Figure 7 shows the resulting orientations and the corresponding grid pattern for each of 9 root fragments from a database of dhfr inhibitors, which contains 397 structures. (9) as can be seen, at the completion of the orientation step, the layout coordinates of molecules with different root fragments are approximately comparable, and the 2d coordinates now encode significant information about shared structural features . Once the fragment tree has been generated and the appropriate steps have been taken to ensure that the layout and orientation of the molecules shows the common fragmentation patterns, there are a number of ways to display this information visually . The most direct method is to display the fragmentation tree as a hierarchical structure, with each root node being a distinct entity . To make examination of the tree practical from a user - interface perspective, it makes sense to allow nodes to be interactively opened or closed, so that parts of the tree can be displayed as necessary . Because molecules are usually grouped together according to common fragmentation patterns this elicits information about the activities of the molecules which are represented by each node, which can be used to identify trends in activity data . Figure 8 shows several subsets of the fragmentation hierarchy for a data set compiled to show activity against various species of dhfr, using the data set described in the previous section. (9) figure 8a shows a single root fragment, and several of the immediately descended fragments . Each of the nodes provides an indication of the activity values against pneumocystis carinii dhfr of the constituent molecules by means of a colored pie chart display at the top left, which is divided up into a number of slices, one for each constituent compound . The color scheme used is green for highly active (<0.1 m), red for inactive (> 10 m) and yellow - orange for moderately active (1 m). As can be seen from the color pattern of the root fragment itself, the constituent molecules exhibit a wide spectrum of activity . (b and c) selected child of the root fragment and a selection of their descendents . By traversing the hierarchy from the root downward, it is possible to reveal groups of structurally related molecules, which show distinctive inhibition patterns . The branch shown in figure 8b shows a high degree of consistency, which suggests that the inhibition capabilities can be ascribed to the core fragments . The branch shown in figure 8c, on the other hand, shows a mixture of active and inactive constituents, which suggests that the inhibition properties are determined to a large extent by the substituents, rather than just the scaffolds . While the hierarchy display is appropriate for detailed analysis of the fragments themselves, it is impractical to examine more than several dozen structures at once with a typical display device . An alternate approach is to display the fragmentation hierarchy of all of the input structures in the form of a dendrogram, where all of the root branches are considered to be descended from a point in the center of a circle, and the branches extend outward from the center. (10) each of the leaf nodes is located at a position about the rim . To ascribe significant structural information to this layout, the ordering of the descendency pattern is chosen to maximize the similarity of any two adjacent leaf nodes . In preparation, fingerprints are calculated for each leaf node structure, using the gpidaph3 scheme. (11) a reference node is selected, which is initially set to the leaf node with the highest average tanimoto similarity to all other nodes . The tree is then traversed recursively, according to the following procedure: (1) for each of the immediate child nodes, assemble a list of all of the leaf nodes of which it is an ancestor . (2) select next - in - sequence child node to be that whose descendent leaf nodes has the highest average tanimoto similarity to the reference node . (3) if the selected node is a leaf node, set the new reference node to this . Once the ordering of the tree is determined, the actual layout of the dendrogram is straightforward . The final position of each node is obtained by evenly spacing about the rim of a disk, in order of tree traversal . The parent of all root nodes is considered to be the center, and the remaining nodes are arranged in between . If the background is chosen so that each pixel within the enclosing circle is assigned a color derived from the value of the average activity of the molecules belonging to the nearest fragment node, it is possible to gain considerable insight into structureactivity relationships . This is shown in figure 9, which plots activity data against p. carinii, using the red / yellow / green activity scheme described in the previous section . It is possible to quickly identify which regions of the fragmentation tree have consistently high or low activity, suggesting that the core fragment is responsible for these properties, as well as branches whose activity is quite mixed, suggesting that the substitution patterns are primarily responsible . Figure 10 shows a cutout section of the dendrogram, where several of the structures of leaf nodes are indicated . The leaf node clustering is evident by comparison of structures (a) and (b), which are descended from a different root fragment, but are almost identical structures, differing only by an additional chlorine atom on (a) and an additional nitrogen into the fused heterocycle scaffold of (b). The structures (c) and (d) have the same core fragmentation sequence, and denote the two ends of a block of very similar compounds clustered together, sharing high activity against p. carinii dhfr . Combined with an interactive user interface which allows facile examination of the structures, which are represented by the fragment nodes, a significant amount of structureactivity information can be inferred from the layout and color - coding . A collection of 467 structures with corresponding activity against the cox-2 enzyme(9) was submitted to the fragmentation and depiction sequence described within . Each of the structures was embedded in 3d, and its atom ordering scrambled randomly to ensure that there was no residual bias from the input sketch . Figure 11 shows a selection of substructure fragments, upon which most of the structures are based . In each case, a certain motif is observed: central 5-membered ring, usually a heterocycle, is decorated by two adjacent substituents, which are either 5- or 6-membered rings, a mixture of phenyl, pyridyl, or a 5-membered heterocycle . Some of the fragments show an additional substituent on the opposite side of the central ring, which is a consequence of the fragmentation ordering . The figure shows each of the fragments with a common orientation, which is chemically intuitive . In all cases, the central heterocycle bears the two adjacent substituents at approximately the 10 oclock and 12 oclock positions . The common orientation is a consequence of the layout methods described in this work: each of the branches of the fragmentation tree are depicted in such a way as to trade off aesthetic depiction against showing common substitution patterns in analogous positions, taking degeneracy into account . Because this data set has a high degree of structural similarity within the core fragments and the substitution patterns, the final spatial orientation using 2d structures reveals the alignment shown . Figure 12 shows a portion of a radial dendrogram, composed from the hierarchy of cox-2 inhibitors, using the same algorithm as described in the previous section . One sub - branch is shown in its entirety, and the structures of six of these compounds are shown . All of these compounds are based on the same 1,2-diphenylimidazole core, with a trifluoromethyl substituent in the 4-position . The activity values vary from 68 to 0.03 m . Compounds a and b, as well as all of the compounds that are arranged in between them in the linear cluster, feature a sulfonamide substituent in the same position on the phenyl ring at the 1-position, and all of them exhibit high activity, as can be seen by the bright green color coding . The remainder of the compounds in this sub - branch have a methylsulfonyl substituent in place of sulfonamide, and their activity is much more varied and is determined by the substitution pattern on the phenyl ring at the 2-position . For example, compounds c and d, which have a small substituent in the meta position, exhibit strong activity, while compounds e and f, which have larger substituents, exhibit weaker activity . Each of the compounds shown has a meta - substituted phenyl ring at the 2-position, and in each case, one of the meta substituents is depicted so that it is oriented at approximately the 2 oclock position . While an unconstrained depiction algorithm will be likely to make these layout choices on the grounds of congestion, the symmetrical phenyl substituents are explicitly mapped to each other on an atom - by - atom basis, in order to maximize the topological similarity of the interstructure mapping . A single depiction is chosen for the precursor fragment, and applied to all descendents, which ensures that the most similar substituents are placed at a consistent position . All algorithms described in this work have been implemented in svl (scientific vector language), running in moe (molecular operating environment). (12) the total processing time for the data set of dhfr inhibitors described in part 1 (397 compounds) is approximately 16 s, measured on an intel 2.0 ghz 32-bit processor . The time taken for each step breaks down roughly as follows: initial depiction (4 s), fragment generation (2 s), deriving common mapping (4 s), colligative redepiction (2 s), and branch orientation (4 s). For the cox-2 inhibitors described in part 2 (467 compounds), the total time taken was approximately 50 s, of which 33 s were devoted to deriving the common mapping scheme . The rate limiting steps scale linearly in proportion to the number of input molecules, with the exception of the mapping step, in the presence of symmetry - containing fragments . An abundance of degenerate partial fragments causes the uqo portion of the common mapping algorithm to dedicate additional computational resources to finding an optimal mapping . This is the principal reason why the cox-2 data set takes longer to analyze than does the dhfr data set, since the dhfr inhibitors are largely based on heterocyclic ring blocks with low symmetry, while the core fragments of the cox-2 inhibitors contain an abundance of substituents, such as phenyl rings, that have rotational symmetry . A method has been demonstrated for treating scaffold - like fragmentation trees so that common ancestor fragments are depicted and oriented in a consistent way that makes common structural features readily evident to the observing chemist . The algorithm operates without supervision and produces aesthetically desirable results using a combination of new and preexisting techniques . With the molecule layout and orientation method established, software applications that present fragmentation trees become significantly more valuable to medicinal chemists . There is no longer a requirement to sketch the input molecules in any particular way, nor is it necessary to postprocess the structures to elicit insight into the structureactivity information that is encoded in such trees . The value of presenting structural data in this way has been demonstrated, by using high - level and medium - level hierarchical tree views, which combine constrained clustering with activity color - coding, and the ability to examine individual structures that have a common depiction and orientation . Such views are effective ways to examine structureactivity data, and the algorithms that have been described essentially remove the manual effort required to produce them . Use of such visualization and grouping tools can allow the user to make intuitive selections of compounds for further development . For example, if a set of compounds which has balanced activity and diverse structure is necessary for producing a qsar validation set, either the tree or dendrogram presentation methods could be used to identify such a subset, either by visual inspection or automated selection . Alternatively, when considering hypothetical new compounds to add to the collection, it may be informative to depict them under the same conditions as for the existing compounds, and insert them into their appropriate locations within the hierarchy according to the ordering scheme used by the visualization method . Examination of the patterns exhibited by neighboring structures may aid in qualitatively assessing the expected properties of the compound . Future research will involve developing new ways to present information - rich, easily comprehensible graphics in a concise area, for example, single computer screen, projected slide or printed page, as well as developing interactive user interfaces for arranging and querying content data . The hierarchical atom - to - atom mapping system developed in this work has been used primarily for depiction purposes, but the assignment can also be used to differentiate scaffolds vs substituents at any level within the hierarchy, which opens up possibilities for fragment - based qsar and clustering studies.
Successful replacement of metal - ceramic dental restorations by all ceramic core - veneer restorations has long been one of the goals of scientific dental research . The durability and effective long term service of all ceramic systems especially in load bearing situations have yet to be proven.234 few long term clinical studies56789 are available to fully evaluate the performance and life span of newer all ceramic systems for both single and multiunit dental restorations . The interfacial integrity and the reliability of the bond between the high strength core and the weaker veneering material are directly related to the mode of failure of all ceramic restorations.101112131415 the physical, chemical, and mechanical properties of core and veneer materials can significantly affect the interfacial bonding.10121617 previous researches have mainly focused on the strength and failure modes of ceramic bilayers utilizing macro - mechanical testing.161819202122 nanoindentation is a research tool with numerous applications in the dental field for both natural dental hard tissues and restorative dental materials.23242526 quantitative data from the resin - dentin interface2327 suggests that nanoindentation is a potentially useful tool for the evaluation of the bond acquired . Nanoindentation derived properties from the interfaces of restorative dental materials can also be utilized in numerical models to aid in the understanding of the load transfer within a restoration.252628 high strength yttria stabilized zirconia (ytzp) core systems are commercially available for manufacturing dental crowns and bridges after veneering with sintered or heat pressed dental glass - ceramics . Core - veneer matching of modulus of elasticity (e) plays a major role in the success of dental ceramic bilayers, as abrupt e and toughness changes can cast adverse effects on the survival of such restorations.1029 depending on the veneering material and/ or on the ytzp core system used, an interlayer is usually used to modify the appearance of the core material and to theoretically improve the bonding between the core and the veneering material . It has, however, been suggested that such interlayers may not enhance the resultant bond.131430 wang et al.30 found that the interface toughness was reduced in ytzp specimens lined with an interlayer prior to veneering . Nanoindentation across such multi - layered ceramics and e mapping at and across these interfaces could provide further important information on the efficacy of these systems . The aim of this study was therefore to test the modulus of elasticity (e) across the interfaces of veneered dental ytzp ceramics using nanoindentation . All - ceramic veneered specimens (ytzp core plus veneer) were produced as follows . Rectangular specimens (10 mm length 5 mm width 1 mm depth) were sectioned from an yttria - stabilized (5 wt% y2o3) zirconia powder blank (zs blank, lot no . La100691445, kavo everest, biberach, germany) using a diamond disc (cutman 100, no 1575202, girbach dental, germany). The sections were sintered in a pre - programmed furnace (kavo everest therm, kavo, biberach, germany) overnight at 1500 according to manufacturer's instructions . One surface of the sintered ytzp sections was manually wet lapped in a special steel mould to 1000 grit silicon carbide paper mounted on a grinder - polisher (struers knuth - rotor 3, struers, ballerup, denmark), to achieve a depth of 0.5 mm . The specimen depth was selected to reflect a realistic dental restoration core thickness . At the end of the lapping procedure, the specimens were ultrasonically cleaned (transsonic 310 elma, singen kn, singen, germany) for 2 minutes and steam cleaned . A ceramic interlayer material (ips e.max zirliner powder, lot no . H29042, ivoclar - vivadent, schaan, liechtenstein) was mixed with ips e.max ceram all - round build up liquid (lot no . H32800, ivoclar - vivadent) and was used to coat the unlapped sintered ytzp surfaces of two ytzp specimens (type-1 specimens). The type-1 specimens were then vacuum fired according to manufacturer's instructions (table 1a) in a porcelain furnace (multimat mcii, dentsply, weybridge, uk). An even layer of wax (s - u modelling wax, schuler - dental gmbh & co, germany) was applied to the surface of the sintered ceramic interlayer (ips e.max zirliner) of the type-1 specimens and the as - sintered surface of the type-2 (no interlayer) ytzp specimen using a special steel mould to a total specimen thickness of 1.65 mm . A wax sprue (3 mm in diameter) was attached with one end to each specimen at a 45 angle and the other end to a large investment ring base fitted with a surrounding silicone cylinder . The specimens were then invested using manufacturer's recommendations for crowns: 200 g of investment material (ips press vest speed powder, ivoclar - vivadent) mixed using 32 ml of investment liquid (ips press vest speed liquid, ivoclar - vivadent) and 22 ml of distilled water in a vacuum mixer (multivac compact, degudent, hanau, germany) for 2.5 minutes . After setting for 40 minutes the refractory investment cylinder was transferred to a preheated burnout furnace (5365, kavo ewl, biberach, germany) at 850 and held for 1 hour . The preheated cylinder was removed from the furnace and two room temperature glass - ceramic ingots (ips e.max zirpress ingots, lot no . H21335, ivoclar - vivadent) and an alumina plunger (23) were inserted into the refractory muffle . The refractory was next transferred to a preheated pressing furnace (cerampress ney, jeneric pentron, wallington, cn, usa), and the glass - ceramic ingots were extruded into the refractory using the manufacturer's heat pressing settings (table 1b). Following heat pressing, the refractory was removed from the pressing furnace and left to cool to room temperature . The pressed components were divested using 50 m glass beads at 2 bar in a grit - blasting machine (renfert basic quattro is, renfert gmbh, hilzingen germany). The specimens were subsequently immersed in hydrofluoric acid solution (ips e - max press invex liquid, ivoclar - vivadent) in an ultrasonic bath (transsonic 310 elma) for 5 minutes to remove any reaction layer formed during heat pressing . After water rinsing for 1 minute, the specimens were separated from the sprues via a diamond disc with the low speed (15000 rpm) under water lubrication . The glass - ceramic surface of all specimens was lapped to 1000 grit silicon carbide paper to a specimen thickness of 1.5 mm and ultrasonically cleaned for 2 minutes . The specimens were then subjected to two stain firing cycles according to manufacturer's instructions (table 1a). The veneer of the specimens was glazed using ips e.max ceram glaze paste (lot h24056, ivoclar - vivadent) and the firing parameters in table 1a . The layered specimens were mounted in epoxy resin separately (epofix, struers, copenhagen, denmark) as follows: a) 1 type-1 specimen (for preliminary nanoindentation test); b) a pair of 1 type-1 and 1 type-2 specimens (for final nanoindentation test). The specimens' core - veneer interfaces were exposed and polished on wet 500, 800, 1000 and 2400 grit silicon carbide papers mounted on a grinder - polisher (struers knuth - rotor 3). Final finish was achieved on a napless cloth impregnated with a 1 m fine particle diamond suspension (hyprez liquid diamond type k standard concentration, batch 2028, engis corporation, wheeling, il, usa) under lubrication (hyprez fluid, engis corporation, wheeling, il, usa) on a polishing unit (kent 3 automatic polishing unit, engis ltd ., the specimens were washed with detergent under running water and ultrasonically cleaned in water for 5 minutes . Preliminary testing took place across the interface of a type-1 specimen to determine the load for the final mappings . An ultra - micro indentation system (umis) csiro 2000 (asi, canberra, australia) was used . Prior to indentation, alignment between the indenter tip and the on - stage microscope was performed using the 20 objective lens to accurately target the area of indentation . The mounted type-1 specimen was bonded to a metal base using a cyanoacrylate adhesive and transferred to the magnetic stage of the umis . The umis applied a force on the tested material through a diamond indenter and recorded its penetration depth . Measurements were performed using a spherical diamond indenter with nominal tip radius of 5 m . The multiple point load - partial unload method was used313233 in which, for each indentation site, the maximum force was reached in 40 increments . An e value was measured from the load - partial unload stress - strain curve of each increment . Forty e values were thus recorded for each indentation site . The partial unloading fraction in this study was set to 75% for every force increment . As explained in detail by field and swain,31 the indentation modulus (e ) is calculated using: (1)e = 0.75 pmax / he where pmax is the maximum load applied, is the radius of contact, and he is the elastic penetration depth . The material modulus (em) is then calculated using the relationship between indentation modulus (e ), indenter modulus (ei), and material modulus:32,33 (2)1 / e = (1 m) / em + (1 i) / ei which solved for em gives: (3)em = (1 vm) / [(1 / e *) (1 vi) / ei] where m and em are the poisson's ratio and the e of the material, respectively, and i and ei are the poisson's ratio and the e of the indenter, respectively . A common value of 0.22 for the poisson's ratio was used for the tested materials.29 the ei and i of the diamond indenter used were taken as 1150 gpa and 0.07.34 an indentation layout file was created, the schematic of which can be seen in fig . 1a to accurately map the desired indentation sites across the interface . The data retrieved were corrected for variations in the diamond indenter tip radius using a tip function previously obtained by calibration using four reference materials (glassy carbon, sapphire, silicon, and fused silica).35 the corrected data were further processed to plot graphs of e versus depth below contact for every indentation site . The test was then repeated for the type-1 and type-2 pair of specimens using the optimal load (50 mn) determined previously . The data retrieved were again corrected for tip function and further processed to plot graphs of e versus distance from the core - veneer interface . A one - way anova test (sigmastat, ver . 2.03, spss inc ., san jose, ca, usa) was used to statistically compare the near - interface changes in e. a compound optical microscope (olympus bx 60, olympus optical co., ltd ., suitable specimens for all the materials used in the study were fabricated and prepared for scanning electron microscopy (sem). Zirliner (ips e.max zirliner, ivoclar - vivadent) powder was used to fabricate a disc specimen by moistening 1 g of powder with ips e.max ceram alround build up liquid (ivoclar - vivadent) and compacting in a hollow cylinder mould with a plunger . The zirliner disc was then sintered by receiving one zirliner firing, followed by two stain and one glaze simulated firings (table 1a). A ytzp section (zs blank, kavo everest) was cut and sintered as previously described . A zirpress specimen (ips e.max zirpress, ivoclar - vivadent) was produced by investing a 3 mm diameter wax sprue, followed by the heat pressing and divesting route described previously . The zirpress specimen then received the same firing cycles as the test specimens (two stain and one glaze firings, table 1a). All 3 specimens were embedded in epoxy moulds and polished sequentially, as previously described, to a final finish of 1 m . The ytzp specimen was then separated from the epoxy in order to be thermally etched . This specimen was placed at room temperature in a furnace (severn furnace 1800, severn thermal solutions, bristol, uk) and heated to 1450 at a rate of 10/min, where it was held for 30 min . It was then quenched to room temperature in air.36 the ips e.max zirliner and the ips e.max zirpress specimens were etched with a 1% hydrofluoric acid solution for 60 seconds and subsequently washed under running water for 1 minutes and dried . All three specimens were gold coated in a sputter coater (balzers scdo5o bal - tec, liechtenstein) at 40 ma for 100 seconds . A field emission scanning electron microscope was used (jeol jsm-6300f, hertfordshire, uk) in the secondary electron imaging mode (10 kv) and images were acquired from all 3 specimens . X - ray powder diffraction (xrd) was carried out on all the specimens produced for the sem . The ips e.max zirliner and the ips e.max zirpress materials were ground into fine powders . The sintered ytzp specimen was examined as a solid specimen to avoid potential phase transformation from the grinding process . Bragg - brentano flat plate geometry / and ni - filtered cu k radiation (1 = 1.54059 and 2 = 1.54444) was used in an x'pert - pro diffractometer (panalytical b.v ., almelo, the netherlands). Data were continuously collected with an x'celerator solid state multistrip detector from 5 to 120 (2 range), with a step time of 200 seconds and a step size of 0.0334. phase analysis was carried out with panalytical x'pert highscore plus software using the international centre for diffraction pdf-4 database . The graphs in fig . 2 are the selected e versus depth of penetration graphs from the preliminary testing of the ytzp side only of the type-1 specimen . Each e data point has been derived from each of the 40 incremental load - partial unload steps . The representative ytzp surface indentation sites tested at 10, 30, and 50 mn loads are presented here, showing how increasing the indenter maximum load influences the scatter of the acquired e values as the indenter proceeds deeper into the tested material . The results of the final e tests across the interfaces of the type-1 and 2 specimens are presented in table 2 . The mean and standard deviation (sd) of e values of the last 20 data points (partial unloadings) from each indentation site (3 indentation sites per distance) were used to plot graphs of e changes across the interface for both type-1 and 2 specimens (fig . E data were analyzed using a oneway analyses of variance (anova) (sigmastat, ver . 2.03, spss inc ., san jose, ca, usa) and the differences found were significant (f test; p <.001). The values were grouped according to distance from the interface for both type-1 and type-2 specimens giving a total of 60 values per distance . The distance groups were compared using tukey's multiple comparison tests (p <.05). The highest mean e values were all the values on the ytzp, which were significantly different (p <.05) to the interface and veneer e values of the type-1 specimen (table 2). There was a statistical difference between e values of the interface and the veneering materials (p <.05). Veneer e values at 120 m (type-1) were found to have no statistical difference (p>.05) from the 80 m veneer values but were statistically different with all other type-1 e values (p <.05) in table 2 . The type-2 specimen (no ips e.max zirliner) produced the highest mean e at the 40, 80, and 120 m values on the ytzp, which were significantly different (p <.05) to all the other type-2 e values (table 2). There was no significant difference amongst the veneer e values (p>.05). The interface value was significantly different (p <.05) when compared to all other values of type-2 specimen . The 40 m e veneer values between type-1 and type-2 specimens were found to be significantly different (p <.05). The mean (sd in parenthesis) thickness of the ips e.max zirliner layer (interlayer) at the area of interest was measured as 48 (4.8) m . The ytzp specimen showed a high area fraction of fine zirconia grains (fig . The ips e.max zirpress specimen showed nanoscale fine whiskers densely dispersed in a glassy matrix (fig . 5d). The xrd pattern of the ytzp specimen revealed a bulk tetragonal zirconia phase (fig . 6). The xrd pattern of the ips e.max zirpress specimens showed a bulk amorphous phase and some very small peaks that could not be clearly identified (fig . The xrd pattern of the ips e.max zirliner showed a bulk amorphous phase (fig . Specimens were produced so that they would reflect clinical practice in terms of the core - veneer thickness ratio, and the multiple firings were done to simulate the thermal history.10 the ytzp core material to be veneered was left as - sintered for the purposes of this study in order to replicate the surface finish determined by its machining and sintering.37 the ytzp surface to be veneered can be greatly affected by heat treatments and surface finishing38 due to transformation toughening,39 resulting in unconventional interfacial properties.38 while these transformed grains may be restored to their tetragonal configuration after veneering heat treatments, the transformation associated grain volume changes can produce a flawed interface.40 one important advantage of spherical indentation is the absence of significant geometrical variations at the indenter tip . This property particularly facilitates e determination as it renders the initial surface contact elastic.41 e can quite accurately be determined using the single point unload method.32 this method requires relatively low indentation loads and penetration depths and preferably large radii indenters (> 20 m) so that the contact is purely elastic.33 using a large radius indenter in this study would have necessitated wider indentation spacings,26 potentially resulting in incomplete mapping of the interfacial compositional gradient . On this basis, a smaller radius spherical indenter combined with the multiple point load - partial unload method was chosen . This method also provides reliable modulus results,3135 by taking advantage of the elastic nature of the unloadings . In order for this to apply, the unloading force fraction has to be set to a value equal to or less than 75%.35 furthermore, as multiple values are acquired from each indentation site, statistical analysis is greatly facilitated.35 according to fischer - cripps,32 spherical indenters are generally sensitive to surface roughness effects that increase the scatter in small penetration depths, and therefore higher loads are recommended . The representative selected e versus depth of penetration graphs from the ytzp side of the preliminary type-1 tested specimen (fig . The 20 last partial unloadings using the highest tested load (50 mn) were therefore considered ample for the determination of the e values for the final testing . Accuracy of nanoindentation derived data is a matter that depends on various parameters and error sources, which are dealt extensively in the study of fischer - cripps.42 in accordance to this study, corrections for machine compliance, initial penetration depth, and tip function were applied in this study . Potential material pile up or sink in should also be evaluated as this can affect measured modulus values.42 the e value reported for kavo everest zirconia (scientific documentation, kavo, germany) was 210 gpa, very similar to the values obtained in this study (table 2). Similarly, guazzato et al.43 obtained values of 220 gpa for a dental ytzp core material (5 wt% y2o3) using the standard astm methodology (by impulse excitation of vibration). A similar range of values with the e values in table 2 has also been reported for other dental glass - ceramics from the studies by lawn et al.44 (between 68 - 69 gpa) and he and swain26 [65.52 (2.89) gpa], with the latter being measured using a pointed indenter (bercovich) on a umis 2000 nanoindenter . Nanoindentation using spherical indentation and the multiple point load - partial unload method may therefore be a useful predictor of e in this category of materials . In terms of precision, while low standard deviations were obtained for all e values, relatively higher standard deviations were obtained for the ytzp e values than for those of the veneering materials (table 2). We believe that this potentially may be correlated with the relationship of the sizes of the tip,26 the hertzian stress field, and the zirconia grains, as well as the orientations of the latter, and thus should be investigated further . Conversely, the low standard deviations obtained for both the interlayer (ips e.max zirliner) and the heat pressed glass - ceramic (ips e. max zirpress) materials in all indentation sites (table 2) suggest that the testing parameters used in this study may be useful for similar applications and materials . The optical microscopy post evaluation of the indentation areas of the type-1 specimen (fig . 4) showed that the 40 m indents were located within the interlayer (ips e.max zirliner). This finding, coupled with the presence of a statistically significant e difference (p <.05) between type-1 and 2 specimens at this distance, suggests that the interlayer has a lower e value than the heat pressed veneer . As this does not favor the e gradation across the interface and thus modulus matching,1029 the interlayer may potentially act as a weak link between the ytzp core and the heat pressed veneer . The nano - phase whiskers 5c) found in the heat pressed veneering glass - ceramic (ips e.max zirpress) were not categorically matched to a specific crystal type due to the very small, ambiguous x - ray diffraction peaks (fig 6). The crystallite size and volume fraction may therefore be the influencing factors in this outcome . The growth of fluorapatite crystals has been reported to follow an oswald ripening mechanism, where larger crystals grow at the expense of smaller crystals,4647 and this may be responsible for the larger particulates found within the microstructure (fig . 5c). Variations in the morphology and volume fraction of these crystals can be expected to affect the optical, mechanical, and thermal expansion characteristics of these materials.46 in this study (type-1, table 2) the heat pressed glass - ceramic material produced consistent e values with low standard deviations illustrating material consistency and the reliability of this testing method . The interlayer (ips e.max zirliner) had similar nano - phase whiskers sparsely distributed in the glassy matrix with no large micro - particulates observed (fig . Therefore further characterization would aid in identifying and quantifying the whisker like phases present in both ips e.max zirpress and ips e.max zirliner materials . Links between the resultant difference in the e (table 2) and the structural characteristics of the two materials may then be possible . The value on the interface for both type-1 and 2 specimens shows a significantly lower value (p <.05) than the one obtained for zirconia from the 40 m distance and a significantly higher value (p <.05) than the one obtained for the veneer from the 40 m distance (table 2). Optical microscopy post indentation evaluation revealed possible interfacial line fluctuation, as a result of the nature of the substrate and its' processing (fig . The values obtained can be attributed theoretically to the fact that the hertzian stress field, underneath the indents close to the interface, is influenced by both the materials that constitute it, thus giving a mixed modulus reading,2333 and the glass penetration into the zirconia grain boundaries.1448 the relatively larger sd obtained for the interfacial e values in contrast to all the other e values in table 2 may support these assumptions . According to aboushelib et al.,14 an inter - diffusion zone exists in bilayer ceramics, where elements (silica, sodium, aluminum, and potassium) migrate into the zirconia substrate extending to a maximum depth of 10 m of decreasing concentration (fig . 4). It would therefore be interesting to see whether the potential glass penetration could have yielded this reduction in the e value compared to the bulk ytzp (table 2). Transmission electron microscopy and energy dispersive x - ray spectroscopy (tem - edx) may be useful in the further characterization of these areas . The width of the hertzian stress field and the subsurface morphology of the interface are however unknown, and need further investigation . The effect of the interlayer (ips e.max zirliner) in the e reduction seems to stop somewhere between the 40 and 80 m distances in the veneer of the type-1 specimen (fig . 3a), since there is no statistically significant difference (p>.05) between the 80 and the 120 m veneer e values (table 2). For the type 2 specimen, (table 2) there is no statistical difference in e values (p>.05) between 40, 80, and 120 m distances on the veneer, indicating consistent e values in the absence of the interlayer (ips e.max zirliner). On the ytzp side of both type-1 and 2 specimens, 40, 80, and 120 m e values were also not statistically different (p>.05), and thus no influence of the interface was evident at these distances . Therefore the interesting zone for further work would be between 40 m or less in the ytzp and a distance equal to a maximum of double the thickness of the interlayer (ips e.max zirliner) in the veneer, since the result in these distances can be technique dependant . In order to sample this zone, where the interlayer material seems to affect e matching, narrower horizontal indent spacings (diagonally arranged in respect to the interface line) could be used, limited by the potential interaction of the stress fields.26 the size of the hertzian stress field can theoretically be minimized using a smaller radius indenter, but this would be limited by the grain size of the zirconia polycrystals to avoid sampling single crystals . The effect of water storage49 and thermocycling on the degradation of the core and thus on the e matching should also be investigated as it would be more clinically relevant . A good control for these experiments would be a metal - ceramic interface of known successful bond strength . Modulus mappings at this small scale could then be utilized for finite element analysis models2833 to simulate the behavior of dental ceramic multilayers giving insight to the potential failure modes in service . The coefficient of thermal expansion (cte) of ips e.max zirpress (9.75 0.25 10 k) (scientific documentation, ivoclar vivadent) may be matched with that of the kavo everest yttrium - stabilized core material (11.5 0.5 10 k) (scientific documentation, kavo). The interlayer (ips e.max zirliner) material presents an intermediate cte (9.8 0.25 10 k) and thus should act as a favorable parameter in the gradation of the interfacial compressive stresses preventing interfacial crack initiation and thus failure . It has however been suggested that the application of the interlayer between the ytzp core and the heat pressed veneer in crown / bridge ceramic systems should be avoided as it reduces the bond strength14 and interfacial toughness.30 our findings also associate a significantly (p <.05) lower e value with this interlayer, which does not favor the gradation of e across the interface, therefore potentially affecting its reliability due to poor modulus matching.1029 all these properties and characteristics, although potentially having a significant effect on the survival of dental ceramic multilayer crown / bridge structures, cannot solely determine their long term in vivo behavior . Many other interfacial characteristics, including wetting,48 microstructure, core - veneer thickness ratio, geometry, processing, thermal history, and porosity,101617 may synergistically affect it . Consequently nanoindentation derived data (e, hardness, fracture toughness50) may render nanoindentation a powerful complementary tool for the evaluation of dental ceramic interfaces . The conclusion of this study associates a significantly (p <.05) lower e value with the presence of an interlayer between the ytzp core material and glass - ceramic veneering material in a multi - layered all ceramic dental restorative system . The present study has shown nanoindentation using spherical indentation and the multiple point load - partial unload method to be a reliable predictor of e and a useful evaluation tool for layered dental ceramic interfaces.
Clinical diagnosis of occlusal caries is challenging due to the complex morphology of pits and fissures and presence of staining . Early diagnosis of incipient and non - cavitated carious lesions is crucial for performing preventive treatments . Visual examination is more efficient for diagnosis of cavitated rather than non - cavitated and incipient lesions . Furthermore, this method is subjective and its reproducibility is low, since it involves the clinical experience and scientific knowledge of the clinician . On the other hand, radiographs have high specificity and low sensitivity for the diagnosis of non - cavitated lesions and underestimate the actual depth of caries . The ekstrand index scores resemble the clinical situations and are based on signs found on the enamel surface such as opacities, white spots, brown spots, presence of cavities or micro - cavities and a combination of these conditions . New devices using new technologies are applied for quantitative and qualitative diagnosis of incipient demineralized lesions . Fluorescence - based methods are among these modalities, in which the sound and decayed surfaces produce different fluorescence when exposed to a certain light or wavelength . Laser fluorescence diagnodent measures the emitted fluorescent infra - red light and shows the result in whole numbers between 099 . The results of in vitro studies show the ability of diagnodent in exploring relatively advanced carious lesions . These findings are consistent with histological evidence, but they have no relationship with the depth of the lesions in dentin . The result of diagnodent is affected by different variables such as dehydration of the lesion, dental plaque and stains in the grooves of the occlusal surface . Evaluated the efficacy of visual examination, radiographic assessment and diagnodent for the diagnosis of occlusal caries . Kouchaji evaluated 156 permanent molars and revealed that the combined use of visual examination and diagnodent laser had the highest sensitivity and specificity . Additionally, rando - meirelles and de sousa applied the fluorescence laser method, radiography and visual examination to assess the extension of occlusal non - cavitated lesions in permanent molars . The aim of this study was to compare the efficacy of bitewing radiography, fluorescence laser and visual examination in the diagnosis of incipient occlusal caries in permanent first molars . This descriptive cross - sectional diagnostic study was conducted on patients aged 7 to 13 years, referred to the department of pediatric dentistry (school of dentistry, shahid sadoughi university of medical sciences, yazd, iran). Patients teeth were visually examined by a dentist and 31 patients who had signs of pit and fissure caries in at least one molar tooth were selected . The proposal of this study was approved by the ethics committee of the university (p.17.1.188736). Clinical examination of each tooth was performed under adequate lighting after cleaning the tooth surfaces by two examiners calibrated in a pilot study . One - hundred - fifteen teeth of 31 patients that were intact or had incipient and inconspicuous caries with or without color change were selected . The teeth with occlusal restorations, enamel hypoplasia, hypomineralization or structural defects, cavitated lesions, fissure sealant, orthodontic bands or brackets or pulp necrosis were excluded . Six of the 115 teeth were also excluded due to patient dropout; therefore, the three diagnostic methods were performed for 109 teeth . Occlusal surfaces of the teeth were cleaned from plaque and debris using water spray and cotton pellets if necessary . Occlusal caries were scored (v0v4) using ekstrand s visual scoring system (table 1). Then, bitewing radiographs were taken (planmeca prostyle, helsinki, finland) at 70kv, 8 ma and 0.36s exposure settings using e speed 2235 mm dental films (kodak, rochester, usa) in xcp film holder and processed using a dental film processor (velopex - extra x, london, england). Criteria used in visual examination, radiographic examination and fissure opening images were initially examined and scored (r0r2) by a pedodontist on a negatoscope and then confirmed by a radiologist (table 1). At this time, the teeth were examined by laser fluorescence pen (diagnodent, kavo, biberach, germany) followed by cleaning with a rubber cup and pumice powder, isolation with cotton rolls, and drying . The laser fluorescence pen was placed parallel to the long axis of the tooth on incipient or suspicious dental caries and moved around following calibration of the device on ceramic and intact enamel surfaces . Cavity preparation, or fissurotomy (fissurotomy miro ntf, ss white, lakewood, nj, usa) was performed in cases with obvious or ambiguous dental caries, respectively to assess and score the actual depth of lesions (table 1). Then, the cavities were restored by composite or amalgam, and the fissures were sealed . Roc curve was used to define the best cut - off point for diagnodent and to compare it with the gold standard (figs . 1 and 2) and the area under the roc curve (az) was calculated; then the values of sensitivity, specificity, and accuracy of this method were determined by calculating the area under the roc curve . Roc curve for dentin threshold roc curve for enamel threshold these values were also calculated for visual and radiographic examinations, and compared with the gold standard (fissure opening). The agreement coefficient for radiography and visual method was measured using a kappa test and inter - examiner reliability was evaluated using icc for diagnodent . Altman s paired samples method for calculating a 95% confidence interval was used to compare the accuracy of the methods . This study was conducted on 109 teeth of 31 children aged 7 to 13 (mean 11.111.34) years . After assessment of information compared to the standard method, seven surfaces were sound, 64 showed enamel caries and 38 showed dentin caries . Table 2 compares the visual method scores with those of the standard method (fissure opening) in enamel and dentin, separately . In table 3, comparison of ekstrand s scoring system and the standard fissure opening method by examiners 1 and 2 we did not find score 4 in any of the teeth . Frequency of radiographic scores compared to the standard fissure opening scores according to examiners 1 and 2 all surfaces showing radiolucency in dentine (score 2 based on table 1) were found to have dentin caries (dentin threshold). The best cutoff point for diagnodent in this study included: sound surfaces: 07, enamel decay: 810, dentin decay: 11 . The az value for laser method was 0.83 and 0.84 for the first and second examiners, respectively, which shows high efficacy of this method . The sensitivity of diagnodent method was higher than that of the other two methods, although its specificity in enamel was lower than the other methods (table 4). Sensitivity, specificity and accuracy of caries detection by each examiner (1 and 2) for all methods (visual inspection, diagnodent and radiography) the numbers inside the parentheses are calculated with 95% confidence interval . Totally, all three methods had high sensitivity . The highest accuracy for detection of enamel occlusal caries belonged to diagnodent, but with no significant difference with the other two methods (table 4). The occlusal surface of the tooth is susceptible to caries . However, making a reliable diagnosis is difficult in some cases . Diagnodent for caries detection has been shown in some studies; however, controversial results have been reported regarding its efficacy of caries detection [8,1214]. In this study, surface of the teeth was dried with oil - free air spray before the examination in order to decrease the refractive index between crystals from 1.33 for demineralized wet to 1 for demineralized dried surfaces . Probes and explorers were not used in this study, because they would not increase the diagnostic power . Furthermore, use of explorer may damage the dental tissues and impair the remineralization potential . More recent studies have used ekstrand index for standardization of the stages to detect caries using the visual method . Our results also showed high sensitivity and specificity using this index for detection of enamel occlusal caries . While in our study the sensitivity of radiographic diagnosis was similar to that of visual inspection for occlusal enamel caries . This difference may be related to the quality of films and the examiner s effects . In our study, each of the radiographic images was examined by an expert radiologist followed by a pedodontist . Unlike the findings of this study, another study found that diagnodent was not suitable for diagnosing incipient enamel caries . Regarding our results, the specificity of diagnodent in the enamel was lower than that of other methods (table 4). Inter - examiner agreement for visual examination was moderately acceptable, which was consistent with the results of earlier studies; it may be attributed to the conduction of a pilot study before the main study . In our study, the sensitivity of radiographic examination was relatively high, which is inconsistent with the results of some studies . Souza et al . Concluded that bitewing radiography was not suitable for caries detection because of its low sensitivity . In a study conducted by neuhaus et al, radiographic diagnosis of occlusal caries of deciduous teeth was not as accurate as laser fluorescence pen, laser fluorescence and icads methods, bader and shugars in a review study reported that diagnodent had higher sensitivity, but lower specificity compared to visual examination, and ricketts found that diagnodent led to a greater possibility of false - positive diagnosis than the visual method . In our study, the diagnostic power of diagnodent was found to be higher in dentin compared to the enamel, which is similar to a study by hasani - tabatabaee et al . They found that as the depth of carious lesions increased, diagnodent showed higher values of sensitivity and specificity . Diagnodent compared to visual and radiographic methods is more sensitive and accurate for the diagnosis of enamel caries (table 4). The specificity of radiography and visual methods for the diagnosis of enamel caries was greater than that of diagnodent . Visual method has a lower cost, is faster and has acceptable sensitivity; therefore, it can still be used as an appropriate method for clinical caries detection . In complicated cases, they stated that although diagnodent was more accurate, the visual method was preferred because it had no significant difference with diagnodent and it required shorter time . One limitation in using laser fluorescence pen is receiving fluorescent waves from stains, highly mineralized structures or malformed teeth . This may cause bias, lead to increased sensitivity and cause false - positive results . Exclusion of these teeth in some studies (unlike ours) such as the study by kouchaji, may lead to superior results with regard to the performance of diagnodent compared to studies that included stained teeth . Several studies have pointed to the reliability of laser diagnodent for occlusal caries detection in permanent teeth . In our study, the icc was used for comparison of the values read by diagnodent and showed a strong agreement between observers . In contrast, rodrigus et al, in their in vitro study reported a low agreement in values read by diagnodent between observers . Their study was a histological study and the samples were stored in thymol; this antimicrobial agent destroys porphyrins and consequently weakens the signals received by diagnodent . The surface below the roc curve in our study was 84% at the cut - off point of eight and 94% at the cut - off point of 11 . This was similar to the rate (92% at the cut - off point of 12) obtained by huth et al . Risk factors such as age, history of dental caries, diet, attitude, and topical fluoride application must be considered before treatment of suspicious cases . Considering the recent emphasis on preventive interventions and avoidance of surgical techniques, applying new technologies to detect even the slightest demineralization (incipient caries) seems necessary . One of the limitations of the current study was lack of recording of the depth of lesions, because the calibration and reliability of the measurement of the depth of lesions are difficult to achieve . Additionally, in diagnodent, the qualitative property, i.e., determining the presence or absence of dentinal caries is superior to the quantitative characteristic, i.e., recording the depth of the dentin lesion . In our study, simple radiographic films were used . Future research is needed to compare the validity of digital radiographs (ccd / cmos) with that of conventional radiography regarding their better contrast and resolution for detection of occlusal caries . Although the visual method was not as accurate as lf, considering the insignificant differences and affordability of visual method, the latter can be the first choice for detection of incipient caries . In suspicious cases, radiography and diagnodent
Salivary gland neoplasms constitute an important area in the field of pathology of head and neck region . They are mainly classified as either hodgkin's or non - hodgkin's lymphoma (nhl). Nhl comprises a heterogeneous group of lymphoid neoplasms with a spectrum of behaviour ranging from relatively indolent to highly aggressive and potentially fatal . The vast majority of nhl represent b cell and less commonly t cell lineage neoplasm . Nhls of b cell origin are known to be relatively common in extra nodal sites and many are thought to originate from mucosa associated lymphoid tissue (malt). This is a low to intermediate grade lymphoma that show a follicular architecture and represents the neoplastic counterpart of germinal center b lymphocytes . Follicular lymphoma is very uncommon under the age - group of 40 years and is extremely rare in children . The t (14;18) (q32;q21) chromosome translocation has been purported to be the cytogenetic hallmark of follicular lymphoma . The molecular consequence is deregulation of bcl2 expression leading to over expression of bcl2 proteins in neoplastic follicles . A 40-year - old female patient reported to our institute with a complaint of swelling in the left side of lower jaw since 2 months [figure 1]. Post its onset, the swelling gradually increased in size over a period of 2 months . The patient is a known diabetic since 5 years and is under oral anti diabetic medication . Concurrent with the history of swelling, the patient gives a history of weight loss in past 2 months . No history of cold, cough or fever . Clinical picture showing the swelling in the left submandibular region examination revealed a swelling of 9 4 cm in size which extended anteriorly 1 cm in close proximity to chin on left side and posteriorly in line with the ear lobe [figure 2]. Margins were well - defined and the swelling was firm, fixed to underlying tissues and non tender on palpation . No local rise of temperature was seen in the concerned area . Left submandibular lymph node was 0.5 cm in size and mildly tender on palpation . Few differential diagnoses such as sarcoidosis, sjogren's syndrome, lymphomas and mikulicz disease were considered . Sjogren's syndrome was ruled out as there was no history of dryness . In both sarcoidosis and sjogren's syndrome mikulicz disease is an autoimmune disease and thus was ruled out . Swelling seen 2 cm below the ear lobe the following investigations were done: fnac gave an impression of chronic non specific lymphadenitis . No evidence of cytological atypia seenultra sonography report showed a circumscribed mixed area of 3 1.5 cm in the anterior region of left submandibular gland . No evidence of cytological atypia seen ultra sonography report showed a circumscribed mixed area of 3 1.5 cm in the anterior region of left submandibular gland . The patient was further subjected to ct scan of head and neck and the impression was of submandibular salivary gland tumourmultiple submental and submandibular lymphadenopthy ., a diagnosis of benign tumour of the left submandibular gland was given . During excision of the tumour, it was found that whole of the gland was involved . So, total submandibular gland excision was done and the specimen was sent for histopathological analysis . Gross finding revealed a single bit of soft tissue measuring 4 6 2 cm, yellowish red in color, firm in consistency . [figure 3] cut sections revealed yellowish to whitish central core surrounded by whitish brown to brownish areas [figure 4]. Specimen showing 2 lymph nodes attached to the soft tissue specimen grossed specimen showing yellowish to whitish central core surrounded by whitish brown to brownish areas histopathological examination revealed hematoxylin and eosin (h and e) stained sections revealed destruction of salivary gland architecture . Photomicrograph showing neoplastic lymphocytes in a follicular pattern. (h&e stain, 100) photomicrograph showing neoplastic lymphocytes with hyperchromatic nuclei and scanty cytoplasm . (h&e stain, 200) based on the histopathological appearances, a provisional diagnosis of low grade lymphoma was made and immunohistochemical profiling was done to confirm the type of lymphoma . The tissue was stained for bcl2 an anti apoptotic marker, cd20 b lymphocyte marker, cd3 which is exclusively a t lymphocyte marker, cytokeratins and ema . Immunohistochemistry revealed that the cells were strongly positive for bcl-2 [figure 7] and cd 20 [figure 8] which are both cytoplasmic markers and negative for cd3 [figure 9]. (ihc stain, 200) photomicrograph showing neoplastic cells expressing strong positivity for cd20 . (ihc stain, 100) photomicrograph showing neoplastic cells with negative expression for cd3 . (ihc stain, 100) based on the results of immunohistochemical profiling, a diagnosis of follicular b cell lymphoma was reached . Patient was referred to higher center where full body mri did not reveal any foci of disease . Nevertheless, considering the indolent course of this disease, she has been on regular follow up and has not reported with any clinically suspicious changes . Lymphomas also involve in the descending order of frequency, the submandibular gland (30%), minor salivary glands and the sub lingual glands . The finding of a lymphoma in a major salivary gland could mean one of two things: either it is part of a disseminated process, or it is the first clinicopathologic evidence of lymphoma . In the latter instance, whether the disease originated in the glandular stroma itself or in a para glandular lymph node and then invaded the stroma, the lymphoma is defined as primary, as long as there is no detectable disease outside the salivary gland . Hyman and wolf have given few criterions for diagnosis of primary lymphoma which are: involvement of gland should be the first clinical manifestation of the diseasehistologically, the disease should involve the gland parenchyma and not the adjacent node or soft tissue alonethere should be confirmation of malignant nature of lymphoid infiltrate . Involvement of gland should be the first clinical manifestation of the disease histologically, the disease should involve the gland parenchyma and not the adjacent node or soft tissue alone there should be confirmation of malignant nature of lymphoid infiltrate . Lichenfield et al ., reserved the diagnosis of primary salivary gland lymphoma, when lymphoma has been ruled out elsewhere in the body . The diagnosis of lymphoma in case of a salivary gland swelling is rarely done preoperatively . Even though the diagnosis of lymphomas, in the algorithm of diagnosis of salivary gland swelling is rare, nevertheless, it should be considered . Follicular lymphoma, although unpredictable and often associated with a prolonged course shows indolent behavior . Overtime, there is a tendency to transform to a higher grade of lymphoma, usually diffuse large cell lymphoma . Although various treatment modalities exist, none are curative and very few have been shown to prolong survival.
Animals: six crossbred dogs with naturally occurring tvt (three males and three females, 25 years of age and weighing 1129 kg) were obtained from a community that promotes the welfare of dogs and dogs family planning (dog chance, ratchaburi province, thailand). The dogs were individually housed in cages at the laboratory animal facility of the faculty of veterinary medicine at kasetsart university and were acclimatized to the environment for one week . The animals were allowed access to a standard diet and drinking water ad libitum throughout the experimental period . A cytological examination and a subsequent histological examination of the biopsies the processes used to monitor the health status of all of the dogs, which included a physical examination, a complete blood cell count (cbc) and a serum biochemistry profile related to renal and hepatic function, were performed weekly throughout the studies . The tumor sizes were also measured using a vernier caliper at the beginning of the experiment and weekly before the administration of vincristine . This study was performed according to the guidelines for animal experiments and approved by the animal ethics research committee of the faculty of veterinary medicine at kasetsart university . Drug administration and sample collection: the dogs were treated with vincristine sulfate at a dosage of 0.7 mg / m of body surface area through intravenous administration . Blood samples for single - dose pharmacokinetic analysis were collected from the cephalic vein through iv catheter using edta tubes at 0, 5, 10, 15, 30 and 45 min and 1, 2, 4, 6, 8, 12, 24 and 48 hr after drug administration . The samples were centrifuged, and the plasma was separated and stored at 20c until analysis . The treatments were performed weekly until the tumor had visibly disappeared up to a maximum of eight treatments . The response to treatment and toxicity chemicals and reagents: the reference standards of vincristine sulfate and vinblastine sulfate (internal standard, is) were purchased from sigma - aldrich (st . Louis, mo, u.s.a . ). Vincristine sulfate (v.c.s. ; 1 mg / ml) for treatment was purchased from boryung pharmaceutical co., ltd . Sample preparation: the samples were prepared according to a published method with slight modifications . Briefly, 500 l of plasma was diluted with 500 l of 4% phosphoric acid, and 20 l of 500 ng / ml is solution was then added to the mixture . After thorough mixing, the mixture was applied to an oasis hlb cartridge (water, milford, ma, u.s.a . ). The cartridge was washed with 5% methanol in water and then with 1 ml of methanol: water (50:50, v / v). The eluate was evaporated and reconstituted in 100 l of a mixture of mobile phase a (5 mm ammonium acetate in water titrated to ph 3.5 with acetic acid and mobile phase b (acetonitrile) (50:50, v / v). The supernatant was transferred to micro insert vials, and 10 l was injected into the lc - ms / ms system for analysis . Chromatographic system: chromatographic separation through an lc system was performed using a poroshell 120 ec - c18, 3.0 50 mm, 2.7-m column (agilent technologies, palo alto, ca, u.s.a . ). The mobile phase a consisted of 5 mm ammonium acetate in water titrated to ph 3.5 with acetic acid, and mobile phase b was 100% acetonitrile . The gradient elution was performed as follows: 0 to 0.5 min, isocratic 90% mobile phase a; 0.5 to 3.0 min, 90% to 5% mobile phase a; 3.0 to 5.0 min, isocratic 5% mobile phase a. the flow rate was 0.5 ml / min . The mass spectrometric analysis was performed using a 6460 triple quadrupole mass spectrometer (agilent technologies) and programmed using the agilent masshunter b.06.00 software (agilent technologies). The ionization source parameters were optimized as follows: capillary voltage, 3500 v; gas temperature, 330c; gas flow rate, 8 l / min; nebulizer, 50 psi . The fragment energy was set to 230 v. the multiple reaction monitoring (mrm) transitions were selected to be m / z 825.4 precursor ion to m / z 765.4 production ion for vincristine and m / z 811.4 precursor ion to m / z 751.3 product ion for vinblastine . Validation procedures: in this study, the lc - ms / ms method was developed and validated for the identification and quantification of vincristine in the plasma of dogs with tvt . The calibration standard concentrations were prepared by spiking the working standard solution into blank plasma to yield final concentrations of 0.5, 1, 2.5, 5, 10, 50, 100 and 150 ng / ml . Five duplicates of the quality control (qc) sample at concentrations of 2, 50 and 100 ng / ml were prepared and used to determine the recoveries, intra - day and inter - day precision, and accuracy of the method . The procedure was repeated five times within the same day to gain an intra - day run precision and accuracy and five times of each concentration over five different days to obtain an inter - day run precision and accuracy . The precision of the assay was assessed by calculating the relative standard deviation (r.s.d .) For each concentration level . The accuracy was calculated by comparing the average measurements with the nominal values and is expressed as a percentage . A blank plasma sample, eight calibrated standard concentrations and five duplicates of the qc samples were included in each run . The quantification was obtained using the multiple reaction monitoring (mrm) transitions for vincristine (m / z 825.4 765.4) and is (m / z 811.4 751.3). The calibration curve exhibited linearity (r=0.990) over the concentration range of 0.5 to 150 ng / ml . The lower limit of quantification (lloq), defined as the lowest concentration yielding a signal - to - noise ratio (s / n) higher than 10, was 0.5 ng / ml . Pharmacokinetic study: the determination of the pharmacokinetic parameters of vincristine was performed using a two - compartmental pharmacokinetic model with pk solution 2.0(summit research services, montrose, co, u.s.a . ). The cp term was the extrapolated plasma concentration time curve at zero time, k12 and k21 were micro - rate constants, auc was the area under the curve, t1/2 was the distribution half - life, t1/2 was the elimination half - life, vdarea was the volume of distribution, cl was the plasma clearance, and mrt was the mean residence time . Method validation: the results of intra - day and inter - day precision and accuracy of the assay are presented in table 1table 1.intra-day and inter - day accuracy and precision of vincristine sulfate in dog plasma (n=5)qc sample concentration (ng / ml)mean sd(ng / ml)accuracy(%)precision(% r.s.d)intra - day (5 days)2.02.05 0.201026.4650.049.9 0.8199.71.59100.099.9 0.9999.90.92inter - day (5 days)2.02.22 the intra - day precision and accuracy ranged from 0.92% to 6.46% and from 99.7% to 102%, respectively . The inter - day precision and accuracy ranged from 0.902% to 4.39% and from 98.9% to 108%, respectively . The extraction recoveries were 86.3 10.54, 77.3 9.88 and 88.9 5.49% for low, medium and high qc levels, respectively (table 2table 2.recovery of vincristine following extraction process (n=5)qc sample concentration(ng / ml)mean extraction recovery(%)coefficient of variation(%)2.0 86.3 10.5412.350.0 77.3 9.8811.8100.0 88.9 5.496.18). Pharmacokinetic analysis: the level of vincristine sulfate in plasma was detectable up to 120 min after drug administration . 1.time profile of vincristine plasma concentration after its intravenous administration to dogs with tvt at a dose of 0.7 mg / m . Shows the mean plasma concentration of vincristine as a function of time after a single intravenous administration at a dose of 0.7 mg / m to dogs with tvt . The pharmacokinetic parameters for vincristine in dogs with tvt are presented in table 3table 3.pharmacokinetic parameters (mean sd) of vincristine sulfate after its intravenous administration at a dose of 0.7 mg / mto dogs with tvt (n=6)pharmacokinetic parameters (units)average sdk12 (min)0.094 0.035k21 (min)0.029 0.012cp (ng / ml)119 18.0t (min)21.5 6.90t (min)47.6 14.2cl (l / min / kg)0.010 0.001vd(area) (l / kg)0.660 0.210mrt (min)55.9 19.3auc (ngmin / ml)2,349 317k12, k21= micro - rate constants; cp= plasma concentration at initial time; t1/2= distribution half - life; t1/2 = elimination half - life; cl = clearance; vd(area) = volume of distribution; mrt = mean residence time; auc = area under the plasma concentration - time curve .. the plasma vincristine concentration immediately after administration (cp) was 119 18.0 ng / ml . The apparent volume of distribution (vdarea) and mean residence time (mrt) were 0.660 0.210 l / kg and 55.9 19.3 min, respectively . The distribution half - life (t1/2), elimination half - life (t1/2) and plasma clearance were 21.5 6.90 min, 47.6 14.2 min and 0.010 0.001 l / min / kg, respectively . Time profile of vincristine plasma concentration after its intravenous administration to dogs with tvt at a dose of 0.7 mg / m . K12, k21= micro - rate constants; cp= plasma concentration at initial time; t1/2= distribution half - life; t1/2 = elimination half - life; cl = clearance; vd(area) = volume of distribution; mrt = mean residence time; auc = area under the plasma concentration - time curve . Clinical responses: tumor regression was determined at weekly intervals by a physical examination and histopathological analysis . Complete remission or complete response means that all of the tumors completely disappeared, and partial remission means that the tumors regressed more than 50%, but less than 100% . In our study, three to eight administrations of vincristine at a dose of 0.7 mg / m were able to induce complete remission in five dogs (83.33%). In addition, these five dogs exhibited more than 50% remission after the first week of drug administration . In contrast, one dog exhibited only a partially response after the drug was administered eight times . The tumor mass (approximately 1 cm in diameter) in this dog continued to appear on the posterior portion of the gland penis . Gastro - intestinal side effects, such as vomiting, anorexia and diarrhea, were observed after the first drug administration in three dogs . Hematological side effects, such as leucopenia, neutropenia and thrombocytopenia, were observed in two dogs after the third drug administration, and anemia was observed in one dog after the fourth drug administration . A high incidence of tvt is associated with a large population of free - roaming dogs with uncontrolled sexual activity . Chemotherapy, particularly vincristine sulfate, has been shown to be the most effective treatment and is thus frequently used . Several analytical methods for the determination of vincristine sulfate in biological samples derived from both humans and animals have been reported . Currently, lc - ms / ms is the method that provides the most sensitive and reliable qualitative and quantitative analyses . The present study was conducted to evaluate the pharmacokinetic parameters of vincristine sulfate in dogs with tvt and to determine their correlation with the clinical effects of the drug using the well - suited lc - ms / ms method to determine the concentration of vincristine sulfate in dog plasma . The results from our study show that three to eight weekly intravenous administrations of vincristine at a dose of 0.7 mg / m produced a good response . This response confirmed the efficacy of vincristine sulfate as a single agent for tvt treatment, which is in agreement with previous reports [1, 16, 20]. For the pharmacokinetic evaluation, the plasma concentration as a function of time was described by a two - compartment model . The t1/2 indicates the overall rate of elimination and allows the prediction of vincristine accumulation with a value for vincristine of 47.6 min after i.v . Administration in dogs with tvt, whereas the value of t1/2 was 22.8 hr in healthy dogs . Thus, about 99% of the vincristine from plasma was cleared within 5 hr after i.v . The distribution half - life (t1/2, 21.5 min) of vincristine obtained in dogs with tvt was longer than in healthy dogs (0.14 hr). This may indicate that vincristine is distributed to the peripheral tissues of dogs with tvt, and this finding is further supported by the high uptake of the drug by tubulin - rich tissues [3, 21]. The above - described characteristics of the penetration of vincristine sulfate exhibited a good correlation with the clinical tumor response, which showed that more than 50% remission was obtained after the first drug injection . The volume of distribution obtained in this study is in agreement with the results from previous investigations in mice, tasmanian devils, children humans and adult humans . In previous studies, the accumulative concentration of the drug was found to be high in many organs, including the pancreas, spleen, thyroid, adrenal, intestinal mucosa, lung, liver, kidney and bone marrow, and the spleen was found to be the major organ in which the drug was accumulated . To the best of our knowledge, this study provides the first demonstration of the clinical pharmacokinetics of vincristine sulfate in dogs with tvt using the lc - ms / ms method . In conclusion, although pharmacokinetic parameters of vincristine sulfate in dogs with tvt showed rapid elimination and therefore no accumulation, it resulted in good clinical effects by dosing once a week . In addition, it is well recognized that auc is one of the most important pk parameters that have a relationship with an efficacy of antitumor drugs . Based on the value of auc of vincristine in this study, it might indicate the penetration of the drug into the tumor mass, which increases the efficacy and antitumor activity of the drug . However, further investigation is needed to clarify the pharmacodynamics of the drug in tvt.
Exposure to noise, environmental or occupational, can bring on a wide array of effects, depending on factors such as level and characteristics of the hazardous noise, the duration of the exposure and individual susceptibility . The investigation of sensorineural, cochlear hearing loss relies upon audiometric data such as pure tone audiometry (behavioral audiometry) and otoacoustic emissions (oae). Audiometric behavioral testing is also the main tool recommended by niosh in recordkeeping and assessing hearing decline at the work place (niosh, p. 54 professional musicians are extensively exposed to sound levels that may be detrimental to their hearing . Research on continued exposure to loud music among musicians, has shown adverse effects on the auditory system . These studies correlate professional hazards to hearing damage in accordance with criteria stemming from noise - induced hearing loss (nihl). The use of criteria originating from industrial measures is common in research literature on exposure to music . States that excessive exposure to loud music causes various hearing symptoms such as tinnitus, and leads to a risk of permanent hearing damage, known as nihl or noise - induced permanent threshold shift (nipts). Puissant et al . Tested 8 college - aged trumpeters with a series of preliminary hearing tests prior to a one hour practice session and at several different stages after practice, using damage risk criteria for nihl . They concluded that trumpeters are at a significantly increased risk of hearing loss over that contributable to age alone . Phillips, henrich and mace showed that overall prevalence of nihl was 45%, with 78% of notches occurring at 6000 hz among 329 student classical musicians aged 18 - 25 who played on a variety of instruments (vocals, brass, wind, strings, and percussion). Daniel reported that temporary and permanent hearing impairment are becoming more common among young adults and children, especially with the increased exposure to portable music players . Other findings supporting a positive correlation between hearing thresholds and extent of exposure to music were shown by schmuziger et al . As well as axelsson and lindgren and khri . The current study examines the extent to which the subgroup of professional pop / rock / jazz musicians is affected by continued exposure to loud, amplified music . Extended exposure to noise or to music can cause not only hearing loss, but also other subjectively related symptoms that do not affect hearing thresholds . Khri et al ., list five disorders that were associated with exposure to amplified music: hearing loss, tinnitus, hyperacusis distortion, and diplacusis . Laitinen and poulsen state that aspects other than hearing loss must be considered and that the most frequent hearing disorders that affect musicians are tinnitus, a sporadic, acute phenomenon of phantom noise that lasts seconds or minutes, or may be a constant, chronic and steady sensation, and hyperacusis, an increased auditory sensitivity to loudness . The current research will focus on these two subjectively reported aspects of hearing damage . Using the questionnaire of khri and pure - tone threshold assessments, khri et al . Showed a 49% hearing loss in 139 rock / jazz musicians aged 26 - 51 . Khri also showed other, self - reported, hearing - related symptoms in 74% of the study subjects, such as tinnitus and hyperacusis . Barrens found that pure - tone hearing thresholds, speech audiometry, and self - reported disability were correlated . While individually they are unreliable, together, these variables present reliable information about hearing problems, and are best suited for assessing nihl . The schedule of professional musicians is characteristically ever changing, balancing between steadily paying performances, freelance work and private lessons . The extent of their exposure to music in general and to harmful sound levels in particular varies greatly depending on several factors: the instrument played (drummers being more susceptible to nihl compared to other professional musicians), the type of ensemble (number of players and musical genre), the extent of amplification, acoustics of the practice / performance venue (concert halls being better acoustically treated in comparison to night clubs), as well as the rate of attendance at concerts as an audience, and as part of their musical education . Relative to other kinds of professional musical environments, professional classical musicians within an orchestra are a population studied to a greater extent within methodical controlled studies due to the structured life that is inherent within an orchestra: a steady work environment with controlled acoustics in a permanent rehearsal hall and permanent seating within a section, which allows for repeated exposure in steady conditions . In contrast, professional pop / rock / jazz musicians fluctuate between extremely busy periods of high demand and periods of unemployment . As a result, there is a relatively large body of research pertaining to classical musicians, whereas research on professional pop / rock / jazz musicians is sparse . Although musicians have the power and ability to protect themselves from the risks of nihl, they are not always consciously ready, socially willing or professionally able to reduce their harmful exposure to amplified music, due to the fact that the use of hearing protection can sometimes hinder the quality of performance . Found ear plug use low among 15 classical musicians due to hampered listening to their own and to their colleagues playing . Musicians reported earplugs affecting their timbre and dynamics, and were found to be uncomfortable . It was reported that support and determination were required in order to become accustomed to hearing protectors . Trumpet players reported lack of acclimatization time, shallow earmold seal leading to a large occlusion effect, and hearing loss of harmonic overtones . Thus, musicians often find themselves risking their health in order to achieve a better musical experience . The purpose of the current study was to examine the relationship between the amount of experience a professional pop / rock / jazz musician has and subjective and objective variables of hearing loss . These were evaluated in two ways: self - reporting by professional pop / rock / jazz musicians using a questionnaire based on laitinen and poulsen and an assessment of air - conduction thresholds at 1, 2, 3, 4, 6, and 8 khz . The research hypotheses were: professional pop / rock / jazz musicians experience will positively predict hearing thresholds of 3 - 6 khz.professional pop / rock / jazz musicians experience will positively predict self - reported symptoms (tinnitus and hyperacusis).professional pop / rock / jazz musicians experience will positively predict the use of hearing protection.self-reported symptoms (tinnitus and hyperacusis) will positively predict hearing thresholds of 3 - 6 khz . Professional pop / rock / jazz musicians experience will positively predict hearing thresholds of 3 - 6 khz . Professional pop / rock / jazz musicians experience will positively predict self - reported symptoms (tinnitus and hyperacusis). Professional pop / rock / jazz musicians experience will positively predict the use of hearing protection . Self - reported symptoms (tinnitus and hyperacusis) will positively predict hearing thresholds of 3 - 6 khz . 44 professional pop / rock / jazz musicians aged 20 - 64 were sampled (mean age 37.45, s.d . Participants played various instruments: drums / percussion (22.7%), guitar (34.1%), bass (electric / upright) (9.1%), piano / keyboard (13.6%), strings (cello / violin) (6.8%), wind instruments (recorders, flute, piccolo, harmonica, xaxophone, electric wind instrument [ewi]) (6.8%), accordion (2.3%), voice (2.3%) and other (bouzouki, mandolin, kora [african harp]) (2.3%). 33 of the 44 musicians reported that in addition to their main instrument, they also play another musical instrument . Slightly more than half of all musicians (56.8%) reported using hearing protection at some time in their lives . Drummers showed a range of 15 - 84 hours playing and a range of 17 - 47 years playing . 70% of drummers reported using hearing protection, in relation to the rest of the participants of the study who reported 42.1% use of hearing protection . Based on guidelines from khri, a professional pop / rock / jazz musician was considered according to the following criteria: played an amplified musical instrument for at least 4 consecutive years and practiced / taught for a minimum of 5 h a weekgave a minimum of 3 performances a yearwas financially compensated for playing the instrument . Played an amplified musical instrument for at least 4 consecutive years and practiced / taught for a minimum of 5 h a week gave a minimum of 3 performances a year was financially compensated for playing the instrument . Musical experience (me) was calculated based on the number of years playing the instrument, multiplied by the number of weekly hours the musician played or was exposed to his / her musical instrument . Sixty - four percent of the participants achieved a minimal playing experience score of 0 - 500 units (years * weekly hours). All participants had a minimum of four years of experience playing their instrument with an average of 22.7 years (s.d . The average weekly exposure of participants to pop / rock / jazz was 23.55 h (s.d . A family history of hearing loss, acoustic trauma acquired prior to musical career, occupational exposure to noise (other than music), a history of chronic otitis media, prior ear surgery performed, known head trauma, and use of ototoxic medication were all criteria for exclusion from the study . Right and left hearing thresholds were measured based on a pure - tone average (pta) calculation of frequencies 3 - 6 khz and 3 - 8 khz, and adjusted for an age and gender - matched otologically normal reference population, using standardized age - related charts (appendix 1). Initially, professional pop / rock / jazz musicians were approached based on personal acquaintance, but the sample was expanded further in two ways: the participants made additional connections to other professional musicians they knew, and renowned israeli musicians were approached through invitation letters sent to their facebook profiles . In total, 89 professional pop / rock / jazz musicians were approached . Therefore, data pertaining to subjective symptoms includes 44 subjects and data pertaining to objective symptoms includes 41 subjects . Hearing thresholds: raw data and data after accounting for age and gender effects (n = 42) a 1 h meeting was held with each participant, during which the questionnaire was completed in the presence of the examiner, to allow for clarifications and to ensure the participant completed all the questions . This was followed by an audiometric hearing threshold assessment of the frequency range of 1 - 8 khz (by ascending intensity, up 5 db down 10 db), in the professional musician's home or studio, using a portable audiometer, manufactured by interacoustics, model as208, with tdh39 headphones, manufactured by telephonics . Hearing threshold evaluations were conducted after 12 hours of rest and with no immediate prior exposure to amplified music, according to the protocol of audiometric testing of occupational hazard . Laitinen and poulsen used a questionnaire for orchestral musicians, comprised of several standardized questionnaires pertaining to subjective symptoms . The questionnaire, comprised of 91 questions was completed by 145 classical musicians from 3 orchestras in finland . The questionnaire for the current study, the pop / rock / jazz musicians questionnaire (prjmq), included 39 questions divided into the following sections: general information, health information regarding hearing disorders, working capacity and awareness of loudness of instruments, hearing protection, and general knowledge regarding musicians understanding of concepts of loudness as expressed by the db scale . Most questions within the questionnaire began with a yes / no question (e.g. Have you ever experienced hyperacusis? ), followed by a series of scale questions for those participants who replied yes . Scale questions required participants to rate the extent to which they experienced a subjective feeling (tinnitus, hyperacusis): never, seldom, sometimes, often, or always within a certain musical setting (private lesson, rehearsal, performance, or musical concert attended), from one to five . All answers on a particular topic were averaged . Thus, the answers of those participants who reported they had experienced hyperacusis were averaged into a value named mean hyperacusis . Use of hearing protection was measured based on a yes / no question (do you use hearing protection in both ears?). This binary question was followed by a series of more specific questions which were not statistically measurable and therefore were not analyzed in the current research . 44 professional pop / rock / jazz musicians aged 20 - 64 were sampled (mean age 37.45, s.d . Participants played various instruments: drums / percussion (22.7%), guitar (34.1%), bass (electric / upright) (9.1%), piano / keyboard (13.6%), strings (cello / violin) (6.8%), wind instruments (recorders, flute, piccolo, harmonica, xaxophone, electric wind instrument [ewi]) (6.8%), accordion (2.3%), voice (2.3%) and other (bouzouki, mandolin, kora [african harp]) (2.3%). 33 of the 44 musicians reported that in addition to their main instrument, they also play another musical instrument . Slightly more than half of all musicians (56.8%) reported using hearing protection at some time in their lives . Drummers showed a range of 15 - 84 hours playing and a range of 17 - 47 years playing . 70% of drummers reported using hearing protection, in relation to the rest of the participants of the study who reported 42.1% use of hearing protection . Based on guidelines from khri, a professional pop / rock / jazz musician was considered according to the following criteria: played an amplified musical instrument for at least 4 consecutive years and practiced / taught for a minimum of 5 h a weekgave a minimum of 3 performances a yearwas financially compensated for playing the instrument . Played an amplified musical instrument for at least 4 consecutive years and practiced / taught for a minimum of 5 h a week gave a minimum of 3 performances a year was financially compensated for playing the instrument . Musical experience (me) was calculated based on the number of years playing the instrument, multiplied by the number of weekly hours the musician played or was exposed to his / her musical instrument . Sixty - four percent of the participants achieved a minimal playing experience score of 0 - 500 units (years * weekly hours). All participants had a minimum of four years of experience playing their instrument with an average of 22.7 years (s.d . The average weekly exposure of participants to pop / rock / jazz was 23.55 h (s.d . A family history of hearing loss, acoustic trauma acquired prior to musical career, occupational exposure to noise (other than music), a history of chronic otitis media, prior ear surgery performed, known head trauma, and use of ototoxic medication were all criteria for exclusion from the study . Right and left hearing thresholds were measured based on a pure - tone average (pta) calculation of frequencies 3 - 6 khz and 3 - 8 khz, and adjusted for an age and gender - matched otologically normal reference population, using standardized age - related charts (appendix 1). Initially, professional pop / rock / jazz musicians were approached based on personal acquaintance, but the sample was expanded further in two ways: the participants made additional connections to other professional musicians they knew, and renowned israeli musicians were approached through invitation letters sent to their facebook profiles . In total, 89 professional pop / rock / jazz musicians were approached . Therefore, data pertaining to subjective symptoms includes 44 subjects and data pertaining to objective symptoms includes 41 subjects . Hearing thresholds: raw data and data after accounting for age and gender effects (n = 42) a 1 h meeting was held with each participant, during which the questionnaire was completed in the presence of the examiner, to allow for clarifications and to ensure the participant completed all the questions . This was followed by an audiometric hearing threshold assessment of the frequency range of 1 - 8 khz (by ascending intensity, up 5 db down 10 db), in the professional musician's home or studio, using a portable audiometer, manufactured by interacoustics, model as208, with tdh39 headphones, manufactured by telephonics . Hearing threshold evaluations were conducted after 12 hours of rest and with no immediate prior exposure to amplified music, according to the protocol of audiometric testing of occupational hazard . Laitinen and poulsen used a questionnaire for orchestral musicians, comprised of several standardized questionnaires pertaining to subjective symptoms . The questionnaire, comprised of 91 questions was completed by 145 classical musicians from 3 orchestras in finland . The questionnaire for the current study, the pop / rock / jazz musicians questionnaire (prjmq), included 39 questions divided into the following sections: general information, health information regarding hearing disorders, working capacity and awareness of loudness of instruments, hearing protection, and general knowledge regarding musicians understanding of concepts of loudness as expressed by the db scale . Most questions within the questionnaire began with a yes / no question (e.g. Have you ever experienced hyperacusis? ), followed by a series of scale questions for those participants who replied yes . Scale questions required participants to rate the extent to which they experienced a subjective feeling (tinnitus, hyperacusis): never, seldom, sometimes, often, or always within a certain musical setting (private lesson, rehearsal, performance, or musical concert attended), from one to five . All answers on a particular topic were averaged . Thus, the answers of those participants who reported they had experienced hyperacusis were averaged into a value named mean hyperacusis . Use of hearing protection was measured based on a yes / no question (do you use hearing protection in both ears?). This binary question was followed by a series of more specific questions which were not statistically measurable and therefore were not analyzed in the current research . In order to examine our first hypothesis claiming that musicians experience (years playing x hours playing per week) will positively predict hearing thresholds of 3 - 6 khz, a pearson correlation test was conducted between me and hearing tests of the right and left ears . Results indicated a significant positive correlation between me and the hearing loss in both the left (r(39) = .46, p = .002) and right (r(39) = .47, p = .002) ears . To better understand the effects of hours playing versus years playing we conducted four hierarchical regression analyses for the hearing thresholds pta . In two regressions, the number of years playing was entered in the first step of the equation and hours playing per week was entered in the second step . In the two other regressions hours playing per week was entered in the first step of the equation and years playing was entered in the second step . Such regressions were done for each ear separately . As can be seen from table 1, hours playing per week significantly contributed to hearing loss of both the right and left ears after controlling for the musicians years playing . Specifically, hours playing per week contributed 11% of the explained variance of right ear hearing loss in addition to the contribution of years playing (= .34, p = .037) and 12% of left ear hearing loss in addition to the contribution of years playing (= .36, p = .024). The analysis shows that only hours playing per week was found to have the contribution to hearing loss for both left and right ears after controlling for the amount of years the musician played . Years playing did not contribute significantly to both left and right ears hearing loss after controlling for the contribution for hours playing per week [table 1]. Hierarchical regression analysis predicting hearing thresholds for left and right ears based on musicians experience (times played per week and years playing) to examine the second hypothesis stating that professional musicians experience (years playing x hours played per week) will positively predict self - reported symptoms (tinnitus and hyperacusis), a pearson correlation test was conducted between the two variables . As stated earlier, none of the participants reported having distortion or diplacusis . Results showed a significant positive correlation between me and mean sporadic tinnitus (r(42) = .30, p = .05) and between me and mean hyperacusis (r(42) = .31, p = .04), confirming a significant positive correlation between musical experience and the reported subjective variables . To examine the third hypothesis stating that musicians experience will positively predict the use of hearing protection, three logistic regressions were conducted (one for each measurement: years playing, hours per week, and years playing x hours per week) on the use of hearing protection as the dependent variable . Results indicated that none of the measurements of experience predicted the use of hearing protection . To examine the fourth hypothesis asserting that self - reported symptoms (tinnitus and hyperacusis) will positively predict hearing thresholds of 3 - 6 khz the results indicated a significant correlation between tinnitus and hearing thresholds only for the left ear (rleft(39) = .34, p = .03; rright(39) = .18, p = .25). The results also indicated no significant correlation between hyperacusis and hearing thresholds for both ears (rleft(39) = .17, p = .28; rright(39) = .026, p = .87). In order to examine whether there will be a difference in hearing thresholds in the left and right ears between drummers and non - drummers, we conducted a mixed model repeated measures analysis of variance (anova). The model showed significant results regarding different hearing thresholds between the right and left ears (f (1,39) = 5.41, p = .025, p = .12) and significant results regarding the comparison between drummer musicians than non - drummers musicians (f (1.39) = 7.53, p = .009, p = .16). There was no significant interaction between being a drummer and hearing thresholds (f (1.39) = 1.11, p = .30, p = .028). Specifically, there was greater damage for the left (m = 5.41, s.d . Table 2 presents the means and s.d . Of tested pta at 3 - 6 khz before and after age and gender adjustments . As will be further discussed, table 2 shows that both right and left ear thresholds are affected by an average 2.80 - 5 db decline that cannot be explained by age or gender . Tested pta at 3 - 6 khz (in db) before and after age and gender adjustments (n = 42) finally, in order to examine whether the hearing threshold is significantly different form zero, each score after age and gender correction was compared to zero (representing intact hearing) using a one - sample t - test . Results regarding both left and right ears were found to be significant (tleftear(40) = 3.32, p = 0.002; tright ear(40) = 2.11, p = 0.041), thus proving that there is significant damage to both left and right ears, among participating professional pop / rock / jazz musicians, when compared with norms . The current research tested the hearing thresholds of professional pop / rock / jazz musicians and their subjective reports of tinnitus and hyperacusis, in relation to their musical experience and extent of exposure to amplified music . The study found a positive correlation between the extent of exposure to amplified music and hearing thresholds of 3 - 6 khz . The more experience professional pop / rock / jazz musicians had (i.e., the more exposure to amplified music), the poorer their hearing thresholds . Hours per week was found to have a greater effect in predicting hearing loss in both ears in addition to the contribution of years playing . However, years playing had no significant contribution in predicting hearing loss for both ears in addition to the contribution of hours per week . Axelsson et al . Conducted a longitudinal study of 40 pop / rock musicians, engineers and managers, whose hearing was initially tested at the average age of 26, and then retested after 16 years at the average age of 41 . They found a statistically significant, more advanced hearing loss over time among those musicians who showed hearing loss in the initial study, the greatest hearing loss suffered by drummers . Over 20% of participants also showed other types of notches (beyond 4 - 6 khz) in one or more frequencies in one or both ears . This data is further supported by poissant et al . Who showed that even 1 h of exposure per day could create an apparent increased risk among trumpet players . This research suggests that 60-year - old male and female musicians exposed to trumpet noise for 4 h per day for forty years would be 85% and 300%, respectively, more likely to have hearing loss than their peers with negative or reduced noise exposure histories . The validity of these outcomes is strengthened by the fact that many professional pop / rock / jazz musicians in the current study reported playing an additional instrument, which put them at an even greater risk of nihl not accounted for in the statistical analysis of the current study . Other studies on pop / rock / jazz musicians found hearing decline similar if not worse than the hearing decline found in the current study . Axelsson et al . Found a hearing decline of 2 - 3db in both right and left ears . Thus, greater emphasis should be given to the fact that even a short exposure to amplified music can cause long lasting damage to hearing and as such the importance of using hearing protection . The study also found a significant positive correlation between professional pop / rock / jazz musicians experience / extent of exposure and subjectively - reported variables of tinnitus and hyperacusis: the greater the exposure to music, the more frequent the subjectively - reported symptoms . Whose research showed that tinnitus and hypersensitivity are related to extent of exposure among nonprofessional steelband musicians . This study hypothesized that there would be a significant positive correlation between self - reported symptoms (tinnitus and hyperacusis) and the hearing thresholds of 3 - 6 khz . This prevalence is higher than the estimate among the normal hearing population (15%). Khri stated that the high percentage of permanent tinnitus could be the product of excessive sound levels (p. 48). The current study found a positive correlation between left ear pta thresholds and sporadic tinnitus . A high prevalence of tinnitus has been found in musicians with hearing loss . This study's correlation between tinnitus and hearing loss was found for the left hear only . This finding supports various studies that have indicated a small hearing asymmetry between right and left hearing thresholds, related to exposure to noise . Previous studies show a slightly higher susceptibility to several kinds of hearing damage in the peripheral hearing mechanism of the left ear, related to nihl . Found that nerve fibers within the efferent nerve system attenuate outer hair cell (ohc) motility more strongly in middle frequencies of the left ear, compared with those of the right ear . Where the left ear ohcs are less functional, fibers within the medial olivary complex (moc) compensate, and may become more functional, especially in mid - range frequencies . Claimed that this phenomenon, which increases with human development and affects middle frequencies of the left ear only, results in the left ear being more susceptible to noise damage and tinnitus . As mentioned in the method, 10 professional drummers participated in the current study and were found to have significantly higher hearing thresholds compared to other musicians within the study . Hoffman et al . Indicated that 315 percussionists aged 18 - 75 had poorer mean hearing thresholds at all frequencies, compared with a reference population . Axelsson et al . Found 10 percussionists to have the poorest hearing thresholds in a longitudinal study across 16 years among a group of 40 musicians, sound engineers, and managers . . Showed significant auditory threshold deterioration among 29 steelband musicians, compared with a control group of 30 non - musicians . However, further statistical analysis found that being a professional drummer did not add a significant variance for explaining hearing loss for both left and right ears . A larger sample of drummers is required in order to establish the etiology . Professional drummers hearing decline in our study might have been a result of being more experienced than other participants in the study (having more hours per week of practice). Finally, our study did not find a correlation between the extent of exposure to amplified music and the use of hearing protection or between self reported symptoms and use of hearing protection . Although in our study, 56% of the professional pop / rock / jazz musicians reported use of hearing protection, juman et al . Showed that the use of personal hearing - protective devices (phpd) in the form of earplugs and earmuffs is not reported as a common habit . Laitinen and poulsen found that most classical musicians do not use hearing protectors regularly in both ears, but tend to use them more frequently in one ear . Many professional pop / rock / jazz musicians reported that the use of hearing protection affected the quality of sound and was uncomfortable . This means that while musicians do use hearing protection devices, they do not use them everywhere or all the time . The tradeoff between the protection and comfort / quality of sound may be too big a price to pay, even for professional musicians who are highly exposed to loud music and who suffer hearing deterioration . Found that tinnitus and hyperacusis were reported by subjects as having minimal impact on their lives . It is possible that the degree of self - reported symptoms was not sufficiently bothersome for the professional musicians to try to treat them or protect against them . Although all our subjects did not report any active ear - nose - throat (ent) pathologies, it would be interesting to study possible effects of noise / music on low frequency thresholds . Since all studies dealing with pop / rock / jazz musicians focused on hearing thresholds and questionnaires, future research is needed in larger samples, and with additional objective tools . Therefore, it is suggested that future research focus on other factors affecting use of hearing protection, such as social conventions, type of phpd and awareness to the dangers of extended exposure to amplified music . Hours and years served as a reference to the amount of exposure within the current research . Further research might attempt to consider other relevant contributing factors, such as periods of more or less musical activity within a musician's professional career, or the extent of attendance to musical concerts . This can be done by focusing on particular musical genres or on certain musical instruments . Beyond quantifying hearing thresholds and subjective variables such as tinnitus and hyperacusis, further research is warranted on other subjectively reported variables and on musician's professional and personal quality of life and wellbeing, following hearing decline . Our finding that the left ear of our subjects was more susceptible to noise, supported by earlier findings concerning music as well as regular noise emphasizes the importance of further research of the mechanisms that play a role in this phenomenon . Damage risk criteria (drc) regulations draw on the approach of noise of equal energy, giving identical magnitude to noise intensity and duration of exposure . Thus, rules of intensity versus exposure in hours can predict the extent of auditory damage . The current research has shown that the effects of practice (measured in hours) are more hazardous than the effects of years playing . Future research is warranted in order to better define factors influencing professional musicians exposure to noise . Our findings of the high prediction - power of professional pop / rock / jazz musicians hours / week exposure to amplified music and hearing loss as well as the non - correlation between hours exposure per week and the use of hearing protection highlights the dramatic importance of education towards awareness among musicians . Awareness to the implications of subjective symptoms of hearing loss will make the possible long - lasting damage to professional musicians hearing more salient among the population in general and particularly among those exposed to amplified music . Furthermore, educating on this matter will promote musicians awareness to the need to use hearing protection at all times, as the damage to their hearing happens even after a short exposure . The current study has shown that professional pop / rock / jazz musicians exposed to loud, amplified music, do suffer from symptoms of nihl . Professional pop / rock / jazz musicians extent of exposure to amplified loud music are related to hearing loss and other subjective effects related to hearing damage such as tinnitus and hyperacusis . In addition the current results have shown, that professional pop / rock / jazz musicians exposure to amplified music per week predicts objective hearing loss after controlling for age, gender, and years of experience.
Minorities in the us are more likely to experience health and health - care disparities than their majority counterparts . While many factors may contribute to these disparities, discrimination has been shown to be one of the most prominent.1,2 the body of literature linking discrimination with health and health - care disparities has traditionally concentrated on african americans, and has only recently begun to focus on the latino population,35 perhaps due to recent census findings indicating that the latino population is the largest minority population in the us.6,7 health - care disparities that latinos experience as a result of discrimination, and the subsequent effects on quality of health care, present issues of great social and economic significance for the entire populace . In addition, perceived discrimination has been established as a correlate of health - care access and health - care behavior in a number of ways . Perceived discrimination has been found to be associated with cancer screening8, health - care under - utilization3, receipt of diabetes management indicators such as the hemogloblin a1c test, foot exam and blood pressure exam9 and reported delays in filling pharmacy prescriptions10 . The existence of racial / ethnic and other disparities in care especially among vulnerable populations gives further impetus to understanding how quality of care is impacted by perceived discrimination . Trivedi and ayanian showed that vulnerable persons who perceived discrimination were less likely to receive testing for cvd, flu shots and eye exams1 . Previous studies have examined perceived discrimination among latinos in the us health - care system.5 the 2004 behavioral risk factor surveillance system survey showed that 5.2% of latinos perceived discrimination in health - care settings, while only 2% of non - latino whites reported discrimination.11 the nature of this discrimination was further elucidated by research5,12 that found that latinos born in the us were more likely to report discrimination based on their sociodemographic and sociocultural factors . However, latinos with higher socioeconomic status (ses) and health insurance reported experiencing less discrimination when receiving health care.12 in contrast, other studies have shown that higher ses is associated with greater perception of discrimination.5 perceived discrimination among latinos has been shown to produce effects both in and out of the provider s office . In the hospital setting, for example, latina patients have rated their obstetrical services experience lower than non - latino whites with regard to respect, information and education, physical comfort and emotional support.13 the effects of this discrimination have extended well beyond hospitals, however, and have prevented latinos from seeking health - care services . For example, nadeem et al . Found that for latinas, an increased stigma in seeking mental health - care services kept them from accessing the services they needed.14 furthermore, perceived discrimination has been related to greater psychological distress and lower self - esteem.15 discrimination has also been associated with conditions such as coronary heart disease, high blood pressure and compromised mental health.4,16,17 despite the existence of the above - mentioned studies, perceived discrimination among the latino population remains a relatively understudied area.3,5,19 awareness and understanding of discrimination - induced health - care disparities are needed to mobilize systematic and systemic reform efforts.20 this study contributes to a growing body of literature examining discrimination among latinos by using a nationally representative database to examine general and health - care - specific discrimination experienced by specific groups of latinos in the us . In this study, we examine the association between perceived discrimination and two measures of quality of care: patient report of quality of care in the past 12 months and quality of doctor - patient communication . Study participants were part of a stratified, random - digit dialing telephone survey (the pew hispanic center / robert wood johnson foundation latino health survey)21 of n = 3,899 adult latinos (aged 18 years) designed to produce a statistically representative sample of latinos in the contiguous us . Respondents were identified as latino if they answered yes to the question, are you, yourself, of hispanic or latino origin or descent such as mexican, puerto rican, cuban, dominican, central or south american, caribbean or some other latin american background? Initial telephone interviews (wave 1) were conducted in summer 2007 and had a response rate of 39.5% . Participants were called again in spring 2008 for a second interview (wave 2), focusing on chronic disorders and medical care received . Subjects for this study are the n = 1,067 persons who completed the wave 2 interview . Post - stratification weight adjustment for wave 2 consisted of an adjustment of wave 1 sampling weights so that the sum of the weights by sex, age, nativity and education matched those of latinos in the march 2007 supplement of the current population survey.22 this post - stratification weight adjustment was minor, and wave 2 participants were not statistically different from other wave 1 participants on any demographic measure . For this study, two measures of discrimination were used: the detroit area study (das) discrimination scale consisting of nine 6-point items,23,24 and a measure of doctor or medical staff discrimination from the commonwealth fund health quality survey consisting of three yes or no items; see appendix for items.25 the das scale had range 039, median 4, mean 6.7, sd 8.4 and cronbach s = 0.87 . For all analyses, the das discrimination scale was scaled to have mean 0 and sd 1, and treated as a continuous measure . The doctor or medical staff discrimination items were converted into a single dichotomous variable with zero indicating no to all three items and one indicating a yes to any one of the items . The first was a 5-point measure of self - reported quality of health care received in the past year, with responses excellent, very good, good, fair and poor . The second outcome measure was a scale comprised of four 5-point items from the interpersonal processes of care survey short form,26 which reflects the quality of communication between doctors and other health professionals and the study participant; see appendix for items . This scale is referred to as the doctor communication scale in this study and had range 016, median 10, mean 10.1, sd 4.5 and cronbach s = 0.75 . Weighted sex - age adjusted means of the das discrimination scale were calculated along with weighted sex - age adjusted percentages of respondents who answered yes to any of the doctor discrimination items in table 1 . Significance tests for proportions were performed using a rao - scott statistic for the pearson test for contingency tables2729, and tests for means were done using design - based wald tests30 . Table 1any reported discrimination from doctors or medical staff and mean das discrimination scale for us latinos (n = 1,067) by demographics and self - reported health status any discrimination from doctors or medical staff in past 2 years % mean das discrimination scaleall persons19 (2)0.00 (0.05)sex female20 (3)0.09 (0.07) male18 (2)0.09 (0.07)(test of any difference)0.50.07age (years) 182922 (4)0.30 (0.13) 304919 (2)0.04 (0.05) 506415 (3)0.27 (0.05) 659 (3)0.42 (0.10)(test of any difference)0.02<0.001foreign born by years in us 0519 (4)0.32 (0.09) 61518 (3)0.29 (0.06) 1618 (5)0.12 (0.11)(test of any difference)1.00.3foreign born, all20 (2)0.23 (0.04)us born16 (3)0.32 (0.09)(test of any difference)0.3<0.001language of interview spanish or mostly spanish18 (2)0.22 (0.06) english or a mix of english and spanish19 (3)0.31 (0.09)(test of any difference)0.8<0.001education 08 years22 (4)0.14 (0.10) 911 years17 (4)0.03 (0.12) high school graduate18 (3)0.05 (0.10) some college19 (4)0.20 (0.10) college degree or more14 (5)0.01 (0.14) (test of any difference)0.80.2self - reported health status excellent11 (4)0.12 (0.12) very good14 (3)0.06 (0.12) good16 (3)0.10 (0.08) fair27 (4)0.12 (0.09) poor39 (12)0.02 (0.23) (test of any difference)0.0050.3age - sex adjusted percentages of respondents who answered yes to any of three questions relating to experiences where doctors or medical staff judged the respondent unfairly or treated the respondent with disrespect because of race or ethnicity, language or financial status . See appendix for items . Significance of any difference of proportions among categories is tested using the rao - scott statistic for the pearson test; see methods.age-sex adjusted mean scores for the das discrimination scale standardized to have mean 0 and sd 1 . Significance of any difference of means among categories is tested using an adjusted wald test; see methods.foreign-born includes us citizens who were born on the island of puerto rico . Any reported discrimination from doctors or medical staff and mean das discrimination scale for us latinos (n = 1,067) by demographics and self - reported health status * age - sex adjusted percentages of respondents who answered yes to any of three questions relating to experiences where doctors or medical staff judged the respondent unfairly or treated the respondent with disrespect because of race or ethnicity, language or financial status . Significance of any difference of proportions among categories is tested using the rao - scott statistic for the pearson test; see methods . Age - sex adjusted mean scores for the das discrimination scale standardized to have mean 0 and sd 1 . Significance of any difference of means among categories is tested using an adjusted wald test; see methods . Foreign - born includes us citizens who were born on the island of puerto rico . Self - reported quality of care was analyzed as the outcome variable in weighted ordered logistic regressions in table 2 . The doctor communication scale was the outcome in weighted linear regressions in table 3 . Standard error estimates for all analyses were adjusted for the sampling design using a first - order taylor series approximation . All analyses were conducted using the svy suite of commands from stata statistical software version 10.131 . Figure 1self - reported quality of care by reported discrimination from doctors or medical staff for us latinos (adjusted for sex, age, education, insurance and self - reported health status).figure 2doctor communication scale by reported discrimination from doctors or medical staff for us latinos (adjusted for sex, age, education and insurance).table 2association of self - reported quality of care * with discrimination measures: ordered logistic regression models for us latinos with any past - year doctor visits us born (n = 249)foreign born (n = 591)odds ratio [95% ci]significance (p)odds ratio [95% ci]significance (p)any discrimination from doctors or medical staff0.5 [0.2, 1.2]0.10.5 [0.3, 0.9]0.03das discrimination scale0.5 [0.3, 0.9]0.0090.9 [0.7, 1.2]0.6male1.4 [0.7, 2.8]0.31.0 [0.7, 1.5]1.0age (years) 18291.2 [0.5, 2.6]0.70.6 [0.4, 1.0]0.04 304911 50640.9 [0.5, 1.9]0.80.9 [0.5, 1.4]0.5 652.1 [0.7, 6.4]0.21.3 [0.7, 2.3]0.4 (test of any difference)0.50.09education 08 years0.2 [0.1, 0.8]0.020.8 [0.5, 1.3]0.4 911 years2.1 [0.6, 6.6]0.21.3 [0.8, 2.3]0.3 high school graduate11 some college0.8 [0.3, 1.9]0.61.0 [0.5, 2.1]1.0 college degree or more0.8 [0.3, 2.1]0.61.8 [0.9, 3.4]0.1 (test of any difference)0.0470.2household income $014,9990.9 [0.3, 2.5]0.81.2 [0.7, 2.0]0.4 $15,00024,9991.5 [0.5, 4.8]0.50.8 [0.5, 1.3]0.3 $25,00034,99911 $35,00059,9990.9 [0.3, 2.8]0.90.7 [0.3, 1.3]0.2 $60,0001.2 [0.3, 4.1]0.81.0 [0.4, 2.4]0.9 (test of any difference)0.90.2health insurance2.0 [0.8, 4.9]0.11.7 [1.1, 2.6]0.01self - reported health status excellent1.6 [0.5, 5.2]0.43.3 [1.3, 8.6]0.01 very good2.0 [0.9, 4.5]0.11.4 [0.9, 2.4]0.2 good11 fair1.1 [0.4, 3.6]0.80.5 [0.3, 0.8]0.002 poor0.4 [0.1, 1.5]0.20.5 [0.2, 1.2]0.1 (test of any difference)0.1<0.001outcome variable is 5-point measure of self - reported quality of health care received in the past 12 months: excellent, very good, good, fair or poorforeign born includes us citizens who were born on the island of puerto ricoany self - reported discrimination from doctors or medical staff in past 2 years; see methodsdas discrimination scale standardized to have mean 0 and sd 1table 3association of doctor communication scale with discrimination measures: linear regression models for us latinos with any past - year doctor visits us born (n = 251)foreign born (n = 584)coefficient [95% ci]significance (p)coefficient [95% ci]significance (p)any discrimination from doctors or medical staff3.5 [5.1, 2.0]<0.0011.2 [2.5, 0.0]0.05das discrimination scale0.7 [1.2, 0.1]0.030.5 [1.1, 0.0]0.06male0.5 [1.7, 0.8]0.50.3 [1.1, 0.6]0.5age (years) 18290.4 [0.9, 1.8]0.50.2 [0.9, 1.2]0.8 304900 50640.8 [2.3, 0.7]0.30.4 [1.5, 0.7]0.4 651.4 [3.4, 0.6]0.21.0 [2.3, 0.3]0.1 (test of any difference)0.30.4education 08 years2.3 [5.5, 0.9]0.20.3 [1.4, 0.9]0.6 911 years0.0 [1.8, 1.9]1.00.6 [0.7, 1.8]0.4 high school graduate00 some college0.5 [1.8, 0.8]0.50.6 [2.0, 0.7]0.3 college degree or more0.7 [2.4, 1.0]0.40.1 [1.4, 1.5]0.9 (test of any difference)0.60.5household income $014,9991.4 [3.8, 1.0]0.20.6 [1.9, 0.7]0.4 $15,00024,9991.4 [3.8, 0.9]0.20.0 [1.3, 1.3]1.0 $25,00034,99900 $35,00059,9990.6 [2.6, 1.3]0.50.2 [1.2, 1.6]0.8 $60,0001.3 [3.5, 0.9]0.30.9 [0.8, 2.6]0.3 (test of any difference)0.60.5health insurance0.2 [1.6, 2.0]0.81.0 [0.0, 1.9]0.046outcome variable is 4-item scale assessing the quality of doctor - patient communications (range 016); see methods . Exact zeros in table indicate reference categoriesforeign born includes us citizens who were born on the island of puerto ricoany self - reported discrimination from doctors or medical staff in past 2 years; see methodsdas discrimination scale standardized to have mean 0 and sd 1 self - reported quality of care by reported discrimination from doctors or medical staff for us latinos (adjusted for sex, age, education, insurance and self - reported health status). Doctor communication scale by reported discrimination from doctors or medical staff for us latinos (adjusted for sex, age, education and insurance). Association of self - reported quality of care with discrimination measures: ordered logistic regression models for us latinos with any past - year doctor visits * outcome variable is 5-point measure of self - reported quality of health care received in the past 12 months: excellent, very good, good, fair or poor foreign born includes us citizens who were born on the island of puerto rico any self - reported discrimination from doctors or medical staff in past 2 years; see methods das discrimination scale standardized to have mean 0 and sd 1 association of doctor communication scale * with discrimination measures: linear regression models for us latinos with any past - year doctor visits * outcome variable is 4-item scale assessing the quality of doctor - patient communications (range 016); see methods . Exact zeros in table indicate reference categories foreign born includes us citizens who were born on the island of puerto rico any self - reported discrimination from doctors or medical staff in past 2 years; see methods study participants were part of a stratified, random - digit dialing telephone survey (the pew hispanic center / robert wood johnson foundation latino health survey)21 of n = 3,899 adult latinos (aged 18 years) designed to produce a statistically representative sample of latinos in the contiguous us . Respondents were identified as latino if they answered yes to the question, are you, yourself, of hispanic or latino origin or descent such as mexican, puerto rican, cuban, dominican, central or south american, caribbean or some other latin american background? Initial telephone interviews (wave 1) were conducted in summer 2007 and had a response rate of 39.5% . Participants were called again in spring 2008 for a second interview (wave 2), focusing on chronic disorders and medical care received . Subjects for this study are the n = 1,067 persons who completed the wave 2 interview . Post - stratification weight adjustment for wave 2 consisted of an adjustment of wave 1 sampling weights so that the sum of the weights by sex, age, nativity and education matched those of latinos in the march 2007 supplement of the current population survey.22 this post - stratification weight adjustment was minor, and wave 2 participants were not statistically different from other wave 1 participants on any demographic measure . For this study, two measures of discrimination were used: the detroit area study (das) discrimination scale consisting of nine 6-point items,23,24 and a measure of doctor or medical staff discrimination from the commonwealth fund health quality survey consisting of three yes or no items; see appendix for items.25 the das scale had range 039, median 4, mean 6.7, sd 8.4 and cronbach s = 0.87 . For all analyses, the das discrimination scale was scaled to have mean 0 and sd 1, and treated as a continuous measure . The doctor or medical staff discrimination items were converted into a single dichotomous variable with zero indicating no to all three items and one indicating a yes to any one of the items . The first was a 5-point measure of self - reported quality of health care received in the past year, with responses excellent, very good, good, fair and poor . The second outcome measure was a scale comprised of four 5-point items from the interpersonal processes of care survey short form,26 which reflects the quality of communication between doctors and other health professionals and the study participant; see appendix for items . This scale is referred to as the doctor communication scale in this study and had range 016, median 10, mean 10.1, sd 4.5 and cronbach s = 0.75 . Weighted sex - age adjusted means of the das discrimination scale were calculated along with weighted sex - age adjusted percentages of respondents who answered yes to any of the doctor discrimination items in table 1 . Significance tests for proportions were performed using a rao - scott statistic for the pearson test for contingency tables2729, and tests for means were done using design - based wald tests30 . Table 1any reported discrimination from doctors or medical staff and mean das discrimination scale for us latinos (n = 1,067) by demographics and self - reported health status any discrimination from doctors or medical staff in past 2 years % mean das discrimination scaleall persons19 (2)0.00 (0.05)sex female20 (3)0.09 (0.07) male18 (2)0.09 (0.07)(test of any difference)0.50.07age (years) 182922 (4)0.30 (0.13) 304919 (2)0.04 (0.05) 506415 (3)0.27 (0.05) 659 (3)0.42 (0.10)(test of any difference)0.02<0.001foreign born by years in us 0519 (4)0.32 (0.09) 61518 (3)0.29 (0.06) 1618 (5)0.12 (0.11)(test of any difference)1.00.3foreign born, all20 (2)0.23 (0.04)us born16 (3)0.32 (0.09)(test of any difference)0.3<0.001language of interview spanish or mostly spanish18 (2)0.22 (0.06) english or a mix of english and spanish19 (3)0.31 (0.09)(test of any difference)0.8<0.001education 08 years22 (4)0.14 (0.10) 911 years17 (4)0.03 (0.12) high school graduate18 (3)0.05 (0.10) some college19 (4)0.20 (0.10) college degree or more14 (5)0.01 (0.14) (test of any difference)0.80.2self - reported health status excellent11 (4)0.12 (0.12) very good14 (3)0.06 (0.12) good16 (3)0.10 (0.08) fair27 (4)0.12 (0.09) poor39 (12)0.02 (0.23) (test of any difference)0.0050.3age - sex adjusted percentages of respondents who answered yes to any of three questions relating to experiences where doctors or medical staff judged the respondent unfairly or treated the respondent with disrespect because of race or ethnicity, language or financial status . Significance of any difference of proportions among categories is tested using the rao - scott statistic for the pearson test; see methods.age-sex adjusted mean scores for the das discrimination scale standardized to have mean 0 and sd 1 . Significance of any difference of means among categories is tested using an adjusted wald test; see methods.foreign-born includes us citizens who were born on the island of puerto rico . Any reported discrimination from doctors or medical staff and mean das discrimination scale for us latinos (n = 1,067) by demographics and self - reported health status * age - sex adjusted percentages of respondents who answered yes to any of three questions relating to experiences where doctors or medical staff judged the respondent unfairly or treated the respondent with disrespect because of race or ethnicity, language or financial status . Significance of any difference of proportions among categories is tested using the rao - scott statistic for the pearson test; see methods . Age - sex adjusted mean scores for the das discrimination scale standardized to have mean 0 and sd 1 significance of any difference of means among categories is tested using an adjusted wald test; see methods . Foreign - born includes us citizens who were born on the island of puerto rico . Self - reported quality of care was analyzed as the outcome variable in weighted ordered logistic regressions in table 2 . The doctor communication scale was the outcome in weighted linear regressions in table 3 . Standard error estimates for all analyses were adjusted for the sampling design using a first - order taylor series approximation . All analyses were conducted using the svy suite of commands from stata statistical software version 10.131 . Figure 1self - reported quality of care by reported discrimination from doctors or medical staff for us latinos (adjusted for sex, age, education, insurance and self - reported health status).figure 2doctor communication scale by reported discrimination from doctors or medical staff for us latinos (adjusted for sex, age, education and insurance).table 2association of self - reported quality of care * with discrimination measures: ordered logistic regression models for us latinos with any past - year doctor visits us born (n = 249)foreign born (n = 591)odds ratio [95% ci]significance (p)odds ratio [95% ci]significance (p)any discrimination from doctors or medical staff0.5 [0.2, 1.2]0.10.5 [0.3, 0.9]0.03das discrimination scale0.5 [0.3, 0.9]0.0090.9 [0.7, 1.2]0.6male1.4 [0.7, 2.8]0.31.0 [0.7, 1.5]1.0age (years) 18291.2 [0.5, 2.6]0.70.6 [0.4, 1.0]0.04 304911 50640.9 [0.5, 1.9]0.80.9 [0.5, 1.4]0.5 652.1 [0.7, 6.4]0.21.3 [0.7, 2.3]0.4 (test of any difference)0.50.09education 08 years0.2 [0.1, 0.8]0.020.8 [0.5, 1.3]0.4 911 years2.1 [0.6, 6.6]0.21.3 [0.8, 2.3]0.3 high school graduate11 some college0.8 [0.3, 1.9]0.61.0 [0.5, 2.1]1.0 college degree or more0.8 [0.3, 2.1]0.61.8 [0.9, 3.4]0.1 (test of any difference)0.0470.2household income $014,9990.9 [0.3, 2.5]0.81.2 [0.7, 2.0]0.4 $15,00024,9991.5 [0.5, 4.8]0.50.8 [0.5, 1.3]0.3 $25,00034,99911 $35,00059,9990.9 [0.3, 2.8]0.90.7 [0.3, 1.3]0.2 $60,0001.2 [0.3, 4.1]0.81.0 [0.4, 2.4]0.9 (test of any difference)0.90.2health insurance2.0 [0.8, 4.9]0.11.7 [1.1, 2.6]0.01self - reported health status excellent1.6 [0.5, 5.2]0.43.3 [1.3, 8.6]0.01 very good2.0 [0.9, 4.5]0.11.4 [0.9, 2.4]0.2 good11 fair1.1 [0.4, 3.6]0.80.5 [0.3, 0.8]0.002 poor0.4 [0.1, 1.5]0.20.5 [0.2, 1.2]0.1 (test of any difference)0.1<0.001outcome variable is 5-point measure of self - reported quality of health care received in the past 12 months: excellent, very good, good, fair or poorforeign born includes us citizens who were born on the island of puerto ricoany self - reported discrimination from doctors or medical staff in past 2 years; see methodsdas discrimination scale standardized to have mean 0 and sd 1table 3association of doctor communication scale with discrimination measures: linear regression models for us latinos with any past - year doctor visits us born (n = 251)foreign born (n = 584)coefficient [95% ci]significance (p)coefficient [95% ci]significance (p)any discrimination from doctors or medical staff3.5 [5.1, 2.0]<0.0011.2 [2.5, 0.0]0.05das discrimination scale0.7 [1.2, 0.1]0.030.5 [1.1, 0.0]0.06male0.5 [1.7, 0.8]0.50.3 [1.1, 0.6]0.5age (years) 18290.4 [0.9, 1.8]0.50.2 [0.9, 1.2]0.8 304900 50640.8 [2.3, 0.7]0.30.4 [1.5, 0.7]0.4 651.4 [3.4, 0.6]0.21.0 [2.3, 0.3]0.1 (test of any difference)0.30.4education 08 years2.3 [5.5, 0.9]0.20.3 [1.4, 0.9]0.6 911 years0.0 [1.8, 1.9]1.00.6 [0.7, 1.8]0.4 high school graduate00 some college0.5 [1.8, 0.8]0.50.6 [2.0, 0.7]0.3 college degree or more0.7 [2.4, 1.0]0.40.1 [1.4, 1.5]0.9 (test of any difference)0.60.5household income $014,9991.4 [3.8, 1.0]0.20.6 [1.9, 0.7]0.4 $15,00024,9991.4 [3.8, 0.9]0.20.0 [1.3, 1.3]1.0 $25,00034,99900 $35,00059,9990.6 [2.6, 1.3]0.50.2 [1.2, 1.6]0.8 $60,0001.3 [3.5, 0.9]0.30.9 [0.8, 2.6]0.3 (test of any difference)0.60.5health insurance0.2 [1.6, 2.0]0.81.0 [0.0, 1.9]0.046outcome variable is 4-item scale assessing the quality of doctor - patient communications (range 016); see methods . Exact zeros in table indicate reference categoriesforeign born includes us citizens who were born on the island of puerto ricoany self - reported discrimination from doctors or medical staff in past 2 years; see methodsdas discrimination scale standardized to have mean 0 and sd 1 self - reported quality of care by reported discrimination from doctors or medical staff for us latinos (adjusted for sex, age, education, insurance and self - reported health status). Doctor communication scale by reported discrimination from doctors or medical staff for us latinos (adjusted for sex, age, education and insurance). Association of self - reported quality of care with discrimination measures: ordered logistic regression models for us latinos with any past - year doctor visits * outcome variable is 5-point measure of self - reported quality of health care received in the past 12 months: excellent, very good, good, fair or poor foreign born includes us citizens who were born on the island of puerto rico any self - reported discrimination from doctors or medical staff in past 2 years; see methods das discrimination scale standardized to have mean 0 and sd 1 association of doctor communication scale * with discrimination measures: linear regression models for us latinos with any past - year doctor visits * outcome variable is 4-item scale assessing the quality of doctor - patient communications (range 016); see methods . Exact zeros in table indicate reference categories foreign born includes us citizens who were born on the island of puerto rico any self - reported discrimination from doctors or medical staff in past 2 years; see methods the first data column of table 1 shows the percentage of respondents who answered yes to any of the three items indicating perception of discrimination from doctors or medical staff in the past 2 years (see appendix). Overall, only 19% of all persons reported any discrimination from doctors or medical staff . Doctor or medical staff discrimination was significantly associated with age (p = 0.02) with younger persons (1829 years) more likely to report discrimination (22%) than persons aged 65 years . The only other significant association of doctor discrimination and the variables shown in table 1 was between doctor discrimination and self - reported health status (p = 0.005). Persons with poor health status reported dramatically more doctor or medical staff discrimination (39%) than those with better health status (11% for those reporting excellent health and 14% for those reporting very good health). Table 1 shows the mean value of the das discrimination scale by demographics and self - reported health status . The overall mean of zero on this scale reflects the standardization of the scale to mean 0 and sd 1 . Unstandardized, the overall das mean was 6.7 (with range 039), which corresponds to an average answer slightly below the level of the response less than once a year for each of the nine items on the scale (see appendix). The das responses were not significantly different by sex, but were highly associated with age (p <0.001) with younger participants reporting the most discrimination . Us - born latinos reported more discrimination than the foreign - born, with the us born far more likely to report discrimination compared to the foreign born (0.32 sd versus 0.23 sd); however, the age - adjusted means for the foreign born did not significantly differ by the number of years in the us . Language of interview was associated with perceived discrimination to a similar degree as nativity, with those who were interviewed in english or a mix of english and spanish more likely to report discrimination than spanish interviewees (0.31 sd versus 0.22 sd). Reported discrimination on the das scale did not differ significantly by education, nor was self - reported health status associated with das . Table 2 shows ordered logistic regression models for self - reported quality of health care in the past year with separate models for us - born and foreign - born latinos . Included in the models are terms for any discrimination from doctors or medical staff, the standardized das discrimination measure (yielding a beta coefficient), demographics, health insurance and self - reported health status . Models for both us - born and foreign - born latinos yielded the same odds ratio (0.5) for doctor discrimination; however, only in the foreign - born model did it reach statistical significance (p = 0.03). The das discrimination scale, however, was significantly associated with lower quality of care for the us born [or = 0.5 (0.3, 0.9); p = 0.009], but was not significant for the foreign born [or = 0.9 (0.7, 1.2); p = 0.6]. A test of the difference for the das odds ratio for the us born compared to the odds ratio for the foreign born was significant at p = 0.03 . In the model for us - born latinos, the only other significant odds ratio was that for very low education (08 years). In contrast, for foreign - born latinos, younger adults (1829 years), health insurance and self - reported health status were all significantly associated with quality of care . Table 3 shows linear regression models for the doctor communication scale (see appendix). For us - born latinos, both any doctor or medical staff discrimination and das terms were significantly associated (p <0.001 and p = 0.03, respectively) with doctor communication, with persons reporting more discrimination having worse doctor communication . No other terms were significant in the model for the us born . For foreign - born latinos, a test of the difference for the doctor discrimination coefficient for the us born [3.5 (5.1, 2.0)] compared to the coefficient for the foreign born [1.2 (2.5, 0.0)] was significant at p = 0.02 . Notably, the only significant term in the foreign - born model was insurance, and only marginally so . Figures 1 and 2 illustrate the effect size of the self - reported experience of doctor or medical staff discrimination . Figure 1 shows the mean level of self - reported quality of care by nativity for persons reporting any doctor or medical staff discrimination and for those reporting no discrimination . Figure 2 is similar except that the outcome plotted is the mean level of the doctor communication scale . Means in figure 2 were adjusted for sex, age and education, and means in figure 1 were adjusted for these variables and self - reported health status as well (see methods). In figure 1, the difference in self - reported quality of care for us - born latinos who report any doctor discrimination compared to those who report none is 0.7 lower [95% ci (0.2, 1.2); p = 0.004], where one unit represents one step on the response (i.e., the difference between very good and good). For foreign - born latinos, the difference is smaller: 0.4 lower self - reported quality of care [95% ci (0.1, 0.6); p = 0.006]. In figure 2, the difference in the doctor communication scale for us - born latinos who report any doctor discrimination compared to those who report none is 4.1 lower [95% ci (2.5, 5.6); p <0.001]. For foreign - born latinos, the difference is much smaller: 1.7 lower on the doctor communication scale [95% ci (0.7, 2.7); p = 0.001]. Perceived discrimination was associated with quality of care measures in this nationally representative sample of us latinos . However, this association was much stronger among us - born latinos than among the foreign born . Of all correlates of quality, discrimination had the strongest effect as one in five persons reporting perceived discrimination from medical personnel within the past 2 years . We looked at two measures of discrimination, one a measure of general discrimination, the detroit area study (das) discrimination scale, and the other a measure of perceived discrimination from doctors or medical personnel . In a model of self - reported quality of care for us - born latinos, with both discrimination measures as independent variables, the das scale was significantly associated with quality of care (after controlling for sociodemographics and health status), but perceived doctor discrimination was not . In the same model for foreign - born latinos, only doctor discrimination was associated with self - reported quality of care . In models of the quality of doctor - patient communication, both discrimination measures were significant for the us born, but for the foreign born, both were marginally nonsignificant . The finding of a greater association between perceived discrimination and self - reported quality of care measures in the us born compared to the foreign born has several possible explanations . The first possibility is that us - born latinos experience more discrimination than foreign - born latinos because they interact more closely with non - latinos in the us and seek health care in the same settings as non - latinos and that the care they receive in these settings is below the level that they perceive non - latinos receive . The second possibility is that us - born latinos because of their english - language abilities and greater understanding of us culture are more vigilant in monitoring their patient - provider relationship and are better able perceive when receipt of lower quality of care is connected to discrimination than are foreign - born latinos . It may be that foreign - born latinos are protected from perceiving discrimination . Other studies have suggested a similar protective factor of lower acculturation.5,32,33 previous authors have suggested ethnic identity as one of many possible buffers of discrimination, and foreign - born latinos may benefit from this factor.34,35 however, in this study, we did not find associations among discrimination and education, age or income that were found in prior studies of discrimination.36 the third possibility is that among us - born latino there is a larger subgroup of pessimists regarding their life in the us than among the foreign born and these pessimists rate their experiences in general more negatively so they have perceptions both of more discrimination and of lower quality of health care . Based on our results we found that levels of perceived discrimination from doctors or medical staff to be similar among the us born and the foreign born . If there were more pessimists among the us born, there should be a larger proportion reporting discrimination in the health - care setting among the us born than among the foreign born . The strongest association that we found was between perceived doctor or medical staff discrimination and doctor - patient communication in the us born . This seems to support the second possibility that us - born latinos are better able to perceive the connection between discrimination and lower quality of care when it exists . The strong association between general discrimination (as measured on the das scale) and self - reported lower quality of care among the us born seems to support the first possibility those with the most interaction with us culture experience both more discrimination and a perceived lower relative quality of care . Still a fourth possible explanation explaining why us born latinos who perceive discrimination report lower quality of care could be that they have switched providers in the previous year . It is likely that many latinos experienced discrimination prior to the year in which the health - care quality questions referred to . This in turn may explain for the higher rates of perceived discrimination as well as the lower rates of perceived quality of health care received . The association this study found between poor self - reported health status and perceptions of discrimination from doctors or medical staff is of concern . In piette et al.s 37 work with diabetic patients, a similar association was found where 14% of the study s participants reported experiencing health - care discrimination during the prior year, including discrimination due to their race (8%), education or income (9%), age (7%) and sex (10% of women). In the present study, respondents with poorer health status reported more than three and a half times the rate of discrimination in the health - care context . The study is cross - sectional, and arguments of causation cannot be made . The mean of general discrimination assessed on the das scale is relatively low; on average, discriminatory experiences are occurring only a few times a year, and only about one in five latinos reported any discrimination from doctors or medical personnel in the past 2 years . However, it may be that attributes of poor quality of care and poor patient - provider communication, such as rushed care, rudeness, or arrogance on the part of doctors or medical staff, are associated in respondents minds with perceived discrimination . The connection between general discrimination not necessarily in a medical setting and quality of care may be even more tenuous . Looking at general discrimination and discrimination in a medical setting jointly in models as we have done may give some insight into possible factors explaining the association . Although the das discrimination scale and the doctor discrimination measure had a strong positive association with each other, they were not so collinear as to create problems in the models presented here; when the discrimination terms were included in the models separately, results were similar . As in all telephone surveys, the representativeness of the study sample to latinos in general can be questioned . Wave 2 follow - up was also limited in this study; due to financial constraints there was a short time window for completing callbacks . However, our analysis of the wave 2 sample compared to the rest of the wave 1 showed no significant differences on any demographic characteristic . Our sample size was insufficient to fully dissect the associations among general discrimination, doctor discrimination and quality of care and to identify the subgroups within the nativity groups that were responsible for the overall associations found . The study is cross - sectional, and arguments of causation cannot be made . The mean of general discrimination assessed on the das scale is relatively low; on average, discriminatory experiences are occurring only a few times a year, and only about one in five latinos reported any discrimination from doctors or medical personnel in the past 2 years . However, it may be that attributes of poor quality of care and poor patient - provider communication, such as rushed care, rudeness, or arrogance on the part of doctors or medical staff, are associated in respondents minds with perceived discrimination . The connection between general discrimination not necessarily in a medical setting and quality of care may be even more tenuous . Looking at general discrimination and discrimination in a medical setting jointly in models as we have done may give some insight into possible factors explaining the association . Although the das discrimination scale and the doctor discrimination measure had a strong positive association with each other, they were not so collinear as to create problems in the models presented here; when the discrimination terms were included in the models separately, results were similar . As in all telephone surveys, the representativeness of the study sample to latinos in general can be questioned . Wave 2 follow - up was also limited in this study; due to financial constraints there was a short time window for completing callbacks . However, our analysis of the wave 2 sample compared to the rest of the wave 1 showed no significant differences on any demographic characteristic . Our sample size was insufficient to fully dissect the associations among general discrimination, doctor discrimination and quality of care and to identify the subgroups within the nativity groups that were responsible for the overall associations found . The results from this study give a better understanding of the relationship between perceived discrimination and perceived quality of care among us latinos . For health - care providers and policymakers seeking to have an impact on health - care quality, focusing on discrimination may prove to be a fruitful endeavor . Discrimination toward both highly acculturated english - speaking us - born latinos and lower - acculturated spanish - speaking latinos should be addressed . Future research is needed to investigate how interventions can be tailored so that they address perceived discrimination in health - care experiences for latinos, perhaps with different approaches for the us born than the foreign born . Reducing discrimination may be difficult to achieve, but the results of this study suggest that quality improvement interventions must take discrimination into account.
In the context of neuroimaging, machine learning approaches have been used so far to address diagnostic problems, where patients were classified into different groups based on anatomical or functional data . By contrast, in cognitive studies, the standard framework for functional or anatomical brain mapping was based on mass univariate inference procedures . Recently, a new way of analyzing functional neuroimaging data has emerged [2, 3], and it consists in assessing how well behavioral information or cognitive states can be predicted from brain activation images such as those obtained with functional magnetic resonance imaging (fmri). This approach opens new ways for understanding the mental representation of various perceptual and cognitive parameters, which can be regarded as the study of the corresponding neural code, albeit at a relatively low spatial resolution . The accuracy of the prediction of the behavioral or cognitive target variable, as well as the spatial layout of predictive regions, can provide valuable information about functional brain organization; in short, it helps to decode the brain system . Many different pattern recognition and machine leaning methods have been used to extract information from brain images and compare it to the corresponding target . Among them, linear discriminant analysis (lda) [3, 5], support vector machine (svm) [69], or regularized prediction [10, 11] has been particularly used . The major bottleneck in this kind of analytical framework is that there are far more features than samples, so that the problem is plagued by the curse of dimensionality, leading to overfitting . Dimension reduction can be used to extract relevant information from the data, the standard approach in functional neuroimaging being feature selection (e.g., anova) [3, 6, 11, 12]. However, by performing feature selection and parameter estimation separately, such approach is not optimal . Thus, a popular combined selection / estimation scheme, recursive feature elimination, may be used . However, this approach relies on a specific heuristic, which does not guarantee the optimality of the solution and is particularly costly . By contrast, there is great interest in sparsity - inducing regularizations, which optimize both simultaneously . In this paper, we assume that the code under investigation is about some scalar parameter that characterizes the stimuli, such as a scale / shape parameters but possibly also position, speed (assuming a 1-d space), or cardinality . Thus, we focus on regression problems and defer the generalization to classification to future work . Let us introduce the following predictive linear model: (1)y = xw+b, where y represents the behavioral variable and (w, b) are the parameters to be estimated on a training set . A vector w can be seen as an image; p is the number of features (or voxels), and b is called the intercept . Each row is a p - dimensional sample, that is, an activation map related to the observation . With n p, the estimation of w is ill posed . To cope with the high dimensionality of the data, one can penalize the estimation of w, for example, based on the 2 norm of the weights . Classical regularization schemes have been used in functional neuroimaging, such as the ridge regression, lasso, or elastic net regression . However, these approaches require the amount of penalization to be fixed beforehand and possibly optimized by cross - validation . To deal with the choice of the amount of penalization, one can use the bayesian regression techniques, which include the estimation of regularization parameters in the whole estimation procedure . Standard bayesian regularization schemes are based on the fact that a penalization by weighted 2 norm is equivalent to setting the gaussian priors on the weights w: (2)w~(0,a1), a = diag (1,,p), i[1,,p], i+, where is the gaussian distribution and i the precision of the ith feature . The first one is bayesian ridge regression (brr), which corresponds to the particular case 1 = = m . By regularizing all the features identically, brr is not well suited when only few features are relevant . The second classical scheme is automatic relevance determination (ard), which corresponds to the case i j if i j. the regularization performed by ard is very adaptive, as all the weights are regularized differently . However, by regularizing each feature separately, ard is prone to underfitting when the model contains too many regressors and also suffers from convergence issues . These classical bayesian regularization schemes have been used in fmri inverse inference studies [10, 14, 21]. However, these studies used only sparsity as built - in feature selection and do not consider neuroscientific assumptions for improving the regularization (i.e., within the design of the matrix a). Indeed, due to the intrinsic smoothness of functional neuroimaging data, predictive information is rather encoded in different groups of features sharing similar information . A potentially more adapted approach is the bayesian regression scheme presented in, which regularizes patterns of voxels differently . The weights of the model are defined by w = u, where u is a matrix defined as set of spatial patterns (one pattern by column) and are the parameters of the decomposition of w in the basis defined by u. the regularization is controlled through the covariance of, which is assumed to be diagonal with only m possible different values cov () = exp (1)i + +exp (m)i . The matrices i are diagonal and defined subsets of columns of u sharing similar variance exp (i). Due to its class - based model, this approach is similar to the one proposed in this paper, but the construction of i relies on ad hoc voxel selection steps, so that there is no proof that the solution is correct . A contrario, the proposed approach jointly optimizes, within the same framework, the construction of the pattern of voxels and the regularization parameter of each pattern . In this paper, we detail a model for the bayesian regression in which features are grouped into k different classes that are subject to different regularization penalties . The estimation of the penalty is performed in each class separately, leading to a stable and adaptive regularization . The construction of the group of features and the estimation of the predictive function are performed jointly . This approach, called multiclass sparse bayesian regression (mcbr), is thus an intermediate solution between brr and ard . It requires less parameters to estimate than ard and is far more adaptive than brr . Another asset of the proposed approach in fmri inverse inference is that it creates a clustering of the features and thus yields useful maps for brain mapping . After introducing our model and giving some details on the parameter estimation algorithms (the variational bayes or gibbs sampling procedures), we show that the proposed algorithm yields better accuracy than reference methods, while providing more interpretable models . We first detail the notations of the problem and describe the priors and parameters of the model . We recall the linear model for regression: (3)y = f(x, w, b)=xw+b . We denote by y the targets to be predicted and x the set of activation images related to the presentation of different stimuli . The integer p is the number of voxels and n the number of samples (images). Typically, p ~ 10 to 10 (for a whole volume), while n ~ 10 to 10 . Priors on the noisewe use classical priors for regression, and we model the noise on y as an i.i.d . Gaussian variable: (4)~(0,1in),~(;1,2), where is the precision parameter and stands for the gamma density with two hyperparameters 1, 2: (5)(x;1,2)=21x11exp x2(1). We use classical priors for regression, and we model the noise on y as an i.i.d . Gaussian variable: (4)~(0,1in),~(;1,2), where is the precision parameter and stands for the gamma density with two hyperparameters 1, 2: (5)(x;1,2)=21x11exp x2(1). Priors on the class assignmentin order to combine the sparsity of ard with the stability of brr, we introduce an intermediate representation, in which each feature j belongs to one class among k indexed by a discrete variable zj (z = {z1,, zp}).all the features within a class k {1,, k} share the same precision parameter k, and we use the following prior on z: (6)z~j=1p k=1kkjk, where is kronecker's, defined as (7)jk={0if zjk,1if zj = k.we finally introduce an additional dirichlet prior on: (8)~dir() with a hyperparameter . By updating at each step the probability k of each class, it is possible to prune classes . This model has no spatial constraints and thus is not spatially regularized . In order to combine the sparsity of ard with the stability of brr, we introduce an intermediate representation, in which each feature j belongs to one class among k indexed by a discrete variable zj (z = {z1,, zp}).all the features within a class k {1,, k} share the same precision parameter k, and we use the following prior on z: (6)z~j=1p k=1kkjk, where is kronecker's, defined as (7)jk={0if zjk,1if zj = k . We finally introduce an additional dirichlet prior on: (8)~dir() with a hyperparameter . By updating at each step the probability k of each class, it is possible to prune classes . Priors on the weightsas in ard, we make use of an independent gaussian prior for the weights: (9)w~(0,a1) with diag (a)={z1,,zp}, where zj is the precision parameter of the jth feature, with zj {1,, k}. We introduce the following prior on k: (10)k~(k;1,k,2,k) with hyperparameters 1,k, 2,k ., we make use of an independent gaussian prior for the weights: (9)w~(0,a1) with diag (a)={z1,,zp}, where zj is the precision parameter of the jth feature, with zj {1,, k}. We introduce the following prior on k: (10)k~(k;1,k,2,k) with hyperparameters 1,k, 2,k . The link between the proposed mcbr model and the other regularization methods, bayesian ridge regression and automatic relevance determination, is obvious . With k = 1, = = zp, we retrieve the brr model, with k = p, that is, zi zj if i j, and assigning each feature to a singleton class (i.e., zj = j), we retrieve the ard model . Moreover, the proposed approach is related to the one developed in . In this paper, the authors proposed, for the distribution of weights of the features, a binary mixture of gaussians with small and large precisions . Our work can be viewed as a generalization of this model to a number of classes k 2 . For models with latent variables, such as mcbr for instance in a mixture of components, a singularity is a component with one single sample and thus zero variance . In such cases, maximizing the log likelihood yields flawed solutions, and one can use the posterior distribution of the latent variables p(z \| x, y) for this maximization . However, the posterior distribution of the latent variables given the data does not have a closed - form expression, and some specific estimation methods, such as variational bayes or gibbs sampling, have to be used . We first estimate the model by the variational bayes, and the resulting algorithm is thus called vb - mcbr . We also detail an algorithm, called gibbs - mcbr, based on a gibbs sampling procedure . The variational bayes (or vb) approach provides an approximation q() of p(\| y), where q() is taken in a given family of distributions and = [w,,, z,]. Additionally, the variational bayes approach often uses the following mean field approximation, which allows the factorization between the approximate distribution of the latent variables and the approximate distributions of the parameters: (11)q()=q(w)q()q()q(z)q(). We introduce the kullback - leibler divergence (q()) that measures the similarity between the true posterior p(\| y) and the variational approximation q(). (y) as (12)log p (y)=(q())+(q()) with (13)(q())=dq()log p(y,)q(),(q())= dq()log q()p(y), where (q()) is called free energy and can be seen as the measure of the quality of the model . As (q()) 0, the free energy is a lower bound on log p (y) with equality if and only if q() = p(\| y). So, inferring the density q() of the parameters corresponds to maximizing with respect to the free distribution q(). In practice, the vb approach consists in maximizing the free energy iteratively with respect to the approximate distribution q(z) of the latent variables and with respect to the approximate distributions of the parameters of the model q(w), q(), q(), and q(). The variational distributions and the pseudocode of the vb - mcbr algorithm are provided in appendix a. this algorithm maximizes the free energy . In practice, iterations are performed until convergence to a local maximum of . With an ard prior (i.e., k = p and fixing zj = j), we retrieve the same formulas as the ones found for variational ard . The resulting algorithm is called gibbs - mcbr; the pseudocode of the algorithm and the candidate distributions are provided in appendix b. the gibbs sampling algorithm is used for generating a sequence of samples from the joint distribution to approximate marginal distributions . The main idea is to use conditional distributions that should be known and possibly easy to sample from, instead of directly computing the marginals from the joint law by integration (the joint law may not be known or hard to sample from). The sampling is done iteratively among the different parameters, and the final estimation of parameters is obtained by averaging the values of the different parameters across the different iterations (one may not consider the first iterations, this is called the burn in). Our model needs few hyperparameters; we choose here to use slightly informative and class - specific hyperparameters in order to reflect a wide range of possible behaviors for the weight distribution . This choice of priors is equivalent to setting heavy - tailed centered student's t - distributions with variance at different scales, as priors on the weight parameters . We set k = 9, with weakly informative priors 1,k = 10, k [1,, k] and 2,k = 10, k [1,, k]. Moreover, we set 1 = 2 = 1 . Starting with a given number of classes and letting the model automatically prune the classes can be seen as a means of avoiding costly model selection procedures . The choice of class - specific priors is also useful to avoid label switching issues and thus speeds up convergence . Crucially, the priors used here can be used in any regression problem, provided that the target data is approximately scaled to the range of values used in our experiments . In that sense, the present choice of priors can be considered as universal . We also randomly initialize q(z) for vb - mcbr (or z for gibbs - mcbr). Our method is evaluated with a cross - validation procedure that splits the available data into training and validation sets . In the following, (x, y) are a learning set (x, y) is a test set, and y^t = f(xtw^) refers to the predicted target, where w^ is estimated from the training set . The performance of the different models is evaluated using, the ratio of explained variance: (14)(yt, y^t)=var(yt)var(yty^t)var(yt). This is the amount of variability in the response that can be explained by the model (perfect prediction yields = 1, while <0 if prediction is worse than chance). In our experiments, the proposed algorithms are compared to different state - of - the - art regularization methods . Elastic net regression, which requires setting two parameters 1 and 2 . In our analyzes, a cross - validation procedure within the training set is used to optimize these parameters . Here, we use 1{0.2,0.1,0.05,0.01}, where =\|\|xty\|\|, and 2 {0.1,0.5,1 ., 10 . Support vector regression (svr) with a linear kernel, which is the reference method in neuroimaging . The c parameter is optimized by cross - validation in the range of 10 to 10 in multiplicative steps of 10 . Bayesian ridge regression (brr), which is equivalent to mcbr with k = 1 and 1 = 2 = 1 = 2 = 10, that is, weakly informative priors . Automatic relevance determination (ard), which is equivalent to mcbr with k = p and 1 = 2 = 1 = 2 = 10, that is, weakly informative priors . All these methods are used after an anova - based feature selection as this maximizes their performance . Indeed, irrelevant features and redundant information the optimal number of voxels is selected within the range {50,100,250,500}, using a nested cross - validation within the training set . We do not directly select a threshold on p value or cluster size, but rather a predefined number of features . The estimation of the parameters of the learning function is also performed using a nested cross - validation within the training set, to ensure a correct validation and an unbiased comparison of the methods . The implementation of elastic net is based on coordinate descent, while svr is based on libsvm . Methods are used from python via the scikit - learn open source package . For vb - mcbr and gibbs - mcbr, in order to avoid a costly internal cross - validation, the number of iterations used is fixed to 5000 (burn in of 4000 iterations) for gibbs - mcbr and 500 for vb - mcbr . Preliminary results on both simulated and real data showed that these values are sufficient enough for an accurate inference of the model . As explained previously, we set k = 9, with weakly informative priors 1,k = 10, k [1,, k] and 2,k = 10, k [1,, k]. Moreover, we set 1 = 2 = 1, and we randomly initialize q(z) for vb - mcbr (or z for gibbs - mcbr). We now evaluate and illustrate mcbr on two different sets of simulated data . We first test mcbr on a simulated data set, designed for the study of ill - posed regression problem, that is, n p. data are simulated as follows: (15)x~(0,1) with ~(0,1),y=2(x1+x2x3x4)+0.5(x5+x6x7x8)+. We have p = 200 features, n = 50 images for the training set, and n = 50 images for the test set . We compare mcbr to the reference methods, but we do not use feature selection, as the number of features is not very high . We average the results of 15 different trials, and the average explained variance is shown in table 1 . Gibbs - mcbr outperforms the other approaches, yielding higher prediction accuracy than the reference elastic net and ard methods . Vb - mcbr falls into the local maximum of and does not yield an accurate prediction . Indeed, it cannot finely adapt the weights of the relevant features, as these features are regularized similarly as the irrelevant ones . Svr has also low accuracy, due to the fact that we do not perform any feature selection . Thus, svr suffers from the curse of dimensionality, unlike other methods such as ard or elastic net, which performs feature selection and model estimation jointly . In figure 2, we represent the probability density function of the distributions of the weights obtained with brr (a), gibbs - mcbr (b), and ard (c). With brr the gibbs - mcbr algorithm creates a multimodal distribution, lots of weights being highly regularized (pink distributions), and informative features are allowed to have higher weights (blue distributions). With mcbr, weights are clustered into different groups, depending on their predictive power, which is interesting in application such as fmri inverse inference, as it can yield more interpretable models . Indeed, the class to the features with higher weights ({x1, x2, x3, x4}) belong which is small (average size of 6 features) but has a high purity (percentage of relevant features in the class) of 74% . We now look at the values of w1 and w2 for the different steps of the two algorithms (see figure 3). We can see that vb - mcbr (b) quickly falls into a local maximum, while gibbs - mcbr (a) visits the space and reaches the region of the correct set of parameters (red dot). The simulated data set x consists of n = 100 images (size 12 12 12 voxels) with a set of four square regions of interest (roi) (size 2 2 2). We call the support of the roi (i.e., the 32 resulting voxels of interest). Each of the four rois has a fixed weight in {0.5,0.5, 0.5,0.5}. We call wi, j, k the weight of the (i, j, k) voxel . The resulting images are smoothed with a gaussian kernel with a standard deviation of 2 voxels, to mimic the correlation structure observed in real fmri data . To simulate the spatial variability between images (intersubject variability, movement artifacts in intrasubject variability), we define a new support of the rois, called such that, for each image lth, 50% (randomly chosen) of the weights w are set to zero . Thus, we have . We simulate the target y for the lth image as (16)yl=(i, j, k)wi, j, kxi, j, k, l+l with the signal in the (i, j, k) voxel of the lth image simulated as (17)xi, j, k, l~(0,1), and l ~ (0,) is a gaussian noise with standard deviation> 0 . We choose in order to have a signal - to - noise ratio of 5 db . The resulting images of weights are given in figure 4, with the true weights (a) and resulting anova f - scores (b). The reference methods can detect the truly informative regions (rois), but elastic net (f) and ard (h) retrieve only part of the support of the weights . Moreover, elastic net yields an overly sparse solution . Brr (g) also retrieves the rois but does not yield a sparse solution, as all the features are regularized in the same way . We note that the weights in the feature space estimated by svr (e) are nonzero everywhere and do not outline the support of the ground truth . Vb - mcbr (c) converges to a local maximum similar to the solution found by brr (g); that is, it creates only one nonempty class, and thus regularizes all the features similarly . We can thus clearly see that, in this model, the variational bayes approach is very sensitive to the initialization and can fall into nonoptimal local maxima, for very sparse support of the weights . Finally, gibbs - mcbr (d) retrieves most of the true support of the weights by performing an adapted regularization . In this section, we assess the performance of mcbr in an experiment on the mental representation of object size, where the aim is to predict the size of an object seen by the subject during the experiment, in both intrasubject and intersubject cases . The size (or scale parameter) of the object will be the target variable y. we apply the different methods on a real fmri dataset related to an experiment studying the representation of objects, on ten subjects, as detailed in . During this experiment, ten healthy volunteers viewed objects of 4 shapes in 3 different sizes (yielding 12 different experimental conditions), with 4 repetitions of each stimulus in each of the 6 sessions . We pooled data from the 4 repetitions, resulting in a total of n = 72 images by subject (one image of each stimulus by session). Functional images were acquired on a 3-t mr system with an eight - channel head coil (siemens trio, erlangen, germany) as t2 * -weighted echo - planar image (epi) volumes . Twenty transverse slices were obtained with a repetition time of 2 s (echo time: 30 ms; flip angle: 70; 2 2 2-mm voxels; 0.5 mm gap). Realignment, normalization to mni space, and general linear model (glm) fit were performed with the spm5 software (http://www.fil.ion.ucl.ac.uk/spm/software/spm5/). The normalization is the conventional method of spm (implying affine and nonlinear transformations) and not the one using unified segmentation . The normalization parameters are estimated on the basis of a whole - head epi acquired in addition and are then applied to the partial epi volumes . The effect of each of the 12 stimuli convolved with a standard hemodynamic response function was modeled separately, while accounting for serial autocorrelation with an ar(1) model and removing low - frequency drift terms using a high - pass filter with a cutoff of 128 s. the glm is fitted separately in each session for each subject, and we used in the present work the resulting session - wise parameter estimate images (the -maps are used as rows of x). The four different shapes of objects were pooled across for each one of the three sizes, and we are interested in finding discriminative information on sizes . This reduces to a regression problem, in which our goal is to predict a simple scalar factor (size of an object). All the analyzes are performed without any prior selection of regions of interest and use the whole acquired volume . The dimensions of the real data set for one subject are p ~ 7 10 and n = 72 (divided in 3 different sizes, 24 images per size). We evaluate the performance of the method by a leave - one - condition - out cross - validation (i.e., leave-6-image - out), and doing so the glm is performed separately for the training and test sets . The parameters of the reference methods are optimized with a nested leave - one - condition - out cross - validation within the training set, in the ranges given before . The dimensions of the real data set for one subject are p ~ 7 10 and n = 72 (divided in 3 different sizes, 24 images per size). We evaluate the performance of the method by a leave - one - condition - out cross - validation (i.e., leave-6-image - out), and doing so the glm is performed separately for the training and test sets . The parameters of the reference methods are optimized with a nested leave - one - condition - out cross - validation within the training set, in the ranges given before . The intersubject analysis relies on subject - specific fixed - effect activations that is, for each condition, the 6 activation maps corresponding to the 6 sessions are averaged together . This yields a total of 12 images per subject, one for each experimental condition . The dimensions of the real data set are p ~ 7 10 and n = 120 (divided into 3 different sizes). We evaluate the performance of the method by cross - validation (leave - one - subject - out). The parameters of the reference methods are optimized with a nested leave - one - subject - out cross - validation within the training set, in the ranges given before . Additionally, we perform an intersubject regression analysis on the sizes . The intersubject analysis relies on subject - specific fixed - effect activations that is, for each condition, the 6 activation maps corresponding to the 6 sessions are averaged together . This yields a total of 12 images per subject, one for each experimental condition . The dimensions of the real data set are p ~ 7 10 and n = 120 (divided into 3 different sizes). We evaluate the performance of the method by cross - validation (leave - one - subject - out). The parameters of the reference methods are optimized with a nested leave - one - subject - out cross - validation within the training set, in the ranges given before . Compared to the results on simulated data, vb - mcbr still falls in a local maximum similar to the bayesian ridge regression which performs well in this experiment . Moreover, both gibbs - mcbr and vb - mcbr are more stable than the reference methods . Compared to the results on simulated data, vb - mcbr still falls in a local maximum similar to the bayesian ridge regression which performs well in this experiment . Moreover, both gibbs - mcbr and vb - mcbr are more stable than the reference methods . As in the intrasubject analysis, both mcbr approaches outperform the reference methods, svr, brr, and ard . However, the prediction accuracy is similar to that of elastic net . In this case, gibbs - mcbr performs slightly better than vb - mcbr, but the difference is not significant.one major asset of mcbr (and more particularly gibbs - mcbr, as vb - mcbr often falls into a one - class local maximum) is that it creates a clustering of the features, based on the relevance of the features in the predictive model . This clustering can be accessed using the variable z, which is implied in the regularization performed on the different features . In figure 5, we give the histogram of the weights of gibbs - mcbr for the intersubject analysis . We keep the weights and the values of z of the last iteration; the different classes are represented as dots of different colors and are superimposed on the histogram . We can notice than the pink distribution represented at the bottom of the histogram corresponds to relevant features . This cluster is very small (19 voxels), compared to the two blue classes represented at the top of the histogram that contain many voxels (746 voxels) which are highly regularized, as they are noninformative.the maps of weights found by the different methods are detailed in figure 6 . The methods are used combined with an anova - based univariate feature selection (2500 voxels selected, in order to have a good support of the weights). As elastic net, gibbs - mcbr yields a sparse solution but extracts a few more voxels . The map found by elastic net is not easy to interpret, with very few informative voxels scattered in the whole occipital cortex . The map found by svr is not sparse in the feature space and is thus difficult to interpret, as the spatial layout of the neural code is not clearly extracted . Vb - mcbr does not yield a sparse map either, all the features having nonnull weights the results obtained with the different methods are given in table 3 . As in the intrasubject analysis, both mcbr approaches outperform the reference methods, svr, brr, and ard . However, the prediction accuracy is similar to that of elastic net . In this case, gibbs - mcbr performs slightly better than vb - mcbr, but the difference is not significant . One major asset of mcbr (and more particularly gibbs - mcbr, as vb - mcbr often falls into a one - class local maximum) is that it creates a clustering of the features, based on the relevance of the features in the predictive model . This clustering can be accessed using the variable z, which is implied in the regularization performed on the different features . In figure 5, we give the histogram of the weights of gibbs - mcbr for the intersubject analysis . We keep the weights and the values of z of the last iteration; the different classes are represented as dots of different colors and are superimposed on the histogram . We can notice than the pink distribution represented at the bottom of the histogram corresponds to relevant features . This cluster is very small (19 voxels), compared to the two blue classes represented at the top of the histogram that contain many voxels (746 voxels) which are highly regularized, as they are noninformative . The maps of weights found by the different methods are detailed in figure 6 . The methods are used combined with an anova - based univariate feature selection (2500 voxels selected, in order to have a good support of the weights). As elastic net, gibbs - mcbr yields a sparse solution but extracts a few more voxels . The map found by elastic net is not easy to interpret, with very few informative voxels scattered in the whole occipital cortex . The map found by svr is not sparse in the feature space and is thus difficult to interpret, as the spatial layout of the neural code is not clearly extracted . Vb - mcbr does not yield a sparse map either, all the features having nonnull weights it is well known that in high - dimensional problems, regularization of feature loadings significantly increases the generalization ability of the predictive model . However, this regularization has to be adapted to each particular dataset . In place of costly cross - validation procedures, we cast regularization in a bayesian framework and treat the regularization weights as hyperparameters . The proposed approach yields an adaptive and efficient regularization and can be seen as a compromise between a global regularization (bayesian ridge regression) that does not take into account the sparse or focal distribution of the information and automatic relevance determination . Additionally, mcbr creates a clustering of the features based on their relevance and thus explicitly extracts groups of informative features . Ard is subject to an underfitting in the hyperparameter space that corresponds to an underfitting in model selection (i.e., on the features to be pruned). Indeed, as ard is estimated by maximizing evidence, models with less selected features are preferred, as the integration is done on less dimensions, and thus evidence is higher . A contrario, mcbr requires far less hyperparameter (2 k, with k p) and suffers less from this issue, as the sparsity of the model is defined by groups . Moreover, a full bayesian framework for estimating ard requires to set some priors on the hyperparameters (e.g., 1 and 2), and it may be sensitive to specific choice of these hyperparameters . A solution is to use an internal cross - validation for optimizing these parameters, but this approach can be computationally expensive . In the case of mcbr, the distributions of the hyperparameters are bound to a class and not to each feature . Indeed, the choice of good hyperparameters for the features is dealt with at the class level . On simulated data, our approach performs better than other classical methods such as svr, brr, ard, and elastic net and yields a more stable prediction accuracy . Moreover, by adapting the regularization to different groups of voxels, mcbr retrieves the true support of the weights and recovers a sparse solution . Results on real data show that mcbr yields more accurate predictions than other regularization methods . As it yields less sparse solution than elastic net, it gives access to more plausible loading maps which are necessary for understanding the spatial organization of brain activity, that is, retrieving the spatial layout of the neural coding . On real fmri data, the explicit clustering of gibbs - mcbr is also an interesting aspect of the model, as it can extract few groups of relevant features from many voxels . In some experiments, the variational bayes algorithm yields less accurate predictions than the gibbs sampling approach, which can be explained by the difficulty of initializing the different variables (especially z) when the support of the weight is overly sparse . Moreover, the vb - mcbr algorithm relies on a variational bayes approach, which may not be optimal, due to strong approximations in model inference . A contrario gibbs - mcbr is more time consuming but yields a better model inference . Finally, the variability in the results may be explained by the difficulty to estimate the model (optimality is not ensured). The question of model selection (i.e., the number of classes k) has not been addressed in this paper . One can use the free energy in order to select the best model, but due to the instability of vb - mcbr, this approach does not seem promising . A more interesting method is the one detailed in, which can be used with the gibbs sampling algorithm . Here, model selection is performed implicitly by emptying classes that do not fit the data well . In that respect, the choice of heterogeneous priors for different classes is crucial: replacing our priors with class - independent priors (i.e., 1,k = 10, k [1,, k]) in the intersubject analysis on size prediction leads gibbs - mcbr to a local maximum similar to vb - mcbr . Finally, this model is not restricted to the bayesian regularization and can be used for classification, within a probit or logit model [35, 36]. The proposed model may thus be used for diagnosis in medical imaging, for the prediction of both continuous or discrete variables . In this paper, we have proposed a model for adaptive regression, called mcbr . The proposed method integrates, in the same bayesian framework, brr and ard and performs a different regularization for relevant and irrelevant features . It can tune the regularization to the possible different level of sparsity encountered in fmri data analysis, and it yields interpretable information for fmri inverse inference, namely, the z variable (latent class variable). Experiments on both simulated and real data show that our approach is well suited for neuroimaging, as it yields accurate and stable predictions compared to the state - of - the - art methods . The variational bayes approach yields the following variational distributions: q(w) ~ (w \|,) with (a.1)a=diag (l1,,lp) withlj=k=1kq(zj = k)l1,kl2,k j{1,,p},(a.2)=(a1a2xtx+a)1,(a.3)=a1a2xty; q(k) ~ (l1,k, l2,k) with (a.4)l1,k=1,k+12j=1pq(zj = k),(a.5)l2,k=2,k+12j=1p(jj2+jj)q(zj = k); q() ~ (a1, a2) with (a.6)a1=1+n2,(a.7)a2=2 + 12(yx)t(yx)+12tr (xtx); q(zj = k) ~ exp with (a.8)jk=12(j2+jj)l1,kl2,k+ln (k)+12((l1,k)log (l2,k)),(a.9)k = exp {(dk)(k=1k = kdk)},(a.10)dk=k+j=1pq(zj = k), where is the digamma function (x) = (x)/(x). The vb - mcbr algorithm is provided in pseudo - code in algorithm 1 . With = [w,,, z,], we have the following candidate distributions (i.e., the distributions used for the sampling of the different parameters): p(w \| {w}) (w \|,) with (b.1)=(xtx+a)1 with a = diag (z1,,zp),(b.2)=xty; p(\| {}) k=1(k \| l1,k, l2,k) with (b.3)l1,k=1,k+12j=1p(zj = k),(b.4)l2,k=2,k+12j=1p(zj = k)wj2; p(\| {}) (a1, a2) with (b.5)a1=1+n2,(b.6)a2=2 + 12(yx)t(yx); p(zj \| {z}) mult(exp j,1,, exp j, k) with (b.7)jk=12wj2k+ln (k)+12log k; p(k \| {}) dir(dk) with (b.8)dk=k+j=1p(zj = k). The algorithm is provided in pseudocode in algorithm 2.
An overarching societal goal is to understand animal and human intelligence and translate that knowledge into technology for prosthetic, assistive, and decision support applications . Traditional research in this field considers the brain to be a specially adapted information - processing system, which can be modeled using mathematical optimization or production rule artificial intelligence systems . Despite many decades of investment in such learning and classification systems one proposed remedy comes from research in social robotics, which attempts to augment the understanding of intelligent behavior by capturing the important dynamics of cognition using robotic interaction with humans (dautenhahn, 2007; scheutz et al ., 2007). However, almost all social robotics systems to date continue to incorporate some mixture of existing machine learning and production rule cognitive systems . For this reason, investigators are now asking whether critical neural dynamics have indeed been left out of the traditional models . Fortunately, the past two decades of neuroscience research has yielded an abundance of quantitative parameters that characterize the brain's interdependent electrophysiological (markram et al ., 1997; schindler et al ., 2006), genomic (toledo - rodriguez et al ., 2004), proteomic (toledo - rodriguez et al ., 2005), researchers now have access to over a hundred neuroscience databases (society for neuroscience, 2007), including automated warehousing collections such as the allen brain atlas (allen institute, 2007) and a new data - sharing website sponsored jointly by the u.s . National science foundation and national institutes for health, called the collaborative research in computational neuroscience (teeters et al ., 2008). A previous limitation to the use of biologically realistic models has been the computational overhead . Fortunately, the past decade has witnessed an order of magnitude increase in computation power of individual computers and cluster configurations with a tremendous drop in cost for system components . A few groups have already reported simulations on the order of one million simplified neural elements (izhikevich et al ., 2004; ripplinger et al ., growth in computational technology has also encouraged nontechnical persons to participate across the internet using avatars in complex virtual reality games and social networking communities (e.g., second life), which may include not only other human participants but also programmed robots . Thus, taken together, advances in computer technology and interactive 3-d software have set the stage not only to facilitate supercomputer modeling of realistic brains, but also to promote acceptance by humans that virtual reality projections may be capable of meaningful cognitive interaction . Developing tenable models to capture the essence of natural intelligence for real - time application requires that we discriminate features underlying information processing and intrinsic motivation from those reflecting biological constraints (such as maintaining structural integrity and transporting metabolic products). Furthermore, despite the large and increasing number of physiological parameters provided by experimental inquiry, most of the data relates either to the very small scale of individual or small groups of neurons (e.g., intracellular, 2-photon, or unit recordings at discrete recording sites), or at the other extreme, the joint effect of thousands or millions of neurons over millimeter (optical imaging) or centimeter fields (fmri and pet). Thus the architecture and response patterns at the middle scale, or mesocircuit, remain largely uncharacterized, requiring that the brain modeler proposes and systematically tests plausible connection patterns and learning dynamics . Another challenge in designing neuromorphic systems is that they must in some way be driven intrinsically by a motivational influence such that the dynamics that subserve information processing are themselves affected by a drive to accomplish the tasks (with neural learning that reinforces successful behavioral adaptation) (oudeyer and kaplan, 2007; oudeyer et al ., 2007; samejima and doya, 2007; schweighofer et al ., 2007). The motivational system must capture the aboutness of its own relationship to other behaving entities (and vice versa) in its environment (i.e., intentionality). Considered together, physiological responsiveness to intrinsic motivation with intentionality should reflect behaviors consistent with emotional drive rather than by rules or objectives specified under the traditional information - processing paradigm . This suggests that intelligence has evolved most directly as a way to better serve emotional drive (rather than in spite of it). We therefore hypothesize that the development of truly intelligent systems cannot occur outside the real - time, emotional interaction of humans with an intentionality - capable neuromorphic system . This does not exclude the possibility that intelligent systems, once refined, could ultimately be cloned at a point in development where they are ready to learn advanced tasks . Hence, to grow intelligent systems we must start with minimalist brain architectures that are capable of being driven by intrinsic motivation and intentionality in scenarios requiring intelligent behavior in a real - world context . One approach to growing human - like intelligence is to recapitulate the way in which children develop cognitive functions over the first several years of social experience . In testing our hypothesis, it would be relevant not only to grow such intelligent systems but also to comprehend, at each step, the differential changes in architecture giving rise to novel and intelligent cognition . To address these objectives, in a recent publication, goodman et al . (2007) proposed a hybridization of neuromorphic brain modeling validation using virtually projected robots interacting with human actors, which we call virtual neurorobotics (vnr). Our proposed definition, open to future collaborative revision, is defined in table 1 . The definition expands upon the definition of neurorobotics, which alone would imply a biologically representative robotic control system (criterion 3), and to test our hypothesis, we additionally require that the robotic system demonstrate sufficient physical and cognitive realism that the human accepts the robot as deserving of emotional reward (criteria 1 and 4) in a real - time interactive loop (criterion 2), with a cognitive architecture potentially extensible to larger cognitive scale (criterion 5). The robot is sufficiently embodied for the human to tentatively accept the robot as a social, emotional partner the human - robot interaction loop operates in real time, with no pre - specified parcellation into receptive and responsive time windows the cognitive control is a neuromorphic brain emulation incorporating realistic neuronal dynamics with time constants that reflect synaptic activation and learning, established membrane and circuitry properties the neuromorphic architecture can potentially provide circuitry underlying intrinsic motivation and intentionality, using emotional rather than rule - based learning & reinforcement the neuromorphic architecture is expandable to progressively larger scale and complexity to support brain model development and validation the components of the real - time loop are further delineated in table 2 . Here, we emphasize that the interaction between human and virtual robot be unscripted, of a spontaneous, action - reaction nature . That is, there is no segmentation of behavioral time into periods wherein the robot is receptive, waiting, analyzing, and/or taking action . The action - reaction requirement implies also that the system operate nearly in real time . In our experiences, human actors readily accept delays of up to 3 or 4 s without becoming frustrated about unrealistic robotic response and loosing cognitive and emotional linkage . Of course, the range of behaviors is indirectly constrained by the sensory and motor capabilities of the robot, the types of behaviors exhibited by the human, and the context (e.g., background activity). Temporal sequencing (timing)computation and communication provide nearly real - time robot response, to maintain cognitive and emotional linkagetime is not segmented a priori for robot or actor receptiveness or reaction computation and communication provide nearly real - time robot response, to maintain cognitive and emotional linkage time is not segmented a priori for robot or actor receptiveness or reaction environment (scene)realistic contents, including sights, sounds, and objectsmay be affected by the actions of robot or actormay include other robots or multiple actors realistic contents, including sights, sounds, and objects may be affected by the actions of robot or actor may include other robots or multiple actors live participant (actor)human, child or adult, depending on type of target intelligencewillingness to accept scene as realistic situationwillingness to assume robot has ability to perceive and respond meaningfullywillingness to attribute intentionality to robot human, child or adult, depending on type of target intelligence willingness to accept scene as realistic situation willingness to assume robot has ability to perceive and respond meaningfully willingness to attribute intentionality to robot neuromorphic system (robot)central nervous subsystem (brain) may include neocortex, hippocampus, basal ganglia, and/or other limbic regions relating to attention, reward, and fearcomputational architecture must incorporate biologically plausible learning algorithms and support for expansion to progressively larger scale and complexityrepertoire of sensors, expressions, and behaviors are commensurate with its physical and brain complexity central nervous subsystem (brain) may include neocortex, hippocampus, basal ganglia, and/or other limbic regions relating to attention, reward, and fear computational architecture must incorporate biologically plausible learning algorithms and support for expansion to progressively larger scale and complexity repertoire of sensors, expressions, and behaviors are commensurate with its physical and brain complexity to - date there is a paucity of literature meeting our criteria (see related work, below). Thus we focus here on our own research (goodman et al ., 2007) as an example of the vnr principles . In that work, we chose an instinctual friend vs. foe response wherein a resting dog responds to movement in its visual field with either (1) a cautious growl while remaining in a lying position, (2) threatening bark while sitting up, or (3) happy breathing and tail - wagging while fully standing . A human actor was told that he / she is visiting a home with a dog unknown to him / her . As shown in figure 1, a robotic dog was projected in pseudo-3d onto the forward screen, with external sensors that enable its simulated brain to see and respond to the actor's movements, in the context of a background scene projected onto the rear screen (for this demonstration, we used a static image of a suburban neighborhood). The robot's eyes (a tracking pan - tilt - zoom camera) and ears (monaural or spaced stereo microphones) capture the actor's movements and voice in the context of the background scene, which is projected independently (and may contain moving elements, including other animals or actors). The brainstem is a supercomputer running threads that synchronously (1) capture and preprocess video images, sound, and touch, (2) convert preprocessed sensory images into probabilities of spiking for each primary neocortical region, (3) upload spike probability vectors to the brain simulator, (4) then from the brain simulator accept motor neuron region output spike density vectors and trigger corresponding dominant motor sequences (e.g., for the virtual dog robot: sitting, lying, barking, walking) via the robotic simulator program (webots / urbi), which makes the corresponding changes in behavior of the projected robot (and incorporates internal sensation such as proprioception and balance). The brain simulator is a neuromorphic modeling program running on a supercomputer, executing a pre - specified spiking brain architecture, which can adapt as a result of learning (using reward stimuli offered by the actor's voice or stroking of the touch pad). Based on successful performance, researchers iteratively plug in alternative or more complex brain architectures . A proposed enhancement would be to couple live in vivo or in vitro neural tissue (brain slice) to the brain simulation using multielectrode arrays and optical imaging, in order to continuously calibrate and constrain synthetic brain dynamics . The simple neuromorphic brain consisted of 64 single - compartment neurons divided into four columns representing pre - motor regions (precursors to coordinated behavioral sequences), each connected to one of the visual field preferences based on gabor filter configurations . According to the probability vector received from brainstem, ncs injected short (1 ms) step current (3 na) pulses sufficient to reach the threshold of 50 mv and the actor in this scenario was told in advance that moving vertically - oriented objects (including body parts) will pose a threat to the robot, whereas moving horizontally - oriented objects will be perceived as friendly gestures; the actor was free to choose any sequence of movements in response to the perceived intent of the robot . Robot behavioral sequences are triggered when the neuromorphic brain output to brainstem has 50 ms of consistent spiking in one pre - motor region compared with another . Periods without domination of one pre - motor region over another trigger the robot to lie down and growl . In cell rasters, each row represents the timing of action potentials (spikes) of a single neuron; darker gray markers indicate clustered bursts of spikes . Figure 2 shows pre - motor action potential spike rasters from a typical 10-s vnr interaction with a human actor . Spike rasters from a 10-s behavior scenario indicating timing of actor (upper row) and robot (lower row) events . Social embeddedness is a key characteristic of the proposed vnr approach, with an emphasis similar to that received initially in the stepwise, ontological development of robotic cognition (breazeal and scassellati, 2000; brooks et al ., 1998) and more recently in epigenetic robotics research focused on the interaction between cognitive and perceptual brain systems (lungarella and berthouze, 2002; schlesinger, 2003). In order to map behavior to robotic cognition, almost all of these models rely on combinations of psychological production rules, fitness functions, and machine learning algorithms . Notably, this includes models aimed at capturing neuronal epiphenomena such as mirror neuronal activity (triesch et al ., 2007). First, we focus on understanding brain physiology at the mesocircuit level, relying on social - emotional robotics to reduce the multitude of potential architectures that could bridge the measurements at the cellular level (e.g., patch clamp and unit recordings) with those at the scales of millions of cells (e.g., optical and fmr imaging). Second, because the stipulation of neuromorphic architecture excludes the use of production rules or hierarchical algorithms as psychological models, any assumptions on motivation, intentionality and behavioral triggering must emerge from the tissue models themselves, and learning from behavioral reinforcement must manifest as synaptic change . Third, since realistic social interaction requires temporal coherence between the simulated robotic brain and that of the human actor, the simulation must incorporate the actual distribution of physiological time constants that characterize membranes, channels, and synapses . Due to the distinguishing characteristics of the proposed vnr approach some groups have reported success in navigational tasks using neuromorphic architectures (banquet et al . 2005; cuperlier et al ., 2005; krichmar et al ., 2005; notable endeavors that share similarities with our work include identification of challenges and opportunities in robot - mediated neurorehabilitation (harwin et al ., 2006), development of prototypes that combine robotics and virtual reality to assist in the rehabilitation of brain - injured patients and support motor control research (patton et al ., 2006), and generation of artificial brains for virtual robots using a new paradigm based on the epigenetic approach (pasquier, 2004, 2005). The rationale for the virtual paradigm in vnr is rooted fundamentally on engineering and human - computer interface considerations, and is similar to that put forward by krichmar and edelman (2005) for robotic instantiation of brain - based devices . Certainly, a closed - loop system could incorporate either real or virtual robots . In our vnr framework, however, we emphasize virtuality for the following reasons: (1) the human actor must find the robotic behavior believable; it is our impression that refined neurorobotic avatars are more readily accepted (perhaps due to the popularity of online virtual reality networking) than clumsy, unreliable physical robotic prototypes; (2) as investigators design and grow more complex neuromorphic brains, robotic behaviors will require additional sensory, motoric, and emotional sophistication, which in turn may entail major changes in the robot's body parts and dimensions, and degrees of freedom of joints and face all of which can be accelerated using software (often in just hours) without the delays and costs of added hardware and its engineering; and, (3) at stages of neurorobotic development at which it would be important to demonstrate the functionality of a physical robot, the software api can be compiled and transferred to a prototype of the hardware robotic system (provided that vnr simulator used a realistic control api). The use of human actors in the vnr approach might be seen as an obstacle in terms of time, resources, and variability . However, there is no other gold standard for realistic, spontaneous, emotionally intelligent interaction . Moreover, it is human - level cognition that we explore and seek to elucidate in our modeling and applications . In addition, the parameters for neuronal membranes, channels, and synapses are given as time constants on the order of milliseconds to seconds, as co - optimized by evolution . This means that, for example, a system that emulates connected neurons but operates at the temporal scale of microseconds cannot interact with the slower responses of humans . Therefore, both the joint distribution of known biological time constants and the need for emotionally intelligent responses require the use of a closed - loop interaction of the brain prototype with an actor . As an alternative, one might consider using animals in place of humans; however, animals rely on many subtle biological sensory cues such as smell, so will readily accept neither embodied nor virtual robots as socially interactive partners . For example, within the webots / urbi environment we are currently developing a social - emotional humanoid robot with functional capabilities motivated by the mds (mobile, dexterous, social) robot under development by the personal robotics group of the mit media lab (http://robotic.media.mit.edu). Our robot will incorporate language understanding and production using corresponding neocortical models based on praise and curiosity . We also plan to calibrate and constrain synthetic brain dynamics by coupling live in vitro (acute slice or sustained culture) or in vivo neural recordings to the brain simulation using multi - electrode arrays and optical stimulation and imaging . First, neuroscience research is expected to directly benefit from vnr in terms of development and validation of new, expandable brain models and architectures as well as study and exploration of various brain disorders and injuries, including strokes and genetic disorders . Second, faster progress in a variety of medical applications areas will likely be enabled by vnr - based research, primary in terms of advancements in neuroprosthetics and new solutions for brain - related assistive technologies . Third, a diversity of other application areas traditionally propelled by developments in artificial intelligence could take advantage of vnr method and tools . These include, but are not limited to, decision - making support driven by human - like behavior and motivation, enhanced robotics - centered navigation and security, and better understanding in the fields of neural development, neurophysiology, and neuropathology . The authors declare that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.
Computers now play an increasingly important role in our daily lives, and their use is associated with lower back pain . The sacroiliac joint (sij) is a widely described source of low back pain . Therapeutic approaches to relieve this pain include the application of a pelvic compression belt (pcb)1 . Researchers have suggested that functional exercises conducted using a pcb have a beneficial effect associated with muscle strengthening2 . Pcb are effective for stabilizing pelvic articulation and enable exercises that address coordination and stabilization3 . Furthermore, evidence shows that application of a pcb can relieve pain and facilitate neuromuscular performance during rehabilitation exercises in patients with lumbopelvic problems4 . In particular, pcbs effectively alter the activation patterns of hip extensor muscles in females with chronic low back pain during prone hip extension5 . Moreover, pcbs offer a conservative measure for the treatment of sacroiliac joint pain and are cheap and considered to be without any adverse side effects6 . In addition, it has been shown that use of a pcb significantly improves health - related quality of life and possibly decreases sacroiliac joint - related pain6 . Hammer et al.7 suggested that pcb application is accompanied by altered rectus femoris activity when walking . Furthermore, pcb improve postural steadiness7 . However, it is unknown whether pcb alter trunk and lower - extremity muscle activities in healthy adults . Therefore, the purpose of this study was to investigate the effects of a pcb on these muscle activities in normal healthy adults . All were given comprehensive information on the study, and all provided written informed consent according to the ethical standards of the declaration of helsinki prior to participation and agreed to participate in the study (table 1table 1.pre- and post - intervention electromyography values of subjects wearing or not wearing a pelvic compression belt (units:% mvic)without pelvic compression beltwith pelvic compression beltchange valueerector spinae101.7 8.8 80.9 5.6 20.8 10.9oblique internus abdominis107.8 20.4 87.0 12.420.8 11.7rectus femoris93.7 15.977.8 8.0 15.8 17.7long head of the biceps femoris94.7 17.383.6 11.0 11.1 13.2mean sd . Their average ages, heights, and weights were 21.60 1.08 years, 171.20 6.23 cm, and 71.23 8.64 kg, respectively . Significant intergroup difference between the gains achieved (p<0.05) the pelvic compression belt (the com - pressor, optp, minneapolis, mn, usa) used was an adjustable body belt with four elastic compression bands that provide stabilizing pressure and was designed to allow the amount of compression to be adjusted at targeted compression sites . The pcb was placed below the anterior superior iliac spine (asis)8, and stabilizing pressure was applied using the elastic compression bands after confirming the location of the compression site . Four surface electromyography signals were processed through the mp150 system when subjects were in the bridge position and transformed into digital signals, which were filtered and processed using acqknowledge software ver . 3.7.3 (biopac systems inc ., goleta, ca, usa) on a personal computer . A 1,000 hz sampling rate was used for electromyography signals, and their amplified waveform was filtered using a 60500 hz band - pass filter and a 60 hz of notch filter . For quantifying collected signals, we used root mean square values9 . In addition, the signals collected from each muscle were normalized versus the maximal voluntary isometric contraction (% mvic). To measure muscle activation at maximal voluntary isometric contraction, after collection of the data for 5 seconds at maximal voluntary isometric contraction for each muscle, the average electromyographic signals as a percentage of mvic for 3 of the 5 seconds, excluding the data for 1 second each from the beginning and end, were used . Muscle activation was measured using electromyogram electrodes fixed to areas of muscle fibers and by pressing on muscle parts and following the direction of muscle texture to find the appropriate positions . The locations of the surface electrodes were as follows: (1) for the elector spinae, 2 cm lateral to the spinous process at the l45 interspace10; (2) for the oblique internus abdominis (oi), in the center of the triangle formed by a horizontal line between the anterior superior iliac spine of the innominate and the umbilicus, midline, and the inguinal ligament11; (3) for the rectus femoris, the midpoint between the upper margin of the patella and asis12; and (4) for the long head of the biceps femoris, the midpoint between the gluteal fold and the knee joint13 . Intragroup comparisons of variables before and after the intervention were performed using the paired samples t - test . Ibm spss statistics ver . 20.0 (ibm corp, armonk, ny, usa) the subjects showed a significant decrease in muscle activation in the erector spinae, oblique internus abdominis, rectus femoris, and biceps femoris while wearing the pcb (p<0.05) (table 1). This study was undertaken to determine how a pcb affects erector spinae (es), oblique internus abdominis (oi), rectus femoris (rf), and long head of the biceps femoris (bf) muscle activation in healthy adults . We observed reduced es, oi, rf, and bf activity with a pcb compared with without a pcb in the bridge position . Several possible explanations exist for less muscle activity in abdominal muscles than in core muscles while wearing the pcb . Stabilizing the core is a dynamic process of maintaining balance . Kaushik et al.14 suggested that the transverse abdominis is the first muscle activated during lower extremity movements, indicating that it is a primary muscle linked to core stability during lower limb movements . In the present study, the decreased oi activity indicated that subjects required less effort to maintain stability when wearing the pcb . Nevertheless, kim et al.15 suggested that decreasing the activation of abdominal muscles on an unstable surface using an external support, such as a pcb, is suitable for improving abdominal muscle control and lumbopelvic stability . In a recent study by hu et al.16, it was found that transverse and oblique abdominal muscles were less active with a pcb in normal subjects because these coordinated muscles are activated to press the ilia against the sacrum, creating a forced closure, and the pelvic belt may have substituted for this stabilizing activity . Therefore, it is thought that the use of a pcb with external pelvic compression might have improved pelvic joint stability and altered neuromotor control of the lumbopelvic and thigh muscles . First, the small sample size may have adversely influenced certain variables and impacted results . Second, the compression force of the pelvic belt was not controlled, although the belt was adjusted by a skilled physical therapist . Furthermore, we recruited healthy adults without a history of low back pain or sacroiliac joint pain, and thus, our findings cannot be generalized to other populations . Finally, we measured emg activity of the trunk and lower extremity, but this is insufficient to represent muscle force directly . Further studies are needed to investigate a more diverse sample of normal healthy subjects.
Infections are common in patients with cirrhosis and have been found in up to 66% of patients with variceal hemorrhage . Prophylactic treatment with third generation cephalosporins is recommended in patients with advanced cirrhosis and gastrointestinal bleeding to cover for the predominant causative gut flora organisms, . Treatment is recommended even if no causative organism is identified as up to 50% of patients with cirrhosis and clinical sepsis have negative cultures . Identifying appropriate antimicrobials can be challenging without a causative organism, and the empiric use of broad spectrum antimicrobials contributes to multidrug resistant organisms . We report the first known case of paracoccus yeeii bacteremia in a patient with decompensated cirrhosis who presented with variceal hemorrhage . This rare gram negative organism may not be susceptible to third generation cephalosporins . This finding may have implications for the management of infected but culture negative patients with cirrhosis . A 42 year old gentleman with decompensated cirrhosis from genotype 1a (g1a) hepatitis c (hcv) had been treated with 24 weeks of ledipasvir and sofosbuvir which ended 4.5 months earlier . The patient presented with new onset jaundice (total bilirubin 23 mg / dl, direct bilirubin 18 mg / dl, alkaline phosphatase 99 u / l, alanine aminotransferase 31 u / l, aspartate aminotransferase 86 u / l) that peaked to total bilirubin 44 mg / dl and direct bilirubin 31 mg / dl . Model for end - stage liver disease (meld) score on admission was 27 . Given the report of subjective fevers in the setting of recently treated k. pneumoniae bacteremia of unknown source, admission blood and urine cultures were obtained but were without growth . Molecular studies revealed hcv relapse (hcv viral load = 4.44 log iu / ml) without ns5b resistance (insufficient sample for ns5a) and undetectable hepatitis b (hbv) and adenovirus . There was no serologic evidence of acute infection with hepatitis e, cytomegalovirus (cmv), or epstein barr virus (ebv). Liver biopsy demonstrated cirrhosis with moderate chronic inflammation, focal severe neutrophilic infiltrates with cholestasis, and ductular proliferation . Magnetic resonance imaging (mri) and magnetic resonance cholangiopancreatography (mrcp) showed no cholangitis or abscess . Although drug induced liver injury from ciprofloxacin was clinically suspected, two sets of blood cultures were obtained daily due to concern for infection and lack of clinical improvement . The patient elected discharge but was readmitted within 24 hours with hematemesis from variceal hemorrhage and portal hypertensive gastropathy . Empiric treatment for undifferentiated septic shock including spontaneous bacterial peritonitis prophylaxis were ceftriaxone 2 g iv every 24 hours, metronidazole 500 mg iv every 8 hours, and vancomycin 2 g iv every 12 hours . Diagnostic paracentesis was again attempted but the patient had minimal ascites . Given the lack of clinical improvement and the finding of bowel edema and severe colitis on computer tomography, infectious disease was consulted . It was recommended to adjust antimicrobials to piperacillin - tazobactam 3.375 mg every 6 hours, metronidazole 500 mg iv every 8 hours, and vancomycin 125 mg po every 6 hours to cover for gut flora including clostridium difficile until it could be ruled out . Blood cultures collected the day before discharge of the previous admission flagged as positive after 5 days of incubation in the aerobic bottle (bactec plus aerobic / f), but no organisms were seen upon gram stain . However, after two days of incubation, small mucoid colonies of oxidase positive gram negative coccobacilli were seen growing on chocolate agar only . The team was notified and antimicrobials were adjusted to only piperacillin - tazobactam 4.5 g every 6 hours . After an additional two days of incubation, an identification of paracoccus yeeii was made by matrix assisted laser desorption ionization time of flight mass spectrometry (maldi - tof ms; vitek ms) with a match of 99.9% to the vitek ms ivd unclaimed database . Identification was confirmed by sequencing of the first 500 bp of the 16s gene . Upon speciation, the patient was switched to ceftazidime 1 g every 12 hours to cover for p. yeeii as well as concern for a hospital acquired pneumonia . The patient completed a 2 week treatment course of gram negative antimicrobial coverage with two beta lactam agents and had clinical improvement with no other culture growth of p. yeeii . Unfortunately, the patient developed end - stage liver disease and eventually succumbed to death due to septic shock from citrobacter freundii pneumonia . To our knowledge, this is the first reported case of p. yeeii in a patient with cirrhosis . Patients with decompensated cirrhosis are at high risk of infection from immune dysregulation, a heightened inflammatory response, altered gut flora, and bowel translocation . However, no causative organism is found in up to 50% of patients with decompensated cirrhosis presenting with clinical sepsis . This case of bacteremia with a rare gram negative organism is instructive regarding optimal diagnosis and treatment of infection in patients with decompensated cirrhosis who have an unclear source of infection as they have an extremely high risk of morbidity and mortality . P. yeeii is an aerobic gram negative coccobacillus that has been isolated in soil and marine sediment, and has been rarely reported as infections in humans . P. yeeii has been identified in the past through commonly available biochemical tests, and this case demonstrates the utility of maldi - tof ms for the identification of this infrequently encountered organism, . The few reported cases of p. yeeii to date have entailed peritonitis among peritoneal dialysis patients,, myocarditis in a heart transplant patient, corneal graft infection, and heart failure with bullous skin lesions . All cases identified p. yeeii through culture of affected tissues . Only 2 cases, including ours, have been associated with bacteremia and ours is the first case of a patient with cirrhosis . The source of bacteremia in our case remains unclear, but it is plausible that the patient s lymphedema and bowel edema could be sources for this environmentally found organism . Over the previous year, the patient had several episodes of bacteremia thought to be from bowel translocation including group g -hemolytic streptococci, streptococcus mitis / oralis, citrobacter braakii, and enterococcus faecium (the patient was not on chronic antimicrobials). Maldi - tof may help with rapid diagnosis and guide appropriate treatment when initial cultures in patients with decompensated cirrhosis and clinical sepsis are negative . It is recommended that patients with advanced cirrhosis who develop gastrointestinal bleeding be treated empirically with third generation cephalosporins, . However, this case illustrates that uncommon gram negative organisms may not necessarily be covered with third generation cephalosporins . Although we did not have antimicrobial susceptibilities performed in our case, the existing literature on p. yeeii suggests that the organism is susceptible to fluoroquinolones and beta - lactams though higher minimum inhibitory concentrations (mics) were reported with third generation cephalosporins . This case reinforces the challenges in isolating rare infections in patients with cirrhosis . In the absence of obvious infectious sources in a patient with decompensated cirrhosis collaboration with infectious disease specialists and microbiologists is warranted in these cases to isolate infectious organisms and to identify appropriate antimicrobials promptly . This research did not receive any specific grant from funding agencies in the public, commercial, or not - for - profit sectors.
Coal is an important basic energy and raw material in china, and it accounts for 70% of primary energy . However, due to the complexity and particularity of the coal mine, safety accidents cannot be well controlled, and it still is the key factor to restrict coal production capacity . On the other hand, the influence of coal mine safety accidents, in particular major accidents, is extremely bad, which can create serious losses to people's life and property . Mine production system is a complex system, and the combined effects on various factors lead to coal mine safety accidents . Many scholars and experts consider that human error or people's unsafe behavior is the main reason for coal mine safety accidents by analyzing cause of accidents, and it accounts for more than 90% in all coal mine safety accidents [35]. Coal underground mining is used as the primary mining method in china, comparing to the surface mining, there are too many affecting factors in the underground, and human factors are the most important affecting factors in these factors . According to hfacs analysis methods in the coal mine accident, human factors are relatively complex and changeable, so more affecting data are unknown; it is a gray system . Low prediction accuracy can be avoided due to historical data lack or inaccuracy by applying the gray scgm(1,1)c model to predict the coal mine safety accidents . At present, main methods for predicting safety accident include experience model, regression model, and the gray prediction method . Xiao - fu and ya - dong had applied regression model to forecast the ship traffic accident . Using the regression models and empirical models to predict accident however, accidents are mainly human error accident in the coal mine . Due to human error is affect by many factors, and in a dynamic time - varying system with low accident data and the non - line random changes, so it is not suitable to use these methods for prediction . In the gray accident prediction, shan et al . Had predicted mine safety accident use of unbiased gray model . Meng and cefeng had applied gray correlation on human error accident prediction in the nuclear power plant . . Had, respectively, applied the gm(1, 1) model and residual gm(1, 1) model to predict coal mine accidents [11, 12]. Appling the gray model to predict accident, however, qualitative analysis intensity should not be impressive enough, and the prediction accuracy should be less if these gray models were individually applied . The main reason is that the model requires data sequence must be exponential distribution, and fitting will be poor when data sequence fluctuations are comparatively large . Based on system cloud gray prediction model features in, combining the advantages of both gray prediction and markov theory, according to the coal mine accident deaths provided by the state administration of coal mine safety, referencing the literature [16, 17], an amended gray markov scgm(1,1)c model is proposed . The gray scgm(1,1)c model is applied to imitate the development tendency of the mine safety accident, and the amended model is to improve prediction accuracy while markov prediction is used to predict the fluctuation along the tendency, so as to further improve the prediction accuracy on random volatile accident data . According to the actual accidents situation in coal mine, human error of the coal mines is not regular; there are some characteristics which include occurrences randomly scattered, raw data samples lack, and imperfect and uncertain information . Gray markov scgm(1,1)c prediction model possesses these characteristic as less information required, easy calculation, high accuracy, and so on . It does not list factors data affecting research object but finds useful information and explores the inherent laws from their own time data sequence establishing model to predict . The gray markov scgm(1,1)c prediction model is the ideal model to forecast coal mine safety incidents . Taking account of randomness of human error data in mines, original time series x of coal mine safety accident deaths first, x is integrated as follows: (2)x(1)=x12,x13,,x1n, here, x1(k)=m=2kx0 m, k=2,3,,n . X-(0) is a close mean value generated sequence for x: (3)x(0)=x02,x03,,x0n, here, x0k+1=x0k+1+x0k2 . Human error is random to lead accidents in coal mine, so majority of accidents are dynamic . Given that integral sequence of safety accident deaths time series is expressed as {x-(1)(k)} that is associated with satisfaction trend of nonhomogeneous index discrete function as fr(k) = be c, thus the data of x-(1)(k) is fit to fr(k). According to gray system cloud forecast method, the system gray scgm(1,1)c prediction model can be expressed as (4)dx(1)(k)dk = ax1(k)+u, k2 . Its time response function can be expressed as (5)x1k = x11+uaeakua . Here, (6)a = lnk=3nx(0)(k1)x(0)(k)k=3nx0k12,b=(n1)k=2nea(k1)x(1)(k)(k=2nea(k1))(k=2nx(1)(k))(n1)k=2ne2a(k1)k=2neak12,c=1n1(k=2nea(k))bk=2nx(1)(k). Given x-(1)(1)=b - c, u = ac, x-(1)(k) is reverted, the system gray scgm(1,1)c prediction model of original data will be expressed as (7)x^0k=2b1ea1+eaeak1,(8)yk = x0kx^0k,(9)k = x^0kx0k, k=kx0k . Y(k), (k), and k are gray fitting accuracy indicators, which reflected the degree of deviation of the predicted values to the original data . Statistics data fluctuations of coal mine accidents deaths are larger, and the regularity is not very strong as uncertainty of the person's behavior . Therefore, the prediction accuracy should not be too good if the scgm(1,1)c model is solely applied to predict accident deaths of coal mine . In order to improve the prediction rate and better meet the actual situation, the prediction model should be corrected to improve the accuracy . Amended principle and steps are as follows. (1)the first time residuals data sequence is got in accordance with the predicted value and actual value: (10)0k = x^0kx0k, k=1,2,,n,(11)0k=01,02,,0n. (2)processing residuals correction sequence . The first time residuals data sequence is got in accordance with the predicted value and actual value: (10)0k = x^0kx0k, k=1,2,,n,(11)0k=01,02,,0n . = (1 + e)(1 e)b, the scgm(1,1)c prediction will be expressed as x^(0)(k)=2ea(k-1)m . If (k) a1, m1, and b1 can be obtained in accordance with a, m, and b used method . The first time residuals corrected scgm(1,1)c model can be expressed as follows: (12)x^10k=2eak1mea1k1m1 . If (k) n), the first time residuals corrected scgm(1,1)c model can be expressed as follows: (13)x^10k=2eak1m+ea1k1m1 . In general, a prediction model can be repeatedly corrected residuals, and the residuals can be negative, the time dimensions are also not equal . If the first time residuals amendment cannot meet the forecast accuracy required, it should do residual correction according to the above amended principles, until the accuracy meets requirements . The scgm(1,1)c prediction fitting curve is essentially an exponential curve, and the prediction result is a relatively smooth curve . Because human error accidents are main part of coal mine accidents, the scgm(1,1)c model solely applied cannot meet forecast accuracy requirements . Markov theory has no aftereffect, that is to say, the future state of the system is only related to the current state, and has nothing to do with the past state . Meanwhile, markov model is adopted to predict states trends through probability transfers, it can adapt to the randomness and variability of state . Applying markov theory to correct the scgm(1,1)c prediction model of coal mine accident deaths can better solve the variability and randomness of accidents caused by human errors to improve the prediction accuracy . The annual change of the number of deaths in coal mine accidents is a dynamic nonstationary random process, and thus the prediction fitting precision indicators also are variability and randomness . Because boundary and connotation of the different annual state are changeable, an adaptive state divided criterion needs to be determined, and the criterion should be consistent with basic timing trend of the coal mine accident deaths . Thus, y(k) was divided into m states, and each state can be expressed as (15)ei1i,2i, i=1,2,,m . Here, 1i = y(k) + ai, 2i = y(k) + bi . In the formula, ei is expressed as i state, 1i and 2i are, respectively, expressed as the upper and lower bounds of the i state, and ai and bi are constants determined according to prediction data . Because y(k) is a time function, 1i and 2i will be changed with time, so the state possesses variability . When the state is divided, the numbers of different intervals are reasonably divided according to the actual situation . If raw data are less, the interval division should be less so as to increase the number of transfers between the various states, and thus the transfer law can be more objectively reflected between states . Conversely, if raw data are more, the interval division should be less in order to excavate more information from a large number of data to improve the prediction accuracy . It is suitable to adapt clustering classification method to determine class number and classification intervals due to less data and uncertain status of human error accidents in the coal mine . The original number of samples is expressed as mij(k) from the state ei transiting to the sate ej by k step, and the number of occurrences of the state ei is expressed as mi, so state transition probability is expressed as follows: (16)pijk = mijkmi, i, j=1,2,,m . M m state transition probability matrix can be obtained as follows: (17)pk = p11kp12kp1mkp21kp22kp2mkpm1kpm2kpmmk . The state transition probability matrix p(k) reflects all statistical regularities of state transition, and the future system state steering can be predicted by investigating the matrix . In the actual analysis of the process, one step transition probability matrix p(1) given that predicted moment object is in the state ek, investigating the k row of p(1) can get the following. (1)if maxpij = pkl, the next time system should most likely shift from the state ek to the state el. (2)if there are two or more probability values identical or similar to k row in the matrix p(1), the future state steering will be difficult to determine; it needs to consider probability transition matrix p(2) or p(n) (n 3). If maxpij = pkl, the next time system should most likely shift from the state ek to the state el . If there are two or more probability values identical or similar to k row in the matrix p(1), the future state steering will be difficult to determine; it needs to consider probability transition matrix p(2) or p(n) (n 3). The system's future state will be determined by investigating state transition probability matrix, and gray change interval of relative prediction value in the future moments also will be determined; it can be expressed as [1i, 2i]. Predicted value of the future moment can be expressed as the interval median as y(k): (18)yk = x0kx^0k=121i+2i = yk+12ai+bi . Chinese coal mining is primarily underground mining; the main mining methods used include blasting mining, general mechanized mining, and comprehensive mechanized mining from 1990 to 2010 . Different methods lead to frequency of the coal mine accidents is not same . In general, blasting mining and general mechanized mining were usually applied in the town local small coal mine; their production capacity is relatively small and mining technology and management level are relatively lower compared to large coal mines, so accidents rate is high . In contrast to these small coal mines, the large state - owned coal mines mainly used comprehensive mechanized mining technology; their production capacity is relatively large, management level is relatively advanced, and the accidents rate is lower . Over the last decade, due to the small and medium sized coal mines integrated and strengthened security management in china, their production capacity and mining technology were gradually improved, so the number of occurrences of accidents in the coal mine was declined . According to statistics data of 19902010 coal mine accident death provided by coal mine safety administration, the trend of accidents deaths in coal mine can be fitted by applying the gray scgm(1,1)c model and prediction accuracy can be improved by the residual modified model . Finally, deaths are validly predicted by using the gray markov scgm(1,1)c model . The raw data are shown in table 1, the trend of coal mine accident deaths and death rate per million ton from 1990 to 2010 are shown in figure 1 . Scgm(1,1)c model of coal mine accident death toll is established by using accidents mortality data of coal mines from 1990 to 2010 in china: (19)x^0k=2b1ea1+eaeak1 . In the prediction model (19), a and b are the most important parameters; they are mainly dependent on the raw data to reflect the development trend of data . Here, coal mine accident deaths for 20 years in china were taken as raw data to predict, in order to find the trend of coal mine deaths . Therefore, it is important to select the sample data; according to the formulas (5) and (6), a and b can be calculated . Here, a = 0.039436, b = 236878 . Applying the formula (19), prediction value of coal mine safety accident deaths can be got, and then the gray fitting accuracy indicators also can be calculated by the formula (8), which reveal overall development trend of safety accident death toll in coal mine . Specific forecast and actual values are shown in table 1 . In accordance with table 1, according to (10), the residual initial sequence can be attained, and then one correction residual prediction model can be expressed as (20)^0k=2ea1k11ea11+ea1b1 . Here, according to the formula (14), a residual modification prediction value of safety accident deaths can be obtained, and the results are shown in table 2 . Comparing tables 1 and 2, it is shown that forecast fitting accuracy of the scgm(1,1)c model has been improved after residual modification, but volatility of the gray fitting accuracy indicators is larger from analyzing table 2 . In order to solve this question, it needs to adopt markov chain prediction for further enhancing accuracy and lowering the volatility . The prediction fitting indicators data in table 2 are divided to 4 states by hierarchical clustering, and the results are shown in table 3 . The corresponding states of each year can be determined according to the divided states, and applying the formula (16), the state transferred introduction matrix can be obtained, and the first step state transition matrix is shown as follows: (21)p1=1000000116161216011031035 . The future coal mine accident deaths toll can be predicted based on the above state transition probability matrix . Accident deaths in 2003 year be in the state e4, according to the state transition probability method, examining the state transition probability matrix p(1) line 4 can get (22)maxp4j = p44, j=1,2,3,4 . After a year transition, under the control of coal mine accidents, the death toll of coal mine accidents in 2004 was most likely in the state e4 . According to table 3, interval of the state e3 is [1.05,1.2], that is, 13 = 1.05 and 12 = 1.2 . Because 1i = y(k) + ai, 2i = y(k) + bi, and y(k) = 1.19 in 2004 according to table 2, ai = 13 y(k) = 1.05 1.19 = 0.14, bi = 23 y(k) = 1.2 1.19 = 0.01 . Using the gray markov scgm(1,1)c model, the death toll of coal mine accidents in 2008 is most likely expressed as (23)yk = yk+12ai+bi=1.19 + 120.14 + 0.01=1.125,x02004=x^02004yk=54331.1256112 . In general, in order to facilitate the calculation, similarly, according to the formula (21) and table 2, accidents deaths of coal mine from 2004 to 2013 can be predicted; prediction error comparison of the amended residuals scgm(1,1)c model and the amended residuals markov scgm(1,1)c is shown in table 4 . Error analysis and predictive value fitting of three methods comparing tables 1, 2, and 4, we know that table 2 was amended by using residuals prediction model on the basis of table 1, and the prediction accuracy had been appropriately improved . For table 4, comparing the residual amended model and markov prediction model can be seen that the residual amended scgm(1,1)c model cannot handle abnormal events, because its prediction parameters are fixed . As china's coal enterprises are affected by the economic situation form abroad and home, their production had been in low period, and some coal was not continuously produced; they were basically in an abnormal state in the last three years, so these factors would have a big impact on mine accident forecast . National and local government paid more attention to safety management in the coal mine, and each coal mine strengthened management and prevention on human error, they would also affect the accuracy of general forecasting methods . However, the markov scgm(1,1)c prediction model is only depend on the previous state, the relationship with other states is very small, so it very better solves the abnormal accident . On the other hand, comparing accidents deaths of coal mine from 2004 to 2013 in china, the number of deaths was 6027 in 2004, and this figure became 1067 in 2013; in the recent ten years, the average decline rate of accidents deaths in coal mine is 16.8% . According to this downward trend, accidents deaths of coal mine in china will be about 889 in 2014 . As figure 2 shows that the fitting degree of the markov scgm(1,1)c model is the best, the amended scgm(1,1)c is better and scgm(1,1)c model is the worst . While error change of the markov scgm(1,1)c is minimal, it illustrates that the markov scgm(1,1)c forecast model can be used as a good method to predict the accident deaths of coal mine in china comparing to the other two methods; its prediction result for accident deaths of coal mine is basically corresponded with deaths decline trend in nearly 10 years . The prediction gained by applying the amended residuals markov scgm(1,1)c model is closer to the actual value; in other words, its error is smaller, and it can better reflect the relationship between the coal mine safety accident death and the number of data series, so the prediction is reliable . The amended residuals markov scgm(1,1)c model combines the advantages of both the single - factor system cloud gray model and markov chain, using residual to amend the system gray cloud; it can take full advantage of the information given by the historical data of coal mine accidents, and overcome the data random volatile effects on prediction precision.
De ridder, vanneste, and focquaert address concerns relating to the definition of a ced and the distinction between treatment and enhancement . They raise a number of problems with the treatment - enhancement distinction and suggest that we need to ask whether we are prepared to change the definition of health used by the medical devices directive. There are in fact three questions raised here . The first is whether our proposal requires a robust characterization of the treatment - enhancement distinction, the second is whether our suggestions, as we intended them, actually involve changing the definition of heath implicit in the mdd (there is no explicit definition), and the third is whether the mere inclusion of ceds within the mdd will have the de facto effect of changing the implicit regulatory concept of health or treatment. In response to the first question, we would maintain that it is in fact an advantage of our approach that it minimizes the importance given to the treatment - enhancement distinction and thus diminishes the need to characterize it in a way that is immune to criticism . On our approach, devices intended for enhancement are regulated in a similar manner to therapeutic medical devices . Thus, if some enhancement devices are misclassified as therapeutic devices, or vice versa, this will not have major implications for their regulation . What matters, from our perspective, are risks and benefits . By contrast, some alternative approaches, including the one recommended by de ridder and collaborators, would regulate ceds and therapeutic medical devices quite differently, with the result that misclassification would have more significant regulatory effects, since devices regulated under the general product safety directive alone are held to less specific and less stringent standards . In short, we agree that the treatment - enhancement distinction is problematic and some of us have elsewhere rejected it but we believe a desire to mitigate the problems it raises counts in favor of our approach, not against it . We would also dispute the suggestion that our approach requires any modification to the concept of health . Our proposal was not to change the definition of health (nor treatment) but, rather, to bring some non - therapeutic devices, which do not aim to improve health problems, within the remit of the mdd . Our proposals involved leaving the definition of a medical device used by the mdd unaltered . Ceds would not be medical devices on the mdd definition . Instead, an ancillary positive list is proposed to bring specific devices with a non - medical purpose within the remit of the mdd alongside those devices defined as medical devices according to the criteria employed in the directive . By proposing an ancillary list for specific cognition - affecting devices without a medical purpose, we in fact reinforce the concepts (ie medical and non - medical) underpinning the european commission's proposed creation of annex xv for implantable and other invasive devices for which the manufacturer claims only a non - medical purpose . We proposed that this list should be extended to include non - invasive neuromodulation for non - medical purposes . However, the third implicit question indeed requires consideration: will there be a de facto change in what is meant by health and treatment if ceds are regulated under the mdd? Arguably, the mere association of ceds with therapeutic medical devices could result in an expansion of the concepts of treatment and health . The possible implications of a shift in what is seen as health - related or there, they suggest that medical device regulation might underscore the illusion that devices are beneficial . However, we suggest that the idea that tdcs and tms techniques can yield cognitive enhancement in healthy adults is not a mere illusion . Further, we suggested that regulatory approval of moderately risky brain stimulation devices should be dependent on evidence of at least some degree of performance. In this respect, it would be important to notify consumers explicitly on which population the ced has shown to be beneficial while reminding that it can be ineffective or detrimental to other populations . Regulation would therefore go some way towards ensuring that effects are not illusory for those devices that are approved (although individuals could believe effects to be bigger than they are). Whilst we concede de ridder, vanneste, and focquaert's point that there is currently no substantive evidence that ceds produce lasting effects outside of research and clinical settings (p. 320), there is a wealth of scientific research that provides proof of concept for the cognitive enhancing effects of tdcs and tms techniques . That the ceds on the market have not been subject to objective assessment is part of the reason why regulation is needed and counts in its favor . They argue that contemporary scholarship shows no serious harms associated with the use of neurofeedback or tdcs . In response to this, we wish to point out particularly in relation to active devices used for tdcs and tms that, whilst devices have indeed been use safely in the laboratory, and the regulated devices that researchers use in such contexts are indeed conducive to safe use, these facts do nothing to ensure that the unregulated devices on sale for enhancement exhibit the same safety profile . First, the exclusion criteria for brain stimulation research are much more conservative than marketed exclusion criteria . Second, while brain stimulation experiments lasts usually between a single session to a dozen (the latter is a relatively rare scenario), there is not safety data on the usage of brain stimulation over a long period of time such as months or years . Essentially, regulation is needed to ensure that devices sold for enhancement are as similar as possible to those devices about which kuersten and hamilton make their safety claims . Indeed and to illustrate the point from another perspective fitz and reiner raised concerns about the foc.us device in their commentary: the internal electrodes exceed the general safety guidelines for current density at all stimulation levels, the voltage limits do not behave as specified in the manual, the device behaves unpredictably when its connection to the head is lost, and under some circumstances the foc.us can generate small voltage current spikes . The internal electrodes exceed the general safety guidelines for current density at all stimulation levels, the voltage limits do not behave as specified in the manual, the device behaves unpredictably when its connection to the head is lost, and under some circumstances the foc.us can generate small voltage current spikes . Accordingly, we emphasize: just because tdcs can be safe does not mean that all of the particular devices on sale are meeting the same level of safety . Further, at the end of their commentary, and perhaps in tension with their assertion that the sorts of ceds we discuss present no significant risks, kuersten and hamilton say that low - risk devices should not be excluded from on - going oversight, since so much concerning the brain, what affects it, and how remains unknown (p. 347). We wish here to emphasize that low - risk devices, as we envisioned them, would typically be non - active devices such as neurofeedback equipment, which they correctly claim pose no risks qua devices (see below). Given their comments on the safety of neurofeeback, especially considered simply as a device, we would be surprised if they would think that such equipment required on - going oversight . Thus, even if there is room to disagree about whether ced is the right label for non - active devices (as we discuss below), we in fact seem to agree on the lack of need for the continual regulation of low - risk devices, such as those that do not transfer energy through the skull . Kuersten and hamilton object to our inclusion of neurofeedback as a ced . However, in offering our definition of ceds, we were not aiming to provide the definitive view on what does and what does not fall within this category . Our interest in offering a definition was only to facilitate identification of a class of non - therapeutic devices that should receive regulatory attention and, on that definition, neurofeedback plausibly does qualify as a ced . Note also that doubts about whether neurofeedback equipment qualifies as a ced on a precise understanding of that term could also be raised regarding tdcs equipment, which kuersten and hamilton believe does qualify . They argue that what actually affects the brain is therapy used with [neurofeeback equipment] (p. 341). But tdcs also only enhances cognitive performance when combined with cognitive training exercises, so could be excluded from the category of ceds on similar grounds . Nevertheless, kuersten and hamilton are right to highlight that tdcs directly modulates brain activity whereas neurofeedback modulates it through psychological mechanisms . We have no problem with the suggestion that this is a significant difference, and indeed it informs our suggestion that tdcs and tms should be classed as iia or iib devices, due to their active nature, and neurofeedback should be in class i, if regulated at all . Kuersten and hamilton next argue that including ceds under the definition of a medical device would not result in our suggested problem of overbroadness (which we suggested would occur if the definition of a medical device were to be altered), since devices, they claim, would be limited by the definition to those that investigate, replace or modify the anatomy or a physiological process. Their claim is based on the assumption that ceds could fall under the definition as it is currently articulated . However, it was the possibility that an altered definition would be overbroad that we thought counted in favor of a supplementary positive list . In setting out our proposals, we worked on the assumption that the current definition did not in fact capture ceds (due to the principal criterion that a medical device be intended to treat, prevent or diagnose) and that changing the definition to remove this principal criterion across the board would have the result that it would then be overbroad . If the principal criterion were simply that a medical device investigate, replace or modify the anatomy or a physiological process then all sorts of devices would fall under the remit of the mdd from earrings to nail extensions . Indeed, the european commission used precisely this argument to support the creation of a positive list of implantable or other invasive devices without a medical purpose . According to their impact assessment on the revision of the regulatory framework for medical devices, the definition of a medical device stipulates that it be intended for treatment, prevention or diagnosis, and the removal of this criterion would result in overbroadness, making a positive list the preferred solution in the case of certain cosmetic devices . Our argument for an analogous list for ceds is based on the assumption that the european commission is correct in its understanding of the mdd and the implications of the current and possible definitions employed therein . Fitz and reiner broadly support our proposals and endorse our central claim, viz . That the regulatory framework for medical devices should be extended to include ceds . Nevertheless, they raise a concern about the ability of ced users to evaluate the risks of ceds, saying that evidence that consumers are in a strong position to evaluate the risks associated with ced use is lacking (p. 323). Whilst we agree that consumers will not be perfect rational calculators, we emphasize that our proposals would eliminate devices from the market that were manifestly dangerous or far more dangerous than needed to serve their intended function . In addition, our liberal view is committed to the contention that allowing some room for error in consumers weighing of benefits vs. risks in relation to their individual well - being can be a justifiable cost of allowing individuals greater autonomy and freedom of action . We stress also that what the risks of devices are their nature and likelihood is something that would, in line with the procedure for medical devices, be assessed by a group of experts prior to approval for the market . So, although we agree that leaving some room for consumer valuation of risk leaves open the possibility that individuals will not factor this carefully into their decision whether to use a product, we contend that this is an acceptable cost of a liberal approach to regulation . Further, we would like to emphasize that the current lack of regulation implicitly signals to home users that there are no real concerns with ceds, as otherwise they would have been regulated ie the perception is likely to be that, given the lack of regulation, there are no risks for consumers to evaluate at all . Thus, even if consumers are not perfect risk calculators, the current situation it likely to be more misleading than one in which risks have been identified by regulators . King, gavaghan, and mcmillan provide an interesting critique of our proposals relating to consumer assessment of ceds, and in particular their risks and benefits . They agree that the concept of medical benefit is not always appropriate for ceds (especially where designed and sold only for enhancement) and that well - being would be the theoretically appropriate construct when assessing such ceds . However, they suggest that well - being, harm and risk are difficult to assess in the pre - market approval process and raise a particular concern about the phenomenon of risk compensation. The central feature of this phenomenon is that some individuals have a propensity for taking a certain level of risk and will increase the riskiness of what they are doing until this propensity is met . They therefore challenge the assumption that pre - market approval will make the use of ceds safer overall, especially in the context of experimental home use . They suggest that, where users are determined to use devices in a risky way, they will do so despite safety standards . In response to this, we raise a point of empirical uncertainty, and emphasize what can still be achieved through regulation, despite the phenomenon of risk compensation instantiated in some individuals . It might be true that people will tend to fulfill their propensity for a particular total amount of risk in their lives, but it is unclear whether they do this by assuming superfluous risk to achieve a particular goal or seeking further risky goals . Perhaps, as king and colleagues suggest, achieving one goal more safely allows one to assume additional risk in relation to other pursuits . However, a risk quota might also be met by increasing or maintaining the level of risk one exposes oneself to in pursuit of one's current primary goal . Which is the case is likely to depend on the number of goals one believes to be valuable to pursue . If ceds are made safer, would users with high risk thresholds seek to obtain riskier devices, or would their risk quota be freed up to pursue additional risky activities? The question is essentially whether individuals tend to increase riskiness across all activities until their risk propensity is met, or whether they increase the riskiness of every activity to some threshold level for each activity, regardless of how superfluous this risk may be . If the former, risk reducing regulation could still have benefits in the form of facilitating pursuit of other risky, but perhaps all - things - considered valuable, activities . Even if individuals with a high risk threshold are inclined to practice riskier use as the device itself becomes safer, we assume that king and colleagues would agree that it is still better that the devices available pose as low a risk as possible to achieve their effects, and that unjustifiably dangerous devices are prohibited from placement on the market . Eg by using failsafe mechanisms, audible warnings, and limits on stimulation duration and strength . An individual's propensity to take risks with a product does not render consideration of that product's safety redundant . By analogy, just because some individuals routinely drive dangerously, this does not negate the need to try to make cars as safe as possible . Even if those engaging in risk compensation reap no overall benefit from making ceds safer, failing to minimize risks would unfairly jeopardize those who do not so compensate . Johnson, gillett, and snelling suggest that the most convincing argument in favor of our position is based on the regulatory assessment of risk, which they believe should be the sole consideration in pre - market assessment . We suggest here that our respective proposals are not as different as they may have seemed, as we proposed that risk should be the primary regulatory concern . However, we diverge from the position advocated by them in our contention that objective improvements to cognitive capacities are amenable and relevant to assessment . Baseline (or, we infer, upper limit) level of risk that society feels an individual should be able to take (eg skydiving or climbing), and then require manufacturers to quantify risks only . They argue for this position by pointing out that when technologies are new, and particularly when new technologies are used for an innovative application, variation in the effects on individuals and useful endpoints my not be obvious ab initio . Our first response is to note that the same applies for risks novel applications may present novel and variable risks that are as difficult to predict as the cognitive improvements one might obtain from such applications . Our second response is to emphasize that the model we proposed is actually quite close to a risk - only model; at least, it is a risk - first model . We suggested using risk as the primary way of categorizing a device and that benefits should only be considered in terms of objective improvements eg demonstrated improvements in a cognitive capacity, such working memory which are precisely the sorts of effects about which manufacturers make their claims . Importantly, this consideration of objective benefit is not intended to downplay the broader benefits that an individual might attain from using a device . Whilst objective benefits can be considered, they must not be thought to constitute the only determinants of well - being . Nonetheless, regulators should still ensure that consumers are provided with information on objective benefits . Indeed, the distinction between thick and thin considerations of well - being drawn by king, gavaghan, and mcmillan is illuminating in this regard . Thin well - being, they say, consists only in all - purpose goods, whereas a thick well - being incorporates the individual agent's conception of value and his particular circumstances . An objective impairment in, say, verbal working memory (that occurs as a trade - off of an objective improvement in, say, visuospatial working memory), will present the very same challenges for quantification, despite such impairment constituting a risk rather than a benefit . To the extent, as the risk only model assumes, that objective risks can be quantified (and impairment in verbal working memory indeed permits such quantification) objective benefits along the same sorts of dimensions can also be quantified). Further, we suggested that room should be made to account for the difficulty of measuring effects (positive or negative) on thick well - being . Our proposal was that, once the manufacturer had made it clear what the objective benefits and risks are for moderately risky devices, the consumer should have the freedom to decide whether that benefit risk ratio is acceptable for them (for example, because they expect it to result in a net gain in thick well - being). On the other hand, we proposed that high - risk devices, presenting risks that might be classed all - purpose bads (eg a high risk of seizure), should be prohibited for sale on the consumer market . Whatever the individual's life plans, in the preponderance of cases of healthy adult use, a seizure is unlikely to promote these plans . Where there is scope for disagreement about the value of an effect for example as to whether a small impairment in verbal working memory is a reasonable price to pay for a similarly sized improvement in visuospatial working memory level of acceptable risk therefore plays a greater role than quantification of benefits in our overall framework . As an objection to our model, king, gavaghan, and mcmillan present the example of the surgical insertion of a ventriculoperitoneal shunt to treat normal pressure hydrocephalus . They note that this would be a high - risk procedure on our proposals, as it comes with the risk of seizures, stroke, paralysis, and death . Moreover, there has never been a comprehensive multicenter randomized controlled trial to demonstrate the performance of this product . This is thus a high - risk procedure for which there is no good evidence of benefit . Nevertheless, they seem to suggest, the use of this procedure should be permitted . It might seem, then, that consistency requires permitting the use of some high - risk ceds as well, calling into doubt our claim that high - risk ceds should not be allowed on the market . In response, we would note that patients with normal pressure hydrocephalus typically have much more to lose from non - intervention than do healthy individuals considering whether to employ ceds . Indeed, one might think that advancing dementia is amongst the worst fates that might befall a person . There is thus a case for tolerating much greater risk in devices intended to treat this condition than in ceds . King and collaborators also use the ventriculoperitoneal shunt example to suggest that it may not be practical to enforce the provision of evidence - based measures of efficacy to consumers for ceds . In response to this, we emphasize that we do not have in mind a requirement such that each manufacturer must perform a randomized controlled trial using their device . Research investigating tdcs and tms in healthy adults is demonstrating efficacy, and we envisage that manufacturers should be required to show how their device complies with design parameters equivalent to those that have been shown in the scientific literature to produce enhancement effects . For example, devices with electrodes positioned in locations on the scalp that have no evidence of producing the particular effect claimed by the manufacturer should not be approved for sale alongside such claims . Manufacturers must be able to identify a credible body of scientific literature that supports the claimed efficacy and safety, given the product's characteristics electrode location, stimulation intensity, duration, and so on . Further, we agree with king, gavaghan, and mcmillan that manufacturers should be required to state clearly the lack of evidence for any unsubstantiated claims, if they are to be allowed to make them at all . Kuersten and hamilton also comment on the risk benefit assessment of ceds, and the standard to which we proposed they should be subjected . They argue that there is a lot of room for maneuver in the mdd to assess acceptable risks when weighed against the benefits (p. 346). This is true, but we nonetheless think a framework for assessment is helpful, especially when the devices in question purport to offer non - medical benefits, a concept for which medical device regulators have little precedent . Indeed, we argued against adopting the approach originally suggested by the mhra for cosmetic devices, which is very risk - averse: according to their approach such devices must present no or the minimum acceptable risk, as they confer no clinical benefit. Conceding that some framework could be helpful, kuersten and hamilton suggest that, because the concept of benefit is nebulous, in the case of ceds, only risk should matter for pre - market approval . In fact, as discussed above, we are broadly in agreement with such a position benefits become increasingly harder to quantify and assess as they move beyond the sorts of benefits that enable individuals to pursue the standard range of activities most people wish to pursue . For example, not being in great pain would count as a benefit for most people, regardless of what they valued, but a small improvement to mathematical ability, for example, would not permit such evaluative consensus . As noted above, our inclusion of regulatory consideration of benefits for moderately risky devices was intended only to refer to objective improvements about which consumers should not be misled . For example, if manufacturers claim that their device improves, say, the user's linguistic fluency, this is a claim that can be tested and measured objectively . Beneficial such an objective improvement is to any particular person will vary depending on their goals, but there will be a fact of the matter about whether the device is able to confer such an improvement aside from the subjective question of how valuable this improvement might be . Certainly, we would advocate regulating the claims that manufacturers make in relation to what their devices can achieve . However, far from our proposals being overly paternalistic, we argued that where there was room for disagreement about how to quantify a benefit in relation to risks, regulation should err on the side of consumer freedom . Further to their concerns regarding individuals abilities to weigh risks, fitz and reiner emphasize the remaining problem of do - it - yourself (diy) users who, in constructing devices from scratch, will be afforded no protection by the regulation of direct - to - consumer devices . One of their proposals, with which we agree, is to bolster our recommendations with additional attempts at active harm reduction. They suggest that members of the professional community could join together to create an inclusive online community, where information could be gathered and disseminated with professional oversight . We are broadly sympathetic with much of what they propose and would support the creation of an inclusive online community, were there to be sufficient expert interest in creating and maintaining it . Indeed, we are currently exploring how expert advice could be dispensed via existing fora, such as reddit . De ridder, vanneste, and focquaert also raise the problem of unsupervised use and suggest potential solutions in the form of age limits, safety - by - design, and requiring user licenses . We agree that the unsupervised use of ceds needs consideration, but do not agree that this should lead to their apparent conclusion that we should regulate the use of ceds rather than their placement on the market . The first task must be to ensure that the devices that people use (unsupervised or otherwise) are not unnecessarily dangerous or simply defective by design . This can be achieved for example through european medical device law, which focuses on premarket requirements and reporting on post - market experience, even though it does not regulate use (apart from requirements related to the instructions the manufacturers must provide). There is no point carefully regulating use if the devices being used are not safe to begin with . Further, the authors suggestion that ceds should be made safe by design requires enforcement beyond that granted by the general product safety directive, and this is precisely the sort of thing that could be achieved by a model of the sort we propose . De ridder, vanneste, and focquaert raise the concern that the societal benefits of enhancement might be lost if devices are in fact effective but regulated . In relation to this, we emphasize that the regulation we advocate is not akin to prohibition . To the extent that our proposals would prevent devices from being placed on the market, this would be limited to very dangerous devices or devices making implausible claims hardly the sorts of devices that would confer net societal benefits . The important point that many other commentators on our model appear to overlook is that the effectiveness of brain stimulation techniques as a type of intervention does not guarantee the effectiveness of particular token devices claiming to be of this effective type . Moreover, as low risk, effective devices are developed, these would be available under our proposal and a vigorous market will emerge . De ridder, vanneste, and focquaert raise concerns about the misuse of resources, which assumes a position markedly different from that which we set out in our paper . They argue, persuasively, that resources should be directed to the most important causes and that enhancements might not be of a high enough priority to warrant use of our limited resources . We agree that limited resources need to be allocated carefully, but our proposal does not threaten such allocation: the devices that would be prohibited under our proposal are ones that would certainly not warrant significant investment of public resources, and our proposal leaves open how resources should be allocated among those that would be permitted . Perhaps de ridder and collaborators assumed us to be advocating the inclusion of ceds under the healthcare funding arrangements, but this is not what we envisaged . The extension to the scope of the mdd, as we recommend it, has no direct implications for the distribution of healthcare resources . Relatedly, the authors ask whether the regulation of ceds requires new regulations regarding who can use them . Again, the answer is for the most part no: our proposals merely affect what can be placed on the market for consumers to purchase . The only exception should be restrictions governing the use of ceds on children or vulnerable adults through contraindication labeling and the criminal law . In contrast, adult consumers will not need a prescription to purchase and use ceds on themselves . Indeed, king, gavaghan, and mcmillan provide a compelling argument in their commentary for why medical practitioners should not serve as gatekeepers to cognitive enhancement . Also of relevance to the question of the correct place of enhancement in healthcare, king, gavaghan, and mcmillan suggest that our reference to formally trained practitioners was not developed . We used this phrase in relation to the use of ceds in children and vulnerable adults, since our proposals envisaged that healthy adults would be able to directly purchase and use ceds, with no medical intermediary . They ask whether we envisage a further tier of regulation and raise a number of interesting points that would need to be addressed if medical professionals or those offering alternative therapies were to serve as gatekeepers to the use of ceds in such populations . They are correct that the regulation of such practice would indeed introduce a further tier of regulation, and we acknowledge that this issue was not addressed in our original paper . However, we believe that the scope for the permissible use of ceds on children may be limited . Further, neuromodulation offered to vulnerable adults overseen by medical professionals will primarily be governed by existing medical ethical guidelines, since such use is likely to be therapeutic and hence occurs within the clinical domain . In relation to children, king, gavaghan, and mcmillan are correct that further thought is needed to establish how use in adolescents should be controlled, and with whose oversight . However, ensuring the safety of devices that might be used for enhancement in children and placing controls on such use are separate issues and both are important . Whilst our proposals were concerned with controlling which devices are sold directly to consumers, we agree that the regulation of services, especially those offered to children needs close attention . Kuersten and hamilton object that we fail to cite much law on the remit of the mdd . The suggestion seems to be that this has left us underinformed . Attempting to clarify the current regulatory situation, they say that instead of the definition of a medical device being the significant factor in the non - application of the mdd to ceds, ceds are instead unlikely to fall under the mdd because manufacturers targeting the general market are discouraged from intending them for a medical purpose (which they argue manufacturers have considerable latitude to do) (p. 344). Manufacturers are deterred, they say, because intending that a device be used for a medical purpose mandates costlier and more time - consuming requirements . We suggest that this is precisely the problem: manufacturers should not be able to evade regulation just because they deem it to be too burdensome . We are fully aware that it is the manufacturers intentions identifiable from the claims they make in relation to their products that are instrumental in bringing a device within the definition of a medical device . We argue that, particularly in the case of brain stimulation devices, opting not to fall under the definition of a medical device should not be a possibility . However, kuersten and hamilton suggest that manufacturers intentions will in fact not matter once a new definition comes in to force, which will specifically define a medical purpose. They cite the mhra's overview of the current proposals for the current revision of medical devices legislation, proposals with which we are very familiar . They quote the document as follows, saying that the definition of medical purpose will remove this decision from manufacturers: [m]edical device means any instrument, apparatus, appliance, software, implant, reagent or other article, intended by the manufacturer to be used alone or in combination, for human beings for one or more of the specific medical purposes of: investigation, replacement or modification of the anatomy or of a physiological process or state (pp . [m]edical device means any instrument, apparatus, appliance, software, implant, reagent or other article, intended by the manufacturer to be used alone or in combination, for human beings for one or more of the specific medical purposes of: investigation, replacement or modification of the anatomy or of a physiological process or state (pp ., we suggest that it cannot be seen how the manufacturer's intentions become irrelevant; on the contrary, they are referred to explicitly . Further, rather than the indents defining medical purpose, the scope of medical purpose is constrained to the specific instances given in the indents . Indeed, the consultation documents from the european commission and the uk's medical and healthcare products agency underscore the continued relevance of the medical / non - medical purpose distinction in relation to devices that replace or modify anatomy: their proposal of a positive list (in annex xv) of implantable or other invasive devices without a medical purpose would be redundant if any replacement or modification of anatomy were to constitute a medical purpose . Again, the indents in the definition of a medical device serve to constrain, rather than define, medical purpose . Thus, we rely not only on the court of justice decision, but also on the interpretation of the mdd offered by the mhra and other european commission documents . Kuersten and hamilton perhaps think that there should not be a gap, or may think that the definitions should be interpreted differently . But the fact is, given the way the definitions are currently being understood by the relevant parties, it will take more than the cited revised definition of a medical device to bring ceds within the scope of the mdd . We were gratified that our paper prompted so much discussion . In reading the commentaries and articulating our responses, it became apparent that the comments and critiques served foremost to highlight the modest scope of our original proposals . Whilst we still believe that pre - market approval is highly important for ceds especially for those that transfer energy across the skull the further proposals made in these commentaries serve to set an agenda for continued discussion of the optimum integrated policy response to ceds and, indeed, other new and emerging technologies designed for enhancement.
Oral hygiene habits are instilled in childhood itself irrespective of the nationality or geographic location of an individual . The most reliable and accepted method of oral hygiene maintenance the world over are mechanical methods of tooth cleaning but adjuvants for decreasing plaque formation and maintaining oral hygiene have been sought . Presently chemotherapeutic agents are used as adjuvant agents to reduce plaque formation but they have their own disadvantages.1 kavala graha or gandoosha2 are procedures recommended for oral hygiene maintenance in ayurveda . It is described as a procedure in which an individual takes a comfortable amount of oil / medicated oil and holds it or swishes it in the mouth . When the oil turns thin and milky white it is spit out without swallowing.2 dr . F. karach popularised this procedure as oil pulling.3 he claimed that oil pulling can cure several illnesses including oral diseases, but his claims were not supported by evidence . Recent studies of oil pulling therapy using sunflower oil4 and sesame oil5 were found to decrease plaque induced gingivitis . Even though coconut oil is used for gargling among the people in coconut farming communities, no studies have been done on the benefits of oil pulling using coconut oil, to date . Coconut oil is an edible oil and is consumed as a part of the staple diet in many tropical countries . Coconut oil is different from most other dietary oils because the predominant composition of coconut oil is a medium chain fatty acid, whereas in the majority of other oils the basic building blocks are almost entirely long chain fatty acids . Human breast milk is the only other naturally occurring substance with such a high concentration of lauric acid . Lauric acid has proven anti - inflammatory effects and antimicrobial effects.678 therefore a study was conducted to assess the effect of coconut oil on plaque formation and plaque related gingivitis . The aim of the study was to evaluate the effect of coconut oil pulling / oil swishing on plaque formation and to evaluate the effect on plaque induced gingivitis . A total of 60 age matched subjects in the age - group of 16 - 18 years with plaque induced gingivitis were included in the study . The use of systemic or topical antibiotics and the history of dental treatment in the past one month were set as exclusion criteria . The study was designed to compare the baseline values and the post intervention values in a single group performing coconut oil pulling in addition to their oral hygiene routine . A thorough history regarding the medical condition and the medication taken in the past 6 months was obtained from the subjects . All the chosen subjects had a habit of brushing once or twice a day with toothbrush and paste . Six subjects had the habit of flossing once in the night along with brushing twice a day . The subjects were advised to routinely perform oil pulling with coconut oil every day in the morning in addition to their oral hygiene routine . Five subjects discontinued from the study as they could not tolerate the taste of the oil and three subjects discontinued from the study because of antibiotic usage during the period . Modified gingival index9 and plaque index10 were used as the clinical measures to assess gingival inflammation and plaque formation respectively . Plaque and gingival indices were measured at baseline that is, and on days 1, 7, 15, 30 after the oil pulling routine was started . Reliability of clinical examination was tested for all the days of assessment and the interexaminer reliability was found to be substantial to good . The kappa coefficient scores were in the range of 65 - 92 [table 1a and b]. The mean gingival index was 0.91 and the plaque index was 1.19 at baseline . In comparison to the baseline values both the gingival and the plaque indices substantially reduced during the period of assessment . There was a steady decline in both the plaque index and the gingival index values from day 7 . The average gingival index score on day 30 was down to 0.401 and the plaque index score was 0.385 [figures 1 and 2]. Statistical analysis using the paired t test showed that the decrease was statistically significant [tables 2 and 3]. Kappa scores for modified gingival index kappa scores for plaque index shows the mean and standard deviation of plaque index scores shows the mean and standard deviation of gingival index scores comparison of plaque index scores between baseline, 7, 15, and 30 days comparison of gingival index scores between baseline, 15, 30 and 45 days dental plaque is defined clinically as a structured, resilient substance that adheres to intraoral hard surfaces and is composed of bacteria in a matrix of salivary glycoprotein and extracellular polysaccharides . Plaque induced gingivitis is the result of an interaction between plaque and the tissues and the inflammatory response of the host . It is associated with the subtle microbial alterations as the plaque matures.1112 oral hygiene measures using chemo mechanical procedures reduce the incidence of plaque related diseases by decreasing the plaque accumulation . Our study aimed at checking the effectiveness of oil pulling with coconut oil as an adjuvant to brushing, in decreasing the plaque accumulation and plaque induced gingivitis . Plaque index by sillness and loe10 and modified gingival index9 were used for clinical assessment in the study as they are the most widely used indices in trials for therapeutic agents.13 oil pulling with sunflower oil was found to significantly reduce plaque index and gingival index after 45 days.4 asokan et al ., found oil pulling therapy with sesame oil was equally effective as chlorhexidine in decreasing plaque induced gingivitis.5 in our study also there was a significant decrease in the plaque and the gingival index at the end of 30 days . There are various hypotheses on the mechanisms by which oil pulling may act in decreasing the plaque and gingival index . In oil pulling, as the oil is swished in the mouth the mechanical shear forces exerted on the oil leads to its emulsification and the surface area of the oil is greatly increased . The oil film thus formed on the surface of the teeth and the gingiva can reduce plaque adhesion and bacterial co aggregation.5 it was also proposed that the alkalis in the saliva can react with the oil leading to saponification and formation of a soap like substance [figure 3] which can reduce the adhesion of plaque.514 coconut oil has a high saponification value and is one of the most commonly used oil in making soaps . The soaps produced with coconut oil can lather well and have an increased cleansing action.15 the lauric acid in the coconut oil can easily react with sodium hydroxide in saliva during oil pulling to form sodium laureate, the main constituent of soap16 which might be responsible for the cleansing action and decreased plaque accumulation . Saponification reaction the significant reduction in gingivitis can be attributed to decreased plaque accumulation and the anti - inflammatory, emollient effect of coconut oil . In animal studies coconut oil was found to be an effective burn wound healing agent and this was attributed to its anti - inflammatory and antiseptic properties.17 coconut oil showed moderate anti - inflammatory effects on ethyl phenylpropiolate induced ear edema in rats, and carrageenin and arachidonic acid - induced paw edema.18 it was found be effective and safe when used as an emollient and moisturiser.19 there are many commercially available mouthwashes . Listerine (phenol compound) and meridol (an amine / stannous fluoride mouthwash) were found to be less efficacious than chlorhexidine in controlling plaque induced gingivitis . After 3 weeks of rinsing, plaque indices remained the lowest in the chlorhexidine group, while subjects using listerine or meridol the score were similar but significantly lower than that of individuals rinsing with the placebo solution . The antimicrobial potential of chlorhexidine was found to be the highest followed by meridol.20 in our study there was a 50% decrease in the plaque and gingival index scores in 4 weeks which is comparable to the decrease produced by chlorhexidine . Chlorhexidine on long term use alters taste sensation and produces brown staining on the teeth which is very difficult to remove . The mucous membranes and the tongue can also be affected and may be related to the precipitation of chromogenic dietary factors on to the teeth and mucous membranes.21 staining is also associated with the of long term use of phenol compound and stannous fluoride containing mouth washes.22 in the present study there were no reported alterations in the taste or noticeable staining from coconut oil at the end of 4 weeks . As an antimicrobial agent, chlorhexidine is effective against both gram positive and gram negative bacteria . Its antibacterial action is due to an increase in cellular membrane permeability followed by coagulation of the cytoplasmic macromolecules.232425 it has also been shown that chlorhexidine can reduce the adherence of porphyromonas gingivalis to epithelial cells.26 pure - culture studies of 10 oral bacteria (eight genera) showed that actinomyces naeslundii, veillonella dispar, prevotella nigrescens, and the streptococci were highly susceptible to chx, while lactobacillus rhamnosus, fusobacterium nucleatum, were less susceptible.27 studies show that coconut oil also has substantial antimicrobial activity . It is shown to have significant antimicrobial activity against escherichia vulneris, enterobcater spp ., including c. albicans, c. glabrata, c. tropicalis, c. parapsilosis, c. stellatoidea and c. krusei728 studies also show that coconut oil is affective against s. mutans and c. albicans in an in vitro oral biofilm model.29 the antimicrobial potency of coconut oil was not tested in our study . Further studies with a have been planned to check the antimicrobial potential of coconut oil . The fact that a control group with a proven chemotherapeutic agent was not used is the major the limitation of our study . Oil pulling has been proven to be an effective method in reducing plaque formation and plaque induced gingivitis . This preliminary study shows that coconut oil is an easily usable, safe and cost effective agent with minimal side effects which can be used as an adjuvant in oral hygiene maintenance . More studies on the antimicrobial potency of coconut oil on microorganisms causing oral diseases is required to authenticate the use of coconut oil as an effective oral antimicrobial agent . Further studies on coconut oil with a large number of subjects and comparative studies using various chemotherapeutic agents can improve the quality of evidence.
Taurodontism is defined as the enlargement of the pulp cavity of a molar tooth at the expense of root length . The name was given because of the apparent similarity between these teeth and those of ungulates, especially bulls . Hence, we have the term taurodont, tauro from the latin term for bull and do nt from the greek term for tooth appears to be a continuous trait . Identification of the taurodont teeth can only be made by radiographic examination as the external morphology of the teeth is within normal configurations . Appearance of a taurodont tooth is very characteristic, and radiographic examination is the only way to visualize a rectangular configuration of the pulp chamber . The pulp chamber is extremely large with a greater apico - occlusal height and there is no cervical constriction of the teeth . These teeth have short roots and the bifurcation or trifurcation may be only few millimeters above the apices of the roots . It has been suggested that the anomaly represents a primitive pattern, a mutation, a specialized or retrograde character, an atavistic feature, an x - linked trait, familial or an autosomal dominant trait . Although taurodontism has been reported in association with certain syndromes and some genetic defects, its true significance is still obscure . Taurodontism appears most frequently as an isolated anomaly, but it has also been associated with several developmental syndromes and anomalies including amelogenesis imperfecta, down's syndrome, ectodermal dysplasia, klinefelter syndrome, tricho - dento - osseous syndrome, mohr syndrome, wolf taurodontism has also been reported to present with other rare syndromes such as smith magenis syndrome, williams syndrome, mccune most studies of its prevalence have, however, employed a categorical approach . Extreme enlargement of the pulp cavity in an ancient population was first reported in neanderthal teeth from the krapina, who described it as a distinguishing feature of this middle palaeolithic human population . The prevalence of taurodontism is reported to range from 2.5% to 11.3% of the human population . This range is likely accounted for by variations in race and differences in diagnostic criteria . Taurodontism is exhibited in 2.5 - 3.5% of the chromosomally normal caucasian population, and most of these teeth are hypotaurodontic . The aim of this retrospective study was to evaluate the frequency of the occurrence of taurodont molars in an indian dental school patient population using full - mouth periapical radiographs . In this paper, we also emphasize the diagnosis, etiology and anatomic and radiographic characteristics of taurodont teeth and also its association with various syndromes and anomalies . A total of 1000 patients retrospective full - mouth periapical radiographs, which were recorded in the department of conservative dentistry and endodontics, from january 2007 to december 2012, were screened . Each of these patients had to have full - mouth periapical radiographs, had to be at least 18 years of age and had to be of indian origin . Personal details including age, sex and race of all these patients were recorded to ensure that all patients were of indian origin . In order to obtain a balanced distribution between male and female patients, an equal number of (500) full - mouth radiographs of male and female patients were studied . The full - mouth radiographs were taken using kodak ultra - speed films (kodak, stuttgart, germany). The radiographs were placed on a viewing box and light surrounding the radiograph was blocked . If disagreement existed, a joint evaluation of all authors was made until a consensus was reached . Shifman and chanannel proposed the following criteria for determining the presence of taurodontism, the distance between the cementoenamal junction and the floor of the pulp chamber is 2.5 mm and if the distance from the lowest point at the occlusal end of the pulp chamber (a) to the highest point at the apical end of the chamber (b) divided by the distance from a to the apex is 0.2 or greater [figure 1]. The relative incidence and the correlations regarding the location of taurodont teeth (maxillary versus mandibular and male versus female) were analyzed [table 1 and figure 2] using the chi - squared test . Measurements based on the study by shifman and chanannel were used to determine the presence of taurodontism . (a) the lowest point at the occlusal end of the pulp chamber and (b) the highest point at the apical end of the pulp chamber . Cej, cementoenamel junction distribution of teeth examined and taurodont teeth in the maxilla and mandible the relative incidence and the correlations regarding the location of taurodont teeth (maxillary and mandibular) in males and females one thousand patients, 500 male and 500 female, between the age of 20 and 70 years (average, 45 years) were included in this study . Maxillary molars comprised 3805 teeth and mandibular molars comprised 3810 teeth [table 2]. Of the 7615 molars examined, 40 (0.53%) teeth were found to have taurodontism . These teeth were detected in 28 (2.8%) of 1000 subjects, with 17 as males (60.71%) and 11 as females (39.28%). Twenty - eight patients were found to have a taurodont molar teeth (24 teeth in male and 16 teeth in female, p = 0.267). Taurodontism was present in 17 of the 500 male patients (3.4%) and 11 of the 500 female patients (2.2%), p = 0.250 [table 3 and figure 3]. Distribution of right and left molars in the maxilla and mandible distribution of total subjects and subjects having taurodont teeth by gender p=0.250 (ns) distribution of total subjects and subjects having taurodont teeth by gender a cluster analysis of total taurodonts in the mandible (45%) versus the maxilla (55%) of both males and females combined showed a statistically significant difference (p <0.05; table 1). Although there have been several studies reporting the prevalence of various dental anomalies, no reported study has been conducted on the prevalence of taurodontism in an indian population . In the present study, patients who reported in the opd of the department of conservative dentistry and endodontics, faculty of dental sciences, who either underwent restorative treatment or an endodontic procedure, providing an estimation of the prevalence of taurodontism in the indian sub - population were analyzed . The present data indicated that the occurrence of taurodontism in the indian population was 2.8% . There is one study in our knowledge that evaluated both maxillary and mandibular molars, reporting the prevalence of taurodontism in indians being 2.49% . In a recent study of various malocclusions, found a higher rate of 8.0% in jordanian dental patients; shifman and chanannel reported a prevalence of 5.6% in israeli patients, whereas macdonald - jankowski and li reported an even higher rate (46.4%) of taurodontism in an adult chinese population . The prevalence rates reported by previous studies range from 0.25% to 48% in different populations . In the present study, men presented a higher prevalence of taurodont teeth than women, although these differences were not statistically significant (p = 0.250). However, the present observation is different from a previous study on asians that reported a higher prevalence in women . The diagnosis of taurodontism using panoramic radiographs is difficult; the anterior regions of both jaws may become distorted and this may result in an incorrect diagnosis of taurodontic molars . Additionally, the differential diagnosis between taurodontic teeth and other teeth exhibiting large pulp chambers (patients with amelogenesis imperfecta seem to have large pulp chambers because of the lack of enamel) is of clinical significance . Nevertheless, a reliable diagnosis of taurodontic molars has been shown to be possible using orthopantomograms . However, to ensure proper identification and the best accuracy of assessment, full - mouth periapical radiographs were used in the present study . On the basis of the external morphology, shaw proposed a classification of taurodont teeth, but later a more accurate approach was established by keen for the categorization of taurodontic teeth using internal morphology . He proposed the taurodont index as an objective method for the assessment and related the height of the pulp chamber to the length of the longest root . This index is used as a biological landmark that undergoes changes during the whole life . To overcome these changes, shifman and chananel used landmarks that are not modified by apposition of reparative dentine or morphological changes of the root anatomy . However, even this ratio can be affected by incomplete root formation or radicular resorption . In addition, root length varies by sex and ethnic groups, which may cast some doubt on the credibility of any metric method as a screening tool for taurodontism between different populations, and limits the comparability of the results of different studies . Recently, a case report highlighted the use of high - end diagnostic imaging modalities such as spiral computerized tomography in making a confirmatory diagnosis of the multiple morphologic abnormalities such as taurodontism, dens invaginatus, pyramidal cusps of the premolars and dens evaginatus . From an endodontist's view, taurodontism presents a challenge during negotiation, instrumentation and obturation in root canal therapy . Magnification devices such as magnifying loupes or surgical microscopes can be helpful to locate canal orifices, evaluate the pulp chamber and obturate the canals . The importance of taurodontism is felt more by endodontists while negotiating the complex internal anatomy of such teeth . Shifman and buchner stressed on the importance of gaining access to the canal orifice, which is not difficult in such teeth due to the absence of reactional dentin that is present in normal teeth; however, this view was contradictory to the study by durr et al ., which says that such a morphology could make the orifice location difficult, creating further difficulty during root canal preparation and obturation . Hayashi reported mandibular taurodont tooth with five canals, wherein only three canals could be instrumented till the working length . He also explained the presence of extra root canals in terms of shape and number . It is therefore stressed that careful exploration of canal orifices and grooves is mandatory under the magnification the diagnosis of taurodont tooth is established . The voluminous pulp present in taurodont teeth needs to be removed completely for the success of treatment; it is here that the importance of irrigation comes into play . Suggested copious irrigation with 2.5% sodium hypochlorite to remove pulpal tissue from the irregular canal walls in such teeth . Grossman and meiman reported that irrigation with 5% sodium hypochlorite for 20 min to 2 h dissolves all pulpal tissue . Moorer and wesselink demonstrated that one of the major factors in pulpal tissue removal was agitation with irrigation . It is highly recommended that agitation must be done with copious irrigation to get rid of the pulpal tissue in taurodont teeth . The proximity of the orifices and deeply situated opening of the canals made it difficult to obturate the canals with any single method of obturation . Therefore, a combination of lateral condensation and warm vertical condensation technique can be performed to achieve the best results . Within the limitations of this study, it was found that: the present study found the prevalence of taurodont molar to be 2.8%male have a higher prevalence rate then female (3.4% vs. 2.2%, respectively), with p = 0.250maxillary molars are most commonly involving teeth than mandibular molars in males, while females have an equal frequency of taurodont teeth . The present study found the prevalence of taurodont molar to be 2.8% male have a higher prevalence rate then female (3.4% vs. 2.2%, respectively), with p = 0.250 maxillary molars are most commonly involving teeth than mandibular molars in males, while females have an equal frequency of taurodont teeth . These variations in prevalence between different populations may be due to ethnic variations, but may also be influenced by differences in criteria used for the interpretation of taurodontism and also the specific teeth examined.
There have always been concerns about the long - term durability of composite resin restorations . These restorations may have a short lifespan due to the degradation of resin dentin interface . Water wet bonding technique was introduced in the beginning of 1990s with the aim of preventing collapse of collagen fibrils after acid etching, improving the penetration of resin into the etched dentin and increasing the durability of resin dentin bond . Consequently, manufacturers increased the concentration of ionic and hydrophilic monomers in order to produce more suitable adhesives to bond to water - saturated etched dentin . The increased concentration of acidic and hydrophilic resin monomers may reduce durability of resin dentin bond due to more water adsorption of hydrophilic resins . In addition to hydrolysis, insufficient penetration of resin into the collagen fibrils due to residual water decreases the durability of resin dentin bond . Recent studies have demonstrated that dentin adhesives cannot replace free and loosely bound water from the interfibrillar spaces, even when resin monomers are able to encapsulate the collagen fibrils . Another reason which reduces the resin dentin bond durability is collagenolysis by endogenous matrix metalloproteinase (mmp) enzymes . Also, acidic resin monomers of self - etch and etch and rinse adhesives increase collagenolytic and gelatinolytic activity in collagen matrix . Several approaches are available to overcome these problems and to achieve the main goal of bonding process which is the complete infiltration of resin monomers into the demineralized collagen fibrils and displacement of water of these spaces . Ethanol wet bonding technique is based on the concept of water replacement from interfibrillar and intrafibrillar spaces . While in the simplified method ethanol with 100% concentration is applied to water saturated acid etched dentin for one minute prior to the application of ethanol solvated hydrophobic resin comonomer blend . In ethanol wet bonding technique, water is replaced by ethanol which alters the demineralized collagen matrix to a dehydrated and fully extended pattern . The ethanol - suspended collagen matrix has less hydrophilicity and prevents phase separation of hydrophobic resin monomers dissolved in ethanol . This technique shrinks the collagen fibrils and enlarges the interfibrillar spaces for hydrophobic monomers to infiltrate the matrix which could extend the longevity of resin sadek et al . Demonstrated that ethanol wet bonding displays a bond strength higher than or equal to water wet bonding . . Showed increased bond strength and durability in ethanol wet bonding compared to water wet bonding . Peumans et al . Studied the clinical success rate of optibond fl over 13 years and showed a 12% failure rate . They also evaluated the success rate of clearfil se bond over eight years and showed 97% success rate . Since the ethanol wet bonding technique has not been compared with other bonding methods clinically, the present study aimed to evaluate and compare the clinical success rate of ethanol wet bonding technique with a three step etch and rinse gold standard adhesive (optibond fl) and a two step self - etch gold standard adhesive (clearfil se bond) in one year clinical service . The null hypothesis tested was that there is no difference between the clinical success rates of restorations using different bonding protocols after one year . This randomized double blind clinical trial included 12 patients with at least three non - carious cervical lesions (nccls). The protocol and consent form were approved by research committee of isfahan university of medical sciences and registered in iranian registry of clinical trials (registration code #irct 201111288242n1). The age of the patients varied between 30 and 60 years (seven males and five females). The patients who referred to the department of operative dentistry at isfahan dental school were screened to see whether they met the inclusion criteria or not . Patients exhibiting severe or chronic periodontitis, untreated periodontal disease, uncontrolled caries, very poor oral hygiene, heavy bruxism and xerostomia were excluded . Other exclusion criteria were wearing removable prosthesis or orthodontics appliances, receiving bleaching treatments or fluoride supplements . Also breast feeding or pregnant women qualified patients who were systemically healthy and had good oral hygiene with normal occlusion were examined in order to identify nccls (abrasion, erosion, abfraction) visually and tacitly . Nccls with at least 1 mm depth on vital teeth or patient reporting thermal sensitivity or both were selected . The incisal or occlusal margin of the cavity should end in enamel and the cervical margins should end in the dentin or cementum . The lesions were observed from lateral, and the cavities were classified into four groups according to the angle of the cavity (<45, 4590, 90135,> 135). The gingivo - occlusal or gingivo - incisal heights of the cavities were measured with a periodontal probe . The age, sex, type of tooth, and arch distribution of the nccls, degree of sclerotic dentine, cervico - incisal height of the cavities, angle of the cavities and other factors which were recorded in this study are summarized in table 1 . All patients were aware about the nature and objectives of the study and the procedures but blinded to the type of restorative technique . Written informed consents were taken from all patients and oral hygiene instructions were given to all patients prior to treatment . Distribution of non - carious cervical lesions according to age and sex distribution, shape, cervico - incisal height of the lesion, degree of sclerotic dentin, presence of antagonist, presence of attrition facets, presence of preoperative sensitivity and tooth and arch distribution in the clinical phase of the present study, each patient received at least one restoration using the three - step etch and rinse protocol, one restoration using the two - step self - etch protocol with enamel etching and one restoration using the ethanol wet bonding protocol with the part of adhesive of optibond fl randomly . The teeth were cleaned with a prophylactic cup and fluoride - free pumice and water followed by rinsing and drying . After local anesthesia administration and shade selection, the teeth were isolated with rubber dam and rinsed with water / air spray . Occlusal or incisal margins were beveled (1 mm with 45 angle) in order to provide more surface area for bonding and to improve esthetics . All cavities were restored with nanohybrid composite resin (grandio, voco, germany) incrementally . Each increment was cured with an led light curing unit (demi, kerr, usa) with an output of 500 mw / cm for 40 s. all restorations were finished and polished with finishing diamond burs (thin taper, jota, swiss), polishing disks (optidisc, kerr, switzerland) and diamond paste (diamond polish product, 1, ultradent, usa). Composition and application procedures of the materials used all patients were recruited for follow - up examinations after 24 h, 6 months, 9 months and 12 months . Two operators whom were blinded about the type of restorative technique examined the restorations separately by use of dental mirror and explorer after tooth prophylaxy and then reached an agreement . Cohen's kappa statistic test which was performed for inter - examiner and intra - examiner calibration was over 72% in all criteria suggesting perfect agreement between examiners . During recalls, . Retention rate, which was evaluated by the presence or loss of the restoration; 2 . Enamel or dentine marginal defect in which the restoration margins were examined with explorer and dental mirror; 3 . Marginal discoloration, which was clinically observed; 4 . Postoperative sensitivity, which was assessed with application of air spray from a distance of 23 cm from the restoration for 3 s while the adjacent teeth were covered by finger . In the present study, the tooth vitality was assessed by a cold spray (frisco spray, ad - arztbedarf gmbh, germany), in addition to the usphs criteria . Finally the data were analyzed with chi - square statistical test using spss software version 12; p value = 0.05 was considered as significant level . In the clinical phase of the present study, each patient received at least one restoration using the three - step etch and rinse protocol, one restoration using the two - step self - etch protocol with enamel etching and one restoration using the ethanol wet bonding protocol with the part of adhesive of optibond fl randomly . The teeth were cleaned with a prophylactic cup and fluoride - free pumice and water followed by rinsing and drying . After local anesthesia administration and shade selection, the teeth were isolated with rubber dam and rinsed with water / air spray . Occlusal or incisal margins were beveled (1 mm with 45 angle) in order to provide more surface area for bonding and to improve esthetics . All cavities were restored with nanohybrid composite resin (grandio, voco, germany) incrementally . Each increment was cured with an led light curing unit (demi, kerr, usa) with an output of 500 mw / cm for 40 s. all restorations were finished and polished with finishing diamond burs (thin taper, jota, swiss), polishing disks (optidisc, kerr, switzerland) and diamond paste (diamond polish product, 1, ultradent, usa). All patients were recruited for follow - up examinations after 24 h, 6 months, 9 months and 12 months . Two operators whom were blinded about the type of restorative technique examined the restorations separately by use of dental mirror and explorer after tooth prophylaxy and then reached an agreement . Cohen's kappa statistic test which was performed for inter - examiner and intra - examiner calibration was over 72% in all criteria suggesting perfect agreement between examiners . During recalls, the following were examined according to the usphs criteria: 1 . Retention rate, which was evaluated by the presence or loss of the restoration; 2 . Enamel or dentine marginal defect in which the restoration margins were examined with explorer and dental mirror; 3 . Marginal discoloration, which was clinically observed; 4 . Postoperative sensitivity, which was assessed with application of air spray from a distance of 23 cm from the restoration for 3 s while the adjacent teeth were covered by finger . In the present study, the tooth vitality was assessed by a cold spray (frisco spray, ad - arztbedarf gmbh, germany), in addition to the usphs criteria . Finally the data were analyzed with chi - square statistical test using spss software version 12; p value = 0.05 was considered as significant level . A total of 36 teeth were restored in which 13 of them were placed in maxillary arch and 23 of them were placed in mandibular arch . Eleven restorations belonged to the anterior segment and 25 restorations belonged to the posterior region (molars and premolars). The recall rate was 100% in 6 months, 9 months and also 12 months . The retention rate at baseline and at 6 months follow up for all three types of bonding protocols was 100%, though the teeth were scored as alfa for the item retention rate . At 9 months follow up, in ethanol wet bonding group, one restoration was lost and the retention rate was 91.67%, although this group was scored as bravo for the item retention rate. The comparison of the three types of techniques used showed no significant difference (p value = 0.358). 100% of restorations in three groups continued to be without any enamel or dentin marginal defects after 12 months and were scored alfa for this item . None of the restored teeth showed marginal discoloration after 12 months (100% without marginal discoloration) and were scored alfa for this item . None of the restorations in optibond fl group showed postoperative sensitivity in different time periods and were scored alfa for this item . One of the teeth restored with ethanol wet bonding showed postoperative sensitivity after 24 h and was scored as bravo for this item . After 6 months, two teeth showed postoperative sensitivity, in which one belonged to the ethanol wet bonding group and the other belonged to the clearfil se bond group and were scored bravo . At 9 months recall, none of the teeth were sensitive, since the restoration of sensitive tooth in ethanol wet bonding group was lost . None of the restored teeth in the three groups showed decay after 12 months (100% without caries); therefore all of them were scored as alfa for this item . Therefore there was no statistically significant difference in the clinical success rate between these three groups after 12 months follow up (p value = 0.358). It is possible to coax completely hydrophobic monomer to acid - etched dentin with an ethanol wet bonding protocol that theoretically improves resin dentin bond durability by minimizing water sorption through polymerized hydrophobic adhesive . Ethanol is a bipolar solvent with less hydrogen bond capacity than water, which leads to chemical dehydration of the demineralized collagen network . Subsequently, interfibrillar and intrafibrillar spaces are filled with ethanol which is a better solvent for resin monomers than water . This fact results in better infiltration of resin monomers into the demineralized collagen network that is suspended in ethanol . In water wet bonding technique, the penetration of bis - gma in collagen network has a gradual decrease but in ethanol wet bonding, a relatively homogenous distribution of hydrophobic bis - gma compounds is seen which inhibits phase separation . Moreover, the interfibrillar collagen spaces contain a hydrogel of proteoglycans, which interferes with resin penetration in the bonding process . Also ethanol shrinks the fibrils; therefore, the interfibrillar spaces are enlarged and allow more resin penetration . Dentin bond formed with ethanol wet bonding and hydrophobic adhesives do not degrade after 18 months water storage without the presence of mmp inhibitors . In the present study, the clinical success rate of ethanol wet bonding technique in comparison to the optibond fl (a three - step etch and rinse adhesive gold standard) and clearfil se bond (a two - step self - etch adhesive gold standard) was evaluated . In this research, evaluated the clinical success rate of optibond fl in non - carious class v cervical lesions over 13 years follow up and stated a high retention rate confirming the bonding efficacy of optibond fl . In this study the retention rate of clearfill se bond after 12 months was 100% which is in agreement with the findings of peumans et al . They evaluated the efficacy of clearfill se bond over 8 years clinical performance with and without selective enamel etching and recorded a retention rate of 100% after 12 months for the etched enamel group; however after 8 years, 2 restorations were lost . It can be concluded from their study that the retention rate decreases over the time and longer recalls is needed to get more reliable results . In their study, the percentage of marginal integrity of the etched enamel over one year was 70% while in our study none of the teeth bonded using clearfill se bond showed marginal defect at the same period . Also in their study, after eight years, none of the teeth showed postoperative sensitivity, while in ours after 6 months one tooth that was bonded with clearfil se bond showed postoperative sensitivity . Two - step self - etch adhesives are expected to cause less postoperative sensitivity compared to etch and rinse adhesives . These adhesives do not remove the smear layer and leave the residual smear plugs which results in less dentinal fluid flow . It must be pointed out that in this study similar to the peumans et al . Study, none of the teeth that restored with clearfill se bond showed caries occurrence which is known as the main reason for failure of composite restorations . The better durability of the mild two - step self - etch adhesives such as clearfil se bond is attributed to the use of a more hydrophobic resin layer on top of the hydrophilic self - etch primer and partial dissolution of apatite mineral which causes a protective effect on collagen degradation . In the present study, the ethanol wet bonding technique showed 91.67% retention rate after 12 months; it may be due to high technical sensitivity of ethanol wet bonding . Studied the durability of resin bonded to water - saturated dentin versus ethanol - saturated dentin and demonstrated that ethanol - saturated dentin had a significantly higher bond strength than water which did not fell over the time . It must be pointed that the selective etching of enamel by use of phosphoric acid was performed in our study, since it improves the durability of bond . Investigated the ethanol wet bonding technique sensitivity by atomic force microscopy (afm). In both ethanol wet bonding protocols, evaporation of water from the water - saturated dentin resulted in the collapse of the collagen matrix . However, osorio et al . Showed that the collapsed collagen matrix was rehydrated and re - expanded with the aid of 50% water present in the 50% ethanol in the progressive method . Thus the progressive method of ethanol wet bonding may be considered as less technique sensitive protocol; however, it is more time consuming . In the present research, the ethanol wet bonding protocol was compared to optibond fl and clearfill se bond and showed high clinical success rate in different aspect of usphs criteria . According to the findings of the present study, there is no statistically significant difference between the clinical success rate of ethanol wet bonding technique with optibond fl and clearfil se bond after 12 months clinical service and the null hypothesis is accepted . Due to this fact that clinical studies have severe limitations in patient selection, unifying the lesions, unifying the degree of sclerotic dentin, type of restored tooth and occlusal loads which differs for posterior and anterior teeth, method of restoration and the number of increments used for the restoration, much more clinical studies with longer recalls are required in order to obtain reliable and practical results and to present ethanol wet bonding as a method for clinical use . It must be considered that class v lesions are not a good model to correlate the results of the present study to class i / ii restorations, or to cavities that are surrounded by enamel margins, since they are primarily dentin lesion and only have a smaller amount of enamel at the incisal side, and also the occlusal loads on class v restorations is not as great for class i / ii restorations . Also the location of class v restorations and the easy accessibility to them is another limitation for correlating the results of the present study to class i / ii restorations . In the present clinical trial, the clinical success rate of ethanol wet bonding technique was compared to a three - step etch and rinse bonding system (optibond fl, kerr, usa) and a two - step self - etch bonding system (clearfil se bond, kuraray, japan) in recalls of 24 h, 6, 9 and 12 months . The difference between the three bonding methods was not statistically significant; therefore, ethanol wet bonding presented equal performance to the optibond fl and clearfil se bond adhesives after 12 months of clinical functioning.
Caesarean section is the delivery of the fetus, placenta, and membranes after the age of viability through an abdominal and uterine incision.1 there is a wide global variation in the incidence of caesarean section . The general range is from 5% to 25% with a continued rise in developed countries while in developing countries the rate is relatively low.12 in nigeria, the rate of caesarean section varies from one center to the other with 10.3% in enugu,3 10.5% (makurdi),4 11.4% (zaria),5 15.8% (jos),6 and 20.3% in birnin - kebbi7 having been reported . The increase in caesarean section rates is largely driven by several factors which include societal demands for improved fetal outcome, protection of pelvic floor, and the aspiration of obstetricians to meet these demand.8 caesarean section can be performed either as an elective or emergency procedure . The former constitutes bulk of the cases.36 the indications for elective caesarean section are many and varying and are often relative rather than absolute . They include contracted pelvis, major degree placenta previa, two or more previous caesarean section, malpresentation, hiv infection in pregnancy, previous vesico - vaginal fistula repair, intrauterine growth restriction, and bad obstetric history.1469 usmanu danfodiyo university teaching hospital (uduth) sokoto, a 500 bed tertiary health center, serves sokoto, kebbi, and zamfara states in nigeria and also receives patients from the neighboring niger republic . The population served is mainly the hausa / fulani ethnic group and of islamic faith . Girls are often married before the age of menarche thus making adolescent pregnancies and their associated complications common finding in the area.8 some of the complications of adolescent childbirth (cephalopelvic disproportion with associated caesarean delivery and vesico vaginal fistula) often necessitate elective caesarean section in subsequent pregnancies.1011 paucity of data in the literature on elective caesarean section in sokoto, north western nigeria informed this study . This was a retrospective analysis of 2284 consecutive caesarean sections performed at uduth sokoto over 9 years (january 2002 to december 2010). The records from the labor room and operating theater were retrieved and checked for caesarean deliveries . The delivery records of patients that had elective caesarean sections were obtained and relevant variables extracted . The variables include age, type of caesarean section, its indication, type of anesthesia used, and the maternal outcome . There were 22,985 total deliveries at the facility out of which 2284 were caesarean sections giving a caesarean section rate of 9.9% . A total of 1784 patients (79.3%) had emergency caesarean section while 498 patients (21.8%) had an elective procedure . General and spinal anesthesia were used in 253 (53.6%) and 219 (46.4%) of cases respectively . The age of the patients ranged from 18 years to 44 years with a mean of 31.23.6 years . Table 1 shows the annual distribution of total, emergency, and elective caesarean section . It demonstrates a gradual rise in the rate of elective caesarean section increasing from 1.7% in 2002 to 3.2% in 2007 . Repeat caesarean section for two or more previous caesarean section (31.1%) and malpresentation (18.4%) were the most common indication for elective caesarean operation . There were 18 maternal deaths from caesarean section and one from the elective caesarean procedure while the rest from emergency caesarean section . The cause of that single death from the former was anaphylactic reaction to general anesthetic agent . Annual distribution of total, emergency, and elective caesarean section indications for elective caesarean section the caesarean section rate of 9.9% in this study is within the range of 6.4 - 32.1% reported in nigeria by previous authors.371214 however, the proportion elective caesarean section (20.7%) relative to its emergency counterpart in the present study is much higher than 5.8 - 16.4% reported in most centers in the country.467 this may be attributed to relatively high rate of cephalo - pelvic disproportion arising from early marriage and teenage pregnancy in the study area10 which would have necessitated two previous emergency caesarean deliveries followed by the elective caesarean procedure . In addition, high rate of vesico - vaginal fistula from prolonged obstructed labor in the study zone15 may be contributory . This is because when the fistula is successfully repaired, subsequent deliveries are usually by elective caesarean operation.16 the rising trend of elective caesarean in these data has similarly been observed by previous authors.46 the reason for this may be the ever increasing list of the indications of elective caesarean delivery, larger cohort of patients with two or more previous caesarean section, and improved patient selection by clinicians with the use of better diagnostic techniques such as ultrasound machine.5 the most common indication in this study, repeat caesarean section, has also been reported in previous studies.4613 however, the second leading indication for the procedure in these data, malpresentation, is in contrast to bad obstetric history and hiv infection in pregnancy reported in jos and makurdi respectivelty.46 in conclusion, the rising trend of elective caesarean section observed in this study, underscores the need to better and improved patient selection together with counseling on its benefits and risks as elective caesarean operation though safer than its emergency counterpart is not entirely free of morbidity or mortality to both the mother and the baby . In addition routine use of spinal anesthesia in performing the procedure should be encouraged.
Various environmental chemicals, industrial pollutants and food additives have been implicated as causing harmful effects . One such food additive is monosodium glutamate (msg) and it is sold in most open market stalls and stores in nigeria as the safety of msg's usage has generated much controversy locally and globally . In nigeria, most communities and individuals often use msg as a bleaching agent for the removal of stains from clothes . There is a growing apprehension that its bleaching properties could be harmful or injurious to the body, or worse still inducing terminal diseases in consumers when ingested as a flavour enhancer in food . Despite evidence of negative consumer response to msg, reputable international organizations and nutritionist have continued to endorse msg, reiterating that it has no adverse reactions in humans . Notably of such is the directorate and regulatory affairs of food and drug administration and control (fda&c) in nigeria, now nafdac has also expressed the view that msg is not injurious to health . Msg improves the palatability of meals and thus influences the appetite centre positively with it resultant increase in body weight . Though msg improves taste stimulation and enhances appetite, reports indicate that it is toxic to human and experimental animals . Msg has a toxic effect on the testis by causing a significant oligozoospermia and increase abnormal sperm morphology in a dose - dependent fashion in male wistar rats . It has been implicated in male infertility by causing testicular hemorrhage, degeneration and alteration of sperm cell population and morphology . It has been reported that msg has neurotoxic effects resulting in brain cell damage, retinal degeneration, endocrine disorder and some pathological conditions such as addiction, stroke, epilepsy, brain trauma, neuropathic pain, schizophrenia, anxiety, depression, parkinson's disease, alzheimer's disease, huntington's disease, and amyotrophic lateral sclerosis . It cannot be stated that msg is the cause of such varied conditions as epilepsy and alzheimer's disease, although there may be concerns of its involvement in its etiology . The fallopian tubes are paired, tubular, seromuscular organs whose course runs medially from the cornua of the uterus toward the ovary laterally at the upper margins of the broad ligaments between the round and utero ovarian ligaments . Millions of tiny hair - like cilia which beats in waves hundreds of times a second catching the egg at ovulation and moving it through the tube to the uterine cavity, line the fimbria and interior of the fallopian tubes . Other cells in the tube's inner lining or endothelium nourish the egg and lubricate its path during its stay inside the fallopian tube .. the tubal wall consists of three layers: the internal mucosa (endosalpinx), the intermediate muscular layer (myosalpinx), and the outer serosa, which is continuous with the peritoneum of the broad ligament and uterus, the upper margin of which is the mesosalpinx . The endosalpinx is thrown into longitudinal folds, called primary folds, increasing in number toward the fimbria and lined by columnar epithelium of three types: ciliated, secretory, and peg cells . In the ampullary and infundibular sections, secondary folds of the tubal mucosa also exist, markedly increasing the surface areas of these segments of the tube . The myosalpinx actually consists of an inner circular and an outer longitudinal layer to which a third layer is added in the interstitial portion of the tube . The serosa of the tube is composed of an epithelial layer histologically indistinguishable from peritoneum elsewhere in the abdominal cavity . This work was carried out to investigate some probable histological effects of msg on the fallopian tubes and its likely involvement in female infertility in nigeria . About 15% cases of female infertility, investigation will show no abnormality . In these cases abnormalities this study was given approval for the methodology and other ethical issues concerning the work by the university of benin research ethics committee . Twenty four, (24) adult female wistar rats with average weight of 230 g were randomly assigned into three groups a, b and c of (n=8) in each group . Groups a and b (n=16) serves as treatment groups while group c (n=8) was the control . The rats were obtained and maintained in the animal holdings of the department of anatomy, school of basic medical sciences, university of benin, benin city, nigeria . They were fed with growers mash obtained from edo feed and flour mill limited, ewu, edo state, and were given water liberally . The monosodium glutamate (3g / sachet containing 99+% of msg) was obtained from kersmond grocery stores, uselu, benin city . Monosodium glutamate administration: the rats in the treatment groups (a and b) were given 0.04mg / kg and 0.08mg / kg of msg thoroughly mixed with the growers mash, respectively on a daily basis . The control group (c) received equal amount of feeds (growers mash) without msg added for fourteen days . The fallopian tubes were quickly dissected and fixed in 10% buffered formaldehyde for 24 hours and embedded in paraffin wax for routine histological techniques . The 0.04mg / kg and 0.08mg / kg msg doses were chosen and extrapolated in this experiment based on the indiscriminate use here in nigeria due to its palatability, and previous work done with this additive . The two doses were thoroughly mixed with fixed amount of feeds (550 g) in each group, daily . Histological study: the fallopian tube tissues were dehydrated in an ascending grade of alcohol (ethanol), cleared in xylene and embedded in paraffin wax . Ph otomicrographs of the desired results were obtained using digital research photographic microscope in the university of benin research laboratory . The fallopian tubes (ft) of the control group showed normal histological features, illustrating a well defined tubal wall which consists of three layers: the internal mucosa (endosalpinx), the intermediate muscular layer (myosalpinx), and the outer serosa (fig . The fallopian tubes of the treated groups showed some cellular hypertrophy (ch) of the columnar epithelium, distortion of the basement membrane separating the endosalpinx from the myosalpinx . There were degenerative and atrophic changes (adc) observed in some parts; these were more pronounced in those that received 0.08mg / kg of msg . There were marked vacuolations (v) and lysed red blood cells (lrbc), (0.08mg / kg treated rats) appearing in the stroma cells (figs . 2 and 3). Adc: atrophic and degenerative changes; ch: cellular hypertrophy; v: vacuolation . Treatment section of fallopian tubes (0.08mg / kg msg); h&e (mag . Adc: atrophic and degenerative changes; v: vacuolation; ch: cellular hypertrophy; lrbc: lysed red blood cells . The results of the hematoxylin and eosin staining (h & e) reactions showed some cellular hypertrophy of the columnar epithelium, distortion of the basement membrane separating the endosalpinx from the myosalpinx . Degenerative and atrophic changes were observed in some parts; these were more pronounced in those that received 0.08mg / kg of msg . There were marked vacuolations and lysed red blood cells (0.08mg / kg treated rats) appearing in the stroma cells . The increase in cellular hypertrophy of the columnar epithelium in the treatment groups as reported in this study may have been as a result of cellular proliferation caused by the improved intake of food which msg influences . The vacuolations observed in the stroma of the fallopian tubes in this experiment may be due to msg interference . Degenerative and atrophic changes and lysed red blood cell which were observed were more pronounced in the groups treated with higher dose (0.08mg / kg) of msg . As a result of the distortion and disruption in the lumen of the fallopian tubes, the ciliary action and other functions of the fallopian tubes may have been highly affected as a result of probable toxic effect of msg . It may be inferred from the present results that higher dose and prolonged administration of msg resulted in degenerative and atrophic changes observed in the fallopian tubes . The actual mechanism by which msg induced cellular degeneration observed in this experiment needs further investigation . Degenerative changes have been reported to result in cell death, which is of two types, namely apoptotic and necrotic cell death . Pathological or accidental cell death is regarded as necrotic and could result from extrinsic insults to the cell such as osmotic, thermal, toxic and traumatic effects . In this experiment msg the process of cellular necrosis involves disruption of membrane's structural and functional integrity which was also a landmark of this experiment . Cell death in response to toxins occurs as a controlled event involving a genetic programme in which caspase enzymes are activated . The results obtained in this study following the administration of 0.04mg / kg and 0.08mg / kg per day of msg to adult wistar rats caused some cellular hypertrophy of the columnar epithelium, distortion of the basement membrane separating the endosalpinx from the myosalpinx . Degenerative and atrophic changes, which were more pronounced in those that received 0.08mg / kg of msg.
Fucosidosis is a rare lysosomal storage disorder, which is inherited in autosomal recessive pattern due to deficient activity of the enzyme alpha l fucosidase . Type i presents in infancy with a very rapid progression of illness and death in early childhood, whereas type ii presents later with lesser severity although it is gradually progressive, and sometimes people survive into adulthood . So far around 100 cases have been reported worldwide with only one case from india . Here, we present two siblings with fucosidosis with characteristic clinical, radiological and laboratory features with a brief review of the literature . An 8-year - old girl presented with features of neuroregression from around 3 years of age . Her developmental milestones were delayed in all domains (social smile - 3 months, head control - 5 months, rolling over - 8 months and walking unassisted and stranger awareness - 2 years). She had steady developmental gains till 3 years of age after which she developed insidious onset global regression of her acquired milestones . She presented to us at 8 years of age in bed bound state with spasticity and generalized dystonia . She had lost her language skills and currently indicates toilet needs and makes sounds without any meaningful words . She had elevated telangiectatic lesions on her palms and soles [figure 1a], which was noticed from 7 years of age . Her younger sibling, a 3-year - old girl, who was delivered preterm has mild developmental delay with autistic traits and mild coarse facies, but has been having good gains with developmental stimulation . (a) sole of the foot of a child showing multiple elevated telangiectatic lesions . Widening of the medial end of clavicles (b), deficiency of the medial end of radial epiphysis (c), inferior beaking of thoraco lumbar vertebrae (d), widening of the acetabulum (e) her blood counts, liver and renal functions were normal . X - rays [figure 1b e] showed widening of medial ends of clavicles, deficiency of medial part of radial epiphysis, inferior beaking of thoracolumbar vertebrae and widening of acetabulum . Magnetic resonance imaging (mri) brain revealed features of hypomyelination with diffuse t2-weighted hyperintensity in subcortical and periventricular cerebral white matter . Globus pallidus (gp), substantia nigra and thalamus were hypointense in t2-weighted images, and t1 showed hyperintensity of the gp [figure 2a d]. There were two hyperintense curvilinear streaks within the lentiform nucleus on t2-weighted images corresponding to the lateral and medial medullary lamina of the gp [figure 3]. Magnetic resonance imaging brain t2-weighted axial image showing hypointensity of globus pallidus bilaterally and the subcortical hyperintensity (a), t1-weighted axial image is showing the hyperintensity of globus pallidi (b), t2 fluid attenuated inversion recovery images shows hypointensity of globus pallidi and hyperintensity of subcortical white matter (c), t2 axial image showing the hyperintensity of subcortical u fibers and deep cerebral white matter suggestive of hypomyelination (d) t2 coronal sections showing the hyperintensity of medial and lateral medullary lamina of the globus pallidi the clinical picture of neuroregression, spasticity, dystonia, coarse facies, dysostosis multiplex, telangiectasia, mri feature of hypomyelination with t2 hypointense and t1 hyperintense gp and substantia nigra was suggestive of fucosidosis . Fucosidase enzyme activity could not be detected in the leukocytes, which confirmed the diagnosis of fucosidosis . The defect leads to intracellular accumulation of fucose containing glycolipids and glycoproteins in various organs leading to the clinical manifestations . The clinical picture consists of progressive mental and motor deterioration, coarse facies, recurrent infections, dysostosis multiplex, angiokeratoma corporis diffusum, visceromegaly and seizures . Our patient had coarse facies, dysostosis multiplex, telangiectatic skin lesions, (which later evolve into angiokeratomas), progressive mental and motor deterioration . Our patients probably had a milder form with slow progression, yet had developmental delay noticed from early infancy . Classical mri finding described in fucosidosis include hypomyelination and demyelination with hyperintensity on t1 and marked hypointensity on t2-weighted images in the gp, diffuse symmetric t2 w hyperintensity of bilateral subcortical and deep white matter . The reason for signal alteration in gp is not exactly known, although several postulates like calcification, iron, manganese, cerebral glycolipid and triglyceride accumulation has been proposed . Our patient had classical mri finding, in addition to that our case had t2-weighted hyperintensity of medial and lateral medullary lamina of gp, which passes between the hypointense gp . Characteristic magnetic resonance spectroscopy findings reported are reduced n - acetyl aspartate / choline with elevated choline / creatine ratios and myoinositol, and a specific spectral peak at 3.8 - 3.9 ppm . Various forms of dysostosis multiplex has been described, which was noted in our case also . Our case had coarse facies with sociocognitive and motor regression, telangiectatic lesions in palms and soles, dysostosis multiplex and characteristic mri features of hypomyelination with gp and substantia nigra hypointensity in t2-weighted images and hyperintensity in t1 images with an undetectable fucosidase enzyme activity in leukocytes . The enzymes levels were not detected in her younger sibling also who has developmental delay with autistic traits . Since she is early in the course of illness, she has been referred for consideration of hematopoietic stem cell transplantation . The treatment options at present are limited to hematopoietic stem cell transplant, which has shown to be curative in anecdotal studies . Have described 22 disease causing mutations in fuca 1 gene, located on the short arm of chromosome 1, which included point mutations, deletions, insertions, missense and inactivating mutations . Early identification might lead to screening of presymptomatic siblings who might potentially be offered stem cell transplantation, which could be curative if done at an appropriate age.
Sequencing the complete genome of mycobacterium tuberculosis h37rv is a major milestone in the genome project and it sheds new light in our fight with tuberculosis . The genome contains around 4000 genes (protein - coding sequences) in the original genome annotation . However, we have found that the intergenic regions can exhibit expression signals, as evidenced by microarray hybridization . We conducted a genome - wide analysis using the affymetrix genechip to explore genes contained in the intergenic sequences of the m. tuberculosis h37rv genome . A working criterion for potential protein - coding genes was based on bioinformatics, consisting of the gene structure, protein coding potential, and presence of ortholog evidence . The bioinformatics criteria in conjunction with transcriptional evidence revealed potential genes with a specific function, such as a dna - binding protein in the copg family and a nickle binding gtpase, as well as hypothetical proteins that had not been reported in the h37rv genome . This study further demonstrated that microarray - based transcriptional evidence would facilitate genome - wide gene finding, and is also the first report concerning intergenic expression in m. tuberculosis genome.
However, oxygen also poses a potential hazard via reactive oxygen species (ros) and reactive nitrogen species (rns), with biological and functional alterations of lipids, proteins, and deoxyribonucleic acid (dna) [13]. Therefore, ros / rns have been initially considered as harmful products of the normal aerobic metabolism . The control of ros / rns production plays physiological roles, especially, in regulating cell signaling to involve cell proliferation, differentiation, and apoptosis [13]. Oxidative stress (os) mediated by free radicals is defined as an imbalance between the production of ros / rns and the antioxidant capacity of the cell [13]. The predominant inhibitory neurotransmitter in the brain is -aminobutyric acid (gaba), and almost all researchers have focused on gaba or the regulation of gaba receptor (gabar) in the brain [48]. Currently, gaba is considered to be a multifunctional molecule with various physiological effects throughout the body [9, 10]. In the brain, many researchers have found that the regulation of gabar has preventive effects against os - induced damage [5, 7, 8]. These results in the brain were mainly explained via specific pathways against os (i.e., inhibition of the response to dna damage [5, 11, 12] and promotion of cell survival [13, 14] or the free radical scavenging system [15, 16]). However, in the liver, the effects of gabar regulation have not been reported . Orthotopic liver transplantation (olt) is an accepted therapy for children and adults with end - stage liver disease, and it currently provides long - term survival and quality lifestyle . However, cold ischemia during organ storage and subsequent reperfusion severely damage the transplanted liver . During cold ischemic preservation, parenchymal cells swell and bleb, and then kupffer and endothelial cells trigger ros / rns production after warm reperfusion . This cold ischemia / warm reperfusion (ciwr) injury is still a major cause of morbidity and mortality after olt due to primary graft dysfunction or a nonfunctioning graft . Reperfusion not only triggers the liver regeneration cascade but also causes fatal damage in the liver graft due to os [18, 19]. Proactive strategies through pharmacological pretreatment to limit graft damage from ciwr injury have the advantage of excellent graft function after olt . A small - for - size graft (sfsg) is also an issue in deceased - donor liver transplantation (ddlt) and living - donor liver transplantation (ldlt). An sfsg is defined as a ratio of graft weight against standard liver volume <40% [20, 21]. An inevitable insufficiency of graft size cannot be avoided in ldlt or split orthotopic liver transplantation (solt) for ddlt . Shear stress not only triggers the liver regeneration cascade but also causes fatal damage in the sfsg by os [22, 23]. An sfsg in ldlt or solt is accompanied by ciwr injury, as well as shear stress with portal hypertension . The choice of a left - side graft is preferred from the viewpoint of greater donor safety and expanded donor candidates in ldlt [20, 24]. Guaranteed solt with successful outcomes resolves a donor shortage in ddlt [24, 25]. Our laboratory has focused on the effect of gabar regulation on liver damage by using rodent models [2628]. We failed to show beneficial effects in gabar regulation ex vivo and in gabar regulation by a specific antagonist [27, 28]. However, gabar regulation in vivo by a specific agonist showed a subtle reduction in liver damage in a murine hepatectomy model involving shear stress with portal hypertension and in a rat orthotopic liver transplantation model with a whole - liver graft involving ciwr injury . Proactive strategies through pharmacological pretreatment to limit graft damage from ciwr injury and shear stress with portal hypertension have advantages for overcoming a current issue . As a final goal of gabar regulation in the liver, we investigated the strategic potential of graft pretreatment in vivo by a gabar agonist in the rat solt model with a 40%-sfsg, and we examined the possible pathways involved . Graft donors and recipients were 812-week - old rats (approximately 250 g). The experimental protocols were approved by the ethical committee of our institution (mayo clinic, institutional animal care and use committee, no . Rats were cared for in accordance with the institutional guidelines for animal welfare based on the national institutes of health guide for the care and use of laboratory animals . A dose of 43.56 nmol / g body weight of gabar agonist (gabaa receptor agonist, muscimol, 114.10 g / mol; 70015, fluka, sigma - aldrich co., st . Four hours before graft harvest, the donor rat intravenously received 1.0 ml of gabar agonist into the penile vein . Comprehensive details of the surgical procedures for rat solt and postoperative care in our institution have been previously described [29, 30]. Briefly, the syngeneic graft had a cold ischemic time of 2 h at 4c in normal ringer's solution . The liver graft was washed twice by 10 ml of normal ringer's solution, immediately after the graft harvest and before graft implantation . The 40%-sfsg was made by the left median and lateral segments at the back table [29, 30]. To avoid any irrelevant signaling, the hepatic artery was reconstructed by ultramicrosurgery in this study [29, 30]. Each rat was housed separately after surgery, and body temperature was maintained by a heating pad . Postoperative observation was performed every 30 min until 6 h after solt, and 1.0 ml of warm lactate ringer's solution was routinely administered every 1 h until 6 h after solt . In this model, we previously demonstrated the importance of a shortened anhepatic phase and exclusion of unreliable samples based on autopsy findings [29, 30]. In this study, the anhepatic phase was maintained within 20 min in each solt, and no surgical complications were observed in each case at autopsy . Recipient rats were divided into three groups according to donor treatments and the recipient's surgery as follows: (i) saline (normal saline, 1.0 ml, i.v .) And laparotomy, (ii) saline (normal saline, 1.0 ml, i.v .) And solt with 40%-sfsg, and (iii) gabar agonist (muscimol, 43.56 nmol (4.98 g)/g body weight, 1.0 ml, i.v .) And solt with 40%-sfsg . First, a survival study was performed (n = 10 in each group). Cell signalings involving cell proliferation, differentiation, and apoptosis were investigated from the early postoperative period [18, 3133], and subsequently, progressive necrosis was observed [18, 3133]. Serum, plasma, and liver samples for histopathological / immunohistological assessment and western blotting analyses were then collected 6 h after solt (n = 5 in each group). Aspartate aminotransferase (ast), alanine aminotransferase (alt), and total bilirubin (t - bil) levels, and the international normalized ratio of prothrombin time (pt - inr) were measured . Serum ast, alt, and t - bil levels were assessed (sgot, sgpt, and total bilirubin reagent, respectively, biotron, hemet, ca, usa). The pt - inr in plasma was measured by the i - stat system (abbott, princeton, nj, usa). Liver tissue was fixed in 10% neutral - buffered formalin, embedded in paraffin, and sliced into 4 m sections . Morphological characteristics and graft injury scores were assessed after hematoxylin - eosin (he) staining . The graft damage score (points) has previously been described elsewhere [30, 34, 35]. Scores were counted in 10 fields (100) in each slide, and then these scores were averaged . Induction of apoptosis was assessed by immunostaining of terminal deoxynucleotidyl transferase - mediated deoxyuridine triphosphate nick - end labeling (tunel) (apoptag peroxidase in situ apoptosis detection kit, s7100, chemicon international, inc ., billerica, ma, usa) and cysteine aspartic acid protease (caspase) 3 (cleaved caspase-3 (asp175) antibody, 9661s, cell signaling technology, inc ., danvers, ma, usa). Tunel - positive nuclei were stained brown, and negative nuclei were counterstained light blue . Slides were scanned with an automated high - throughput scanning system (scanscope xt, aperio technologies, inc ., vista, ca, usa). To quantify the immunohistological findings, positively stained nuclei were counted by aperio imagescope software (aperio technologies, inc . ). All nuclei were classified into four color intensity levels, and the higher two levels were considered as positive . The ratio of positively stained nuclei to all nuclei was calculated, and the mean ratio per mm was determined . The primary antibodies for 4-hydroxynonenal (4-hne) (4 hydroxynonenal antibody, ab46545, abcam, cambridge, ma, usa), ataxia - telangiectasia mutated kinase (atm) (phospho - atm / atr substrate rabbit mab, cell signaling technology), phosphorylated histone h2ax (phospho - histone h2ax antibody, 2577, cell signaling technology), phosphatidylinositol-3 kinase (pi3k) (phospho - pi3k p85/p55 antibody, 4228, cell signaling technology), akt (phospho - akt rabbit mab, 4058, cell signaling technology), superoxide dismutase (sod) 1 (cu / zn superoxide dismutase, ls - b2907, lifespan biosciences, seattle, wa, usa), sod 2 (mn superoxide dismutase, ls - c62194, lifespan biosciences), and catalase (catalase, ls - b2554, lifespan biosciences) were used . Liver samples were collected, homogenized, and centrifuged at high speed for 10 min at 4c . The supernatant was then collected and used for bicinchoninic acid protein determination (bca protein assay reagent, thermo fisher scientific, rockford, il, usa) and western blot analysis . Forty micrograms of protein were run on 420% tris - glycine gels and transferred onto 0.45 m nitrocellulose membranes . The membranes were then blocked with 5% nonfat milk made up in a tris - buffered saline solution . After blocking, the next day, the membranes were washed three times for 10 min with tris - buffered saline solution and then incubated with the peroxidase - conjugated secondary antibody for 1 h, with shaking at room temperature . After incubation, the membranes were once again washed three times for 10 min with tris - buffered saline solution and then developed using chemiluminescence . Signals were quantified by using imagequant 5.0 software (molecular dynamics, sunnyvale, ca, usa). The student's t - test was used for the comparison of unpaired continuous variables between groups . Survival curves were constructed by the kaplan - meier method (log - rank test). Solt with a 40%-sfsg clearly showed poorer survival than laparotomy (p <0.0001), and graft pretreatment by gabar agonist prolonged survival after solt (p = 0.0369). Inflammatory cell infiltration, vacuolization, hepatocyte ballooning, and necrosis were confirmed after solt with a 40%-sfsg . Actual histopathological findings in h - e staining are shown in each group in figures 1(b)1(d). There were significant differences between laparotomy and solt with saline (0.0 0.0 versus 5.8 1.1 points, p <0.0001) and between solt with saline and solt with gabar agonist (5.8 1.1 versus 4.1 1.0 points; p = 0.0280) (figure 1(e)). There were significant differences in serum ast levels between laparotomy and solt with saline (45.4 10.3 versus 387.4 36.8 u / l; p <0.0001) and between solt with saline and solt with gabar agonist (387.4 36.8 versus 296.0 32.3 there were significant differences in serum alt levels between laparotomy and solt with saline (54.2 9.2 versus 354.2 32.1 u / l; p <0.0001) and between solt with saline and solt with gabar agonist (354.2 32.1 versus 272.4 31.3 there were significant differences in serum t - bil levels between laparotomy and solt with saline (0.28 0.04 versus 1.37 0.29 mg / dl; p <0.0001) and between solt with saline and solt with gabar agonist (1.37 0.29 versus 1.02 0.15 mg / dl; p = 0.0453) (figure 2(c)). There were significant differences in pt - inr between laparotomy and solt with saline (0.99 0.04 versus 1.22 0.06; p = 0.0001) and between solt with saline and solt with gabar agonist (1.22 0.06 versus 1.13 0.06; p = 0.0456) (figure 2(d)). The ratio of tunel - positive nuclei was significantly different between laparotomy and solt with saline (0.001 0.002 versus 0.166 0.052; p <0.0001) and between solt with saline and solt with gabar agonist (0.166 0.052 versus 0.092 0.038; p = 0.0324) (figure 3(d)). The ratio of caspase 3-positive nuclei was significantly different between laparotomy and solt with saline (0.0001 0.0001 versus 0.115 0.019; p <0.0001) and between solt with saline and solt with gabar agonist (0.115 0.019 versus 0.080 0.024; p = 0.0347) (figure 4(d)). Normalized 4-hne showed significant differences between laparotomy and solt with saline (1.00 0.06 versus 1.38 0.22; p = 0.0068) and between solt with saline and solt with gabar agonist (1.38 0.22 versus 1.05 0.15; p = 0.0276) (figure 5(b)). Actual intensities of atm and h2ax in each group are shown in figure 6(a). Normalized atm showed significant differences between laparotomy and solt with saline (1.00 0.11 versus 1.21 0.11; p = 0.0131) and between solt with saline and solt with gabar agonist (1.21 0.11 versus 0.90 0.28; p = 0.0477) (figure 6(b)). Normalized h2ax showed significant differences between laparotomy and solt with saline (1.00 0.10 versus 2.59 0.66; p = 0.0007) and between solt with saline and solt with gabar agonist (2.59 0.66 versus 0.83 0.25; p = 0.0005) (figure 6(c)). Actual intensities of pi3k and akt in each group are shown in figure 7(a). Normalized pi3k showed significant differences between laparotomy and solt with saline (1.00 0.11 versus 0.59 0.27; p = 0.0139) and between solt with saline and solt with gabar agonist (0.59 0.27 versus 0.92 0.13; p = 0.0443) (figure 7(b)). Normalized akt showed significant differences between laparotomy and solt with saline (1.00 0.12 versus 0.34 0.24; p = 0.0006) and between solt with saline and solt with gabar agonist (0.34 0.24 versus 1.11 0.22; p = 0.0007) (figure 7(c)). Actual intensities of sod 1, sod 2, and catalase in each group are shown in figure 8(a). Normalized sod 1 showed significant differences between laparotomy and solt with saline (1.00 0.10 versus 0.81 0.16; p = 0.0445) but not between solt with saline and solt with gabar agonist (0.81 0.16 versus 0.82 0.12; p = 0.8248) (figure 8(b)). Normalized sod 2 showed significant differences between laparotomy and solt with saline (1.00 0.13 versus 0.79 0.14; p = 0.0361) but not between solt with saline and solt with gabar agonist (0.79 0.14 versus 0.84 0.15; p = 0.5765) (figure 8(c)). Normalized catalase showed no significant differences between laparotomy and solt with saline (1.00 0.14 versus 0.95 0.14; p = 0.6904) and between solt with saline and solt with gabar agonist (0.95 0.14 versus 0.96 0.26; p = 0.9764) (figure 8(d)). Based on the current situation in the clinical field, the 40%-sfsg needs to be investigated in detail because successful solt overcomes a donor shortage in ddlt, and the shift to a left - lobe graft provides donor safety in ldlt [20, 24, 30]. However, the 40%-sfsg is prone to ischemia / reperfusion injury and shear stress with portal hypertension, and therefore, the os - induced damage after solt is more fatal [18, 3638]. In our study, a survival study, biochemical assays, and histopathological assessment showed that the 40%-sfsg received the liver injury enough . Os causes dna damage and subsequent apoptosis [13], and in our study, immunohistochemistry showed that solt induced apoptosis in the 40%-sfsg . Ros / rns can attack and damage a variety of critical biological molecules [13], and the products of lipid peroxidation reliably and rapidly reflect sensitive and specific signals due to os occurring in vivo [39, 40]. The fatty aldehyde 4-hne is an end product of lipoperoxidation [39, 40]. Therefore, os after solt with a 40%-sfsg resulted in apoptotic induction and subsequent necrosis . Os mediated by free radicals is defined as an imbalance between the production of ros / rns and antioxidant capacity [13]. Ros / rns have been suggested as a major contributing factor for dna damage in the progression of os . As a sensor of dna damage responses, the protein kinase atm can be initiated through rapid intermolecular autophosphorylation induced by dna damage [12, 41]; it phosphorylates various proteins, and subsequently amplifies the responses to dna damage . H2ax is required for cell cycle arrest and dna repair following double - stranded dna breaks [5, 42]. Dna damage results in the rapid phosphorylation of h2ax by atm at sites of dna damage [5, 4345]. Our study showed that this response to and repair of dna damage via atm / h2ax was clearly triggered after solt with a 40%-sfsg and that this cascade is a possible pathway in the process of os - induced injury after solt with sfsg . Our preliminary data in the rat olt model with whole - liver grafts (i.e., a model for only ciwr injury) suggested that gabar regulation by a specific agonist showed differences in atm / h2ax . We consider that gabar regulation may have a beneficial effect against ciwr injury via the atm / h2ax pathway in the liver . From the viewpoint of the production of ros / rns in the process of os, akt also plays a critical role in controlling apoptosis [41, 46, 47] and promotes cell survival [4750]. Apoptotic machinery is inhibited by the activation of akt [46, 51, 52]. Akt is a component of the antiapoptotic process related to the activation of pi3k, and pi3k is upstream from akt [47, 53]. The cell survival pathway via pi3k / akt is also considered as an important signaling pathway to control apoptotic induction in the liver [54, 55]. Our study showed that this promotion of cell survival via pi3k / akt was disturbed after solt with a 40%-sfsg and that this cascade could be one of the possible pathways in the process of os - induced injury after solt with sfsg . Our preliminary data in the murine hepatectomy model (i.e., a model for only shear stress with portal hypertension) suggested that gabar regulation by a specific agonist showed differences in pi3k / akt . Therefore, we consider that gabar regulation may have a beneficial effect against shear stress with portal hypertension via the pi3k / akt pathway in the liver . From the viewpoint of antioxidant defenses, free radical scavenging enzymes, such as sod and catalase, also play an important role in reducing dna damage and subsequent apoptosis [2, 3, 56]. Normal cells are able to defend themselves against os through this scavenging system [3, 56]. Our study showed a decrease in sod 1 and sod 2 levels after solt with a 40%-sfsg, although we initially expected that antioxidant enzymes would increase . Our results appear to be consistent with a previous opinion that os impairs mitochondrial importing and processing of sod . However, another possible explanation for our results may be that this scavenging system failed, and some reactive molecules evaded the detoxification process and damaged potential targets because of drastic damage after solt with a 40%-sfsg, even though these scavenging enzymes can handle large amounts of ros / rns . Our results of the survival study, histopathological assessment, and biochemical assays showed that pretreatment for sfsg by gabar regulation in vivo affected graft damage after solt . Moreover, immunohistochemistry showed that this pretreatment reduced apoptotic induction after solt . In the field of brain research, the effect of gabar regulation on the prevention of os has been reported [57]. Although gaba was initially thought to be confined to the central nervous system, gaba is currently considered to be a multifunctional molecule with various physiological effects throughout the body [9, 10]. Although the liver contains gaba and hepatic gabar [10, 17], the effects of gabar regulation in the liver are unknown . Our study suggests that gabar regulation may have a strategic potential for 40%-sfsgs as a pharmacological pretreatment for reducing os - induced damage after solt, although solt with a 40%-sfsg involves fatal os due to dual damage (i.e., ciwr injury and shear stress with portal hypertension). Whether gabar regulation ex vivo (i.e., a procedure during organ storage) is more suitable for ldlt is unknown . Although our results showed the strategic potential of gabar regulation in vivo as a pretreatment for liver grafts, we failed to confirm a positive effect of gabar regulation ex vivo . Therefore, some innovations are still required for clinical application . In previous reports on the brain, many investigators have suggested that gabar regulation by a specific agonist or antagonist affects the response to reduce os - induced injury [5, 7, 8]. Their preventive effects in the brain have been mainly explained via specific pathways against os (i.e., inhibition of the response to dna damage [5, 11, 12] and promotion of cell survival [13, 14] or the free radical scavenging system [15, 16]). Many previous investigators have suggested that gabar regulation in the brain has certain effects on the response to and repair of dna damage via the atm / h2ax pathway in vivo and in vitro in the process of os [5, 11, 12]. Our study showed that the regulation of hepatic gabar also appeared to reduce os - induced dna damage via the atm / h2ax pathway as well as to have effects in the brain . With regard to the effects of gabar regulation on os in the brain, the pi3k / akt pathway promotes cell survival against dna damage [5, 13, 14, 46, 59]. Our study showed that regulation of hepatic gabar appeared to promote cell survival via the pi3k / akt pathway against os - induced dna damage as well as to have effects in the brain . However, antioxidant enzymes reduce os - induced damage . From the viewpoint of this scavenging system, some researchers have shown that gabar regulation in the brain has preventive effects against os - induced damage via antioxidant enzymes [15, 16]. Although sod 2 plays an important role in preventing dna damage in the sfsg, our results suggested that the effects of the regulation of hepatic gabar against os did not depend on this scavenging system . Overall, we speculate that the regulation of hepatic gabar has a preventive effect against os, by reducing dna damage via the atm / h2ax pathway and by promoting cell survival via the pi3k / akt pathway . However, antioxidant enzymes might be important for gabar regulation in the brain [15, 16]. In conclusion, regulation of gabar by a specific agonist in vivo works well in the liver, as well as the brain . Even though ciwr injury and shear stress with portal hypertension affect 40%-sfsgs after solt and results in fatal os, graft pretreatment in vivo by gabar regulation clearly improves graft damage after solt . This strategy may be advantageous for overcoming current issues in the ddlt and ldlt fields . The effects of gabar regulation on graft damage after solt with a 40%-sfsg appear to prevent os by reducing dna damage via the atm / h2ax pathway and by promoting cell survival via the pi3k / akt pathway.
We aimed to compare the international association of diabetes and pregnancy study groups (iadpsg) and the world health organization (who) criteria to diagnose gestational diabetes mellitus (gdm) in chennai, india . We reviewed the retrospective data of 1351 pregnant women who underwent screening for gdm at four selected diabetes centers at chennai (three private and one government). All women underwent an oral glucose tolerance test using 75 g glucose load and fasting, 1-h, and 2-h samples were collected . A total of 839 women had gdm by either the iadpsg or the who criteria, of whom the iadpsg criteria identified 699 and the who criteria also identified 699 women as having gdm . However, only 599/839 women (66.6%) were identified by both criteria . Thus, 140/839 women (16.7%) were missed by both the iadpsg and the who criteria . 687/699 (98.2%) of the women with gdm were identified by the who criteria . In contrast, each value of iadpsg criteria i.e., fasting, 1 h, and 2 h identified only 12.5%, 14%, and 22%, respectively . A single who cut - point of 2 h> 140 mg / dl appears to be suitable for large - scale screening for gdm in india and other developing countries . Along with the rising tide of the current epidemic of diabetes in india, the prevalence of gestational diabetes mellitus (gdm) is also rising in india . However, the diagnosis of gdm has always been beset with problems related to differing diagnostic criteria with conflicting evidence regarding the maternal and fetal outcomes . Till recently, the gdm diagnostic criteria proposed by world health organization (who) or the american diabetes association (ada) were followed in most countries . Though the who recommendation was not based on studies with maternal and fetal outcomes, the 2-h cut - off value of> 140 mg / dl for diagnosis of gdm was found to predict the neonatal outcomes in a fairly robust manner . Recently, the international association of diabetes and pregnancy study groups (iadpsg) based on the hyperglycemia and adverse pregnancy outcome study has introduced new gdm criteria in an attempt to unify the gdm criteria throughout the world . The iadpsg criteria require three samples i.e., fasting, 1 h, and 2 h after 75 g glucose, whereas the who criteria need two samples namely the fasting and 2 h, although in practice, only the 2 h is used . In this paper, we have applied the who and the iadpsg cut - off values and compared the two criteria with respect to their impact on diagnosing gdm among pregnant women seen at four diabetic clinics in chennai city in southern india . We reviewed the retrospective data of 1351 pregnant women who underwent screening for gdm at four selected diabetes centers at chennai (three private and one government). At diabetes centers, pregnant women with a high index of suspension with elevated glucose levels hence, the prevalence of gdm at such center would be very high and hence they do not reflect the prevalence of gdm in the community . All women underwent an oral glucose tolerance test (ogtt) using 75 g glucose load and fasting, 1-h, and 2-h samples were collected . The iadpsg and who criteria were compared for diagnosis of gdm . According to the iadpsg criteria, any one of the following criteria was used for diagnosis of gdm, i.e., fasting 92 mg / dl (5.1 mmol / l), 1 h 180 mg / dl (10.0 mmol / l), or 2 h 153 mg / dl (8.5 mmol / l). According to the who criteria, either fasting 126 mg / dl (7.0 mmol / l) or 2-h value 140 mg / dl (7.8 out of a total of 1351 pregnant women who underwent screening for gdm at four diabetes clinics in chennai, 839 women were diagnosed to have gdm either by the iadpsg or by the who criteria . The iadpsg criteria identified 699/839 (83.3%) of the total number of women classified as gdm . The who criteria, again, identified 699/839 (83.3%) of the total number of women with gdm . However, as shown in the venn diagram [figure 1], only 559 women of the total 839 women with gdm (66.6%) were identified by both the iadpsg and who criteria . Thus, 140/839 (16.7%) of the gdm women would have been missed if iadpsg criteria alone were used . Conversely, 140/839 (16.6%) of the gdm women would have been missed if who criteria alone was used . Venn diagram showing the gestational diabetes cases identified by both international association of diabetes and pregnancy study groups and world health organization criteria and by either criteria we next compared the usefulness of the who 2-h criteria alone in comparison to the full who criteria namely 2 h and fasting who criteria and the results are shown in table 1 . Use of the who 2-h criteria was found to identify 687/699 (98.2%) of the gdm cases identified by the full who criteria . Comparison of the components of the international association of diabetes and pregnancy study groups and world health organization criteria to identify gestational diabetes mellitus table 1 also compares the percentage of gdm women identified by the fasting, 1 h and 2 h iadpsg compared to the full iadpsg criteria i.e., any 1 elevated value . It can be seen that in contrast to the 2-h who criteria where one value could pick up the majority (> 98%) of gdm cases, each value of the iadpsg criteria identified much lower percentages of gdm cases compared to using all three values . Using the iadpsg criteria, the fasting value alone identified 12.5%, 1-h alone identified 14% and the 2-h alone identified 22% cases and using any two values identified only 51% of the gdm cases . Hence, it is clear that all three values are needed to identify gdm cases by the iadpsg criteria . Of the 88 women with fasting value alone elevated according to the iadpsg criteria, only 30 (34%) were picked up by who 2-h value . Among the 98 individuals who had elevated 1-h value on iadpsg, only 47 (48%) were picked up by who 2-h value . Comparing the 2-h cut - points of 140 mg / dl (who) and 153 mg / dl (iadpsg), it is seen that 113 women had a 2-h cut - point between 140 and 153 mg / dl . This study shows that the number of gdm cases identified at four selected diabetes centers in chennai is the same by who criteria and the iadpsg criteria . However, only 66.6% of the gdm cases identified by the who and iadpsg criteria are the same individuals, whereas 16.7% each of the gdm women identified by iadpsg and who criteria are different individuals . The who first proposed criteria for gdm using a 75 g ogtt in the 1980s . In its technical report published in 1994, it defined gdm as diabetes mellitus (dm) first recognized during pregnancy, and gestational impaired glucose tolerance (gigt) as impaired glucose tolerance (igt) first recognized during pregnancy . In 1998, who recommended new criteria . With regard to gdm, pregnant women who met the who criteria for dm or igt were classified as having gdm and, therefore, the term gigt disappeared . Some studies have been published taking fasting plasma glucose (fpg)> 126 mg / dl as the criteria for gdm . However, the more recent studies have altogether ignored the fpg criteria and have used only the 2-h> 140 mg / dl criteria of the who . When the ada lowered the fpg to 100 mg / dl from the previous 110 mg / dl for diagnosis of impaired fasting glucose in non - pregnant adults, the fpg level of 126 mg / dl in pregnancy started looking too high and most people just chose to ignore the fpg level for the diagnosis of gdm . However, till date, there is no official recommendation from who to drop fpg criteria and to follow only the 2-h value of 140 mg / dl . It appears an anomaly that in the who criteria, the fasting cut - off had been set at 126 mg / dl which is diagnostic of diabetes in non - pregnant adults, whereas the 2-h cut - off was set at 140 mg / dl, which is the diagnostic cut - point for igt in non - pregnant adults . Probably because of this inherent contradiction in the diagnostic criteria, the fasting values in the who criteria are not particularly useful to diagnose gdm and this might explain why the who 2-h value alone picked up over 98% of all cases diagnosed by both fasting and 2-h who criteria in this study . Another point to be noted is that if a pregnant woman has a fpg 126 mg / dl, it is considered overt diabetes complicating pregnancy, and not as gdm, by the iadpsg criteria . Another issue of concern is whether too many women would get diagnosed as gdm because of the low fpg cut - off in the iadpsg criteria . Indeed, of the 88 women who were diagnosed as gdm by virtue of their fpg abnormality alone using iadpsg criteria, only 30 (34%) were classified as gdm by the who criteria . A similar comparison with those with gdm according to the iadpsg 1-h cut - off value showed that only 47/98 (48%) had gdm by who criteria . It is thus possible that by reducing the fpg cut - point to 92 mg / dl, we could be over - diagnosing gdm in normal pregnant women . We have earlier reported that the sensitivity of the 2-h value in the glucose tolerance test (gtt) is much higher than the fasting plasma glucose among non - pregnant indian adults . Thus, it is reasonable to assume that since the iadpsg has raised the 2-h value in the iadpsg to 153 mg / dl, many cases of gdm could be missed . One of the limitations of the study is that with our present data, we cannot conclude whether iadpsg or who criteria is better for indian pregnant women as we do not have data on the maternal and fetal outcomes . In the absence of the outcome data, which however, was beyond the purview of the study, it was not possible to comment on the suitability of diagnosing gdm by either of the two criteria in this population . Nonetheless, this study compared the ease of use of two criteria in the population studied . Future studies should compare the outcomes of the gdm cases diagnosed by iadpsg and who criteria as this would provide the final answer as to which criteria is more suitable for indians . The second limitation is that as the cases were selected from diabetes centers where women had been referred with suspected diabetes, this study cannot be used to study the prevalence rates of gdm . This should be done in a maternity clinic or a general population where the entire population of pregnant women is screened to study the prevalence rates of gdm . This study however reflects the usefulness of the who and iadpsg criteria at a diabetes centers . However, the strength of the study is that it is the first to our knowledge, to directly compare the iadpsg and the who criteria, especially in an asian indian population . The who 2-h criteria of> 140 mg / dl alone appears to be sufficient to diagnose gdm, as it picks up the majority of gdm cases diagnosed by both the whole who criteria as well as the same number of cases as the three sample iadpsg criteria . This could have great benefit especially in rural areas in india where obtaining three blood samples as required by the iadpsg criteria, could be a major challenge.
Bronchial provocation testing may be employed for a variety of purposes, but it is most commonly used to assess the tendency of the bronchi to narrow in response to exogenous stimuli . Testing can be done by direct and indirect challenges using a number of different agents (table 1).table 1agents commonly used in bronchial provocation testing indirect challengesdirect challengesadenosine monophosphate (amp)acetylcholineeucapnic voluntary hyperventilation (evh)carbacholexercisehistaminehypertonic salinemethacholinemannitolnot yet approved in the us agents commonly used in bronchial provocation testing not yet approved in the us methacholine aerosol inhalation is the prototype of a direct inhalation challenge . Bronchoconstriction following inhalation results from the direct action of methacholine on acetylcholine receptors in airway smooth muscle . Methacholine bronchoprovocation challenge is highly sensitive in identifying bronchial hyperreactivity, and a negative test is often used to exclude asthma . Indirect challenges simulate airway responses to specific physiologic situations such as exercise [1, 2]. The most recent indirect challenge is the mannitol (man) dry powder challenge, which is available as a standardized test kit in some countries . It is currently in review, but not approved, by the food and drug administration for use in the us . The kit includes prefilled capsules with mannitol in escalating doses, to be administered in an easy - to - use, handheld dry powder device . The safety and efficacy of man dry powder inhalation challenge have been established in large phase iii trials involving healthy subjects and patients with asthma [3, 4]. Mannitol is a hypertonic stimulus, and inhalation is thought to exert an osmotic effect within the airway that subsequently leads to the release of inflammatory mediators from mast cells, basophils, and human lung fragments . In asthmatic patients this leads to airway narrowing similar to that observed with hypertonic saline and exercise challenge . A positive response to mannitol is used to identify patients who have exercise - induced bronchoconstriction (eib), with or without chronic symptoms of asthma . A negative response suggests that asthma is not present or, in some cases, that it is mild with intermittent symptoms or well - treated [3, 4, 6]. The simplicity of the mannitol challenge suggests that there may be some advantages over other bronchoprovocation tests . Mannitol is given by a handheld dry powder inhaler (dpi), so there is no specialized equipment required for the doctor s office . Use of the dpi also makes the test easy for patients to learn and permits the test to be conducted quickly [4, 6]. These advantages were recently confirmed in a study assessing the sensitivity and specificity of mannitol to identify eib as a manifestation of bronchial hyperreactivity in a highly selected population of possible asthmatics with a normal fev1 . Mannitol and methacholine were therapeutically equivalent to identify eib and establish a clinician s diagnosis of asthma . However, in addition to not requiring specialized equipment, the mannitol challenge was more reproducible and took less time to perform than the methacholine test . Only 43.5% of the subjects had a positive response to exercise as defined by at least a 10% reduction in fev1 on at least one of two challenges . To better understand the mechanism associated with a mannitol challenge, brannan et al . Studied 12 asthmatic and 9 nonasthmatic subjects for evidence of mast cell activation and leukotriene release . They measured urinary excretion of leukotriene (lt) e4 and prostaglandin (pg) d2 metabolites (and mast cell markers) n - methylhistamine and 9,11, pgf2 at 60 min before and 90 min after mannitol inhalation . Airway narrowing provoked by mannitol challenge in asthmatic subjects was associated with increased urinary excretion of 9,11, pgf2 . Excretion of this pgd2 metabolite is an index of mast cell activation, and following mannitol challenge it is probably secondary to increased osmolarity of the airway surface liquid . The data support earlier work showing changes in airway sensitivity to mannitol in the presence of various mediator antagonists . Interestingly, in subjects with asthma the histamine antagonist fexofenadine (2 180 mg over 14 h) reduced airway sensitivity to mannitol, while the leukotriene antagonist montelukast (3 10 mg over 36 h) caused a faster recovery of lung function to baseline post - challenge but had no effect on sensitivity . The same mediators and time sequence of release have been reported with exercise - induced asthma, suggesting a similar mechanism of action . The mast cell stabilizer nedocromil sodium (8 mg, 10 min prechallenge), also has been reported to significantly reduce bronchoconstriction to mannitol inhalation in subjects with exercise - induced asthma . More recently, both cromolyn sodium (40 mg, 15 min prechallenge), another mast cell stabilizer, and formoterol (24 mcg, 15 min prechallenge), a long - acting bronchodilator, were shown to protect against the change in 9,11, pgf2 concomitant with a reduction in the mannitol - induced fall in fev1 (by 35 and 95%, respectively). One of the early observations in the development of the mannitol challenge was that it has a tussive effect . Like other indirect bronchoprovocation challenges, asthmatics cough more than healthy controls in response to mannitol, and the tussive effect is independent of the bronchoconstrictive effect, suggesting different physiological mechanisms . However, the simplicity of the mannitol challenge compared to other indirect bronchoprovocation tests makes it a potentially useful tool for evaluating airways hyperresponsiveness and cough sensitivity . A pilot study looked at the dose of mannitol needed to provoke two or five coughs in 13 subjects with nonasthmatic chronic cough compared to 16 healthy subjects . The subjects with chronic cough showed a heightened cough response to challenge (i.e., less mannitol needed to provoke cough); none had airway responsiveness to mannitol . The findings support the hypotheses that (1) mannitol - provoked cough is increased in patients with nonasthmatic chronic cough, and (2) mannitol - provoked cough is independent of mannitol - provoked bronchoconstriction . The cough response may involve indirect activation of mast cells in the superficial airway and subsequent release or mediators that, in turn, activate local cough receptors . The separate observation that nedocromil sodium failed to attenuate mannitol - induced cough, in contrast to mannitol - induced bronchoconstriction, supports the lack of direct involvement of mast cell pathways . Similar findings have been reported for capsaicin, a common test for studying induced cough . Capsaicin, the hot ingredient in red pepper, is known to stimulate unmyelinated c fibers in the sensory nervous system . Studies have shown that coughing can be induced by capsaicin inhalation in a dose - dependent manner in healthy subjects and those with mild asthma, and that capsaicin challenge does not cause dyspnea and has no effect on the fev1 . It has been demonstrated that capsaicin - induced cough can be blocked by local anesthetic [1416] but not by pretreatment with the mast cell stabilizer cromolyn sodium . These data support the separation of neuronal pathways of coughing from bronchoconstrictive airway responses directly related to inflammatory mediators . Mannitol inhalation challenge may be useful for diagnosing and/or managing at least three types of patients described in the literature . Koskela et al . Analyzed the cough response to three airway challenges in order to clarify whether the recording of the provoked cough would be beneficial in the management of asthma . They used isotonic histamine, hypertonic saline solution, and hypertonic histamine, all delivered by an ultrasonic nebulizer using the 2-min tidal breathing method . Coughing during isotonic histamine challenge seemed to be a manifestation of bronchoconstriction, but recording the cough did not provide additional information to airflow measurements . Sensitivity to the cough - provoking effect of hypertonic challenges was enhanced in patients with asthma and was unrelated to airway hyperresponsiveness . These investigators, therefore, advocated that cough assessment during hypertonic challenges could help to identify those patients with cough - variant asthma . Johansson et al . Investigated the relationship between cough sensitivity to inhaled capsaicin and odor sensitivity using a chemical sensitivity scale for sensory hyperreactivity (css - shr), dividing subjects into four groups with different odor sensitivity according to their css - shr score . Of these, 137 individuals randomly received a capsaicin inhalation test in which the number of coughs encountered 10 min from the start of each inhalation was registered . More than 80% of subjects with positive capsaicin inhalation scores also had positive css - shr scores; only 5% of subjects with negative css - shr scores had a positive capsaicin inhalation test . The direct relationship between sensitivity to inhaled capsaicin and a high css - shr score makes it possible to relate subjective data to objective findings . Mannitol challenge of patients with odor insensitivity and dyspnea may support the diagnosis and lead to early and appropriate treatment . Mannitol - induced coughing was evident in 93% of 419 subjects studied for suspected eib as diagnosed by mannitol and methacholine challenges . Of these, 204 (49%) had occasional cough that did not interfere with the challenge, 178 (42%) had frequent cough that delayed administration of the next dose, and 9 (2%) had severe cough that interfered with the challenge . The studies cited above have shown heterogeneity in the cough response to mannitol . Using a cough counter supplied by karmel sonics - israel, my coworkers and two individuals had no cough at any time during the challenges, while the other had a cough throughout; all three had positive bronchoprovocation testing with drops in fev1 by more than 20% at premaximal doses . The variation in the cough responses probably reflects differences in the underlying mechanisms of airway hyperresponsiveness in these individuals, but more patients should be studied before drawing any conclusions . Similar findings have been reported by dicpinigaitis et al . Who reviewed the safety of capsaicin cough challenges over 20 years of clinical experience . Like the initial results to date with mannitol, no serious adverse events were associated with capsaicin cough - challenge testing, only minor and transient descriptions of throat irritation . However, heterogeneity in the cough response was observed: asthmatics without cough did not differ from healthy volunteers in terms of cough reflux sensitivity to capsaicin, though differences were evident in airway reactivity . The results from mannitol and capsaicin thus support the suggestion that cough and bronchial responsiveness are distinct phenomena mediated through unique neural pathways . Cough monitoring during bronchial challenges may help sort out subtypes of asthma and airway reactivity.fig . Cough monitoring from four studies on three asthma patients undergoing bpt demonstrates the cough - response variability among patients . The patients were monitored with the pulmotrack (model 3010-cc, karmelsonix ltd, haifa, israel) throughout the test . A, b patient whose exposure to mannitol triggered extensive coughing that diminished after albuterol (arrow). All patients had positive bpt with reductions in fev1> 20% at premaximal dose cough monitoring during mannitol bronchial provocation test (bpt). Cough monitoring from four studies on three asthma patients undergoing bpt demonstrates the cough - response variability among patients . The patients were monitored with the pulmotrack (model 3010-cc, karmelsonix ltd, haifa, israel) throughout the test . A, b patient whose exposure to mannitol triggered extensive coughing that diminished after albuterol (arrow). All patients had positive bpt with reductions in fev1> 20% at premaximal dose koskela et al . Analyzed the cough response to three airway challenges in order to clarify whether the recording of the provoked cough would be beneficial in the management of asthma . They used isotonic histamine, hypertonic saline solution, and hypertonic histamine, all delivered by an ultrasonic nebulizer using the 2-min tidal breathing method . Coughing during isotonic histamine challenge seemed to be a manifestation of bronchoconstriction, but recording the cough did not provide additional information to airflow measurements . Sensitivity to the cough - provoking effect of hypertonic challenges was enhanced in patients with asthma and was unrelated to airway hyperresponsiveness . These investigators, therefore, advocated that cough assessment during hypertonic challenges could help to identify those patients with cough - variant asthma . Johansson et al . Investigated the relationship between cough sensitivity to inhaled capsaicin and odor sensitivity using a chemical sensitivity scale for sensory hyperreactivity (css - shr), dividing subjects into four groups with different odor sensitivity according to their css - shr score . Of these, 137 individuals randomly received a capsaicin inhalation test in which the number of coughs encountered 10 min from the start of each inhalation was registered . More than 80% of subjects with positive capsaicin inhalation scores also had positive css - shr scores; only 5% of subjects with negative css - shr scores had a positive capsaicin inhalation test . The direct relationship between sensitivity to inhaled capsaicin and a high css - shr score makes it possible to relate subjective data to objective findings . Mannitol challenge of patients with odor insensitivity and dyspnea may support the diagnosis and lead to early and appropriate treatment . Mannitol - induced coughing was evident in 93% of 419 subjects studied for suspected eib as diagnosed by mannitol and methacholine challenges . Of these, 204 (49%) had occasional cough that did not interfere with the challenge, 178 (42%) had frequent cough that delayed administration of the next dose, and 9 (2%) had severe cough that interfered with the challenge . The studies cited above have shown heterogeneity in the cough response to mannitol . Using a cough counter supplied by karmel sonics - israel, my coworkers and two individuals had no cough at any time during the challenges, while the other had a cough throughout; all three had positive bronchoprovocation testing with drops in fev1 by more than 20% at premaximal doses . The variation in the cough responses probably reflects differences in the underlying mechanisms of airway hyperresponsiveness in these individuals, but more patients should be studied before drawing any conclusions . Similar findings have been reported by dicpinigaitis et al . Who reviewed the safety of capsaicin cough challenges over 20 years of clinical experience . Like the initial results to date with mannitol, no serious adverse events were associated with capsaicin cough - challenge testing, only minor and transient descriptions of throat irritation . However, heterogeneity in the cough response was observed: asthmatics without cough did not differ from healthy volunteers in terms of cough reflux sensitivity to capsaicin, though differences were evident in airway reactivity . The results from mannitol and capsaicin thus support the suggestion that cough and bronchial responsiveness are distinct phenomena mediated through unique neural pathways . Cough monitoring during bronchial challenges may help sort out subtypes of asthma and airway reactivity.fig . Cough monitoring from four studies on three asthma patients undergoing bpt demonstrates the cough - response variability among patients . The patients were monitored with the pulmotrack (model 3010-cc, karmelsonix ltd, haifa, israel) throughout the test . A, b patient whose exposure to mannitol triggered extensive coughing that diminished after albuterol (arrow). All patients had positive bpt with reductions in fev1> 20% at premaximal dose cough monitoring during mannitol bronchial provocation test (bpt). Cough monitoring from four studies on three asthma patients undergoing bpt demonstrates the cough - response variability among patients . The patients were monitored with the pulmotrack (model 3010-cc, karmelsonix ltd, haifa, israel) throughout the test . A, b patient whose exposure to mannitol triggered extensive coughing that diminished after albuterol (arrow). All patients had positive bpt with reductions in fev1> 20% at premaximal dose as a hypertonic agent, mannitol inhalation may have a beneficial effect on mucus elimination in patients with cf . An initial study demonstrated an improvement in lung function related to small airway obstruction and a significant improvement in respiratory symptoms and quality of life after a 2-week treatment with mannitol in 38 patients . The suggested mechanism for the positive action of mannitol in cf is the same as that for nebulized hypertonic saline treatment, i.e., osmotically induced water influx into the bronchial lumen thereby increasing the hydration of airway mucus so that it is cleared more effectively and expectorated . Enhanced ciliary clearance of mucus also should result in a sustained reduction in mucus load, thus providing less opportunity for proliferation of bacteria and infection leading to less antibiotic use and fewer hospitalizations . Long - term studies have been proposed and are presently being assessed by investigative protocols . In summary, mannitol is a simple, easy - to - administer indirect, inhalation challenge test . Sensitivity to mannitol is increased in patients with asthma and in patients with chronic cough, but mannitol - provoked cough occurs via different pathways than mannitol - provoked bronchoconstriction . Mannitol challenge testing may help sort out the heterogeneity in cough and bronchoconstrictive responses in certain patient populations . This article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
Saudi arabia (sa) is a large country with an area of about 2.2 million square kilometers and total population exceeding 27 million, of which 69% are saudis . Less than one quarter of cancer patients in sa present with localized disease . Indeed, most cancer patients present with advanced disease with regional or distant extension . The crude and age standardized cancer incidence rates in 2007 were 52.3 and 82.1 per 100,000 populations, respectively . The five most common cancers, in descending order, are colorectal, non - hodgkin lymphoma, leukemia, lung, and liver in males; and breast, thyroid, colorectal, non - hodgkin lymphoma, and leukemia in females . The palliative care (pc) program at king faisal specialist hospital and research center (kfshrc), riyadh, was established more than 20 years ago as the first program of its kind in the country . The program delivers services for inpatients, outpatients, as well as for those at home through a home health care service . The program has also established a structured postgraduate fellowship for physicians pursuing subspecialty in palliative medicine . Awareness of symptomatology pattern in cancer patients enables health care professionals to place appropriate emphasis on various challenges in a prioritized manner . Prioritization process may include patient care plans, professional education plans, and the needs of health care services . Furthermore, the quality of life of patients with advanced cancer could be significantly improved if specialized palliative care teams were involved in addressing the variety of symptoms that are common among these patients . Therefore, impeccable identification and management of symptoms are two crucial aspects of palliative care . A recent report from 11 european countries suggests that there are variations between countries in the number of inadequately treated cancer - related symptoms . To the best of our knowledge, no previous report has described the prevalence and severity of nonpain symptomatology among patients with advanced cancer in saudi arabia (sa). This paper aims at determining the prevalence and severity of 10 nonpain symptoms in cancer patients visiting the pc outpatient clinic at kfshrc, riyadh, sa . This is a cross - sectional survey exploring the pattern of 10 common nonpain symptoms in an outpatient palliative care clinic . The research advisory council (rac) of kfshrc have approved the research project (rac number 2101053) based on the approval of the research ethics committee . Adult cancer patients who were oriented to person, place, and time were consecutively included in the study . The 10 nonpain symptoms included tiredness, nausea / vomiting, anxiety, depression, shortness of breath, drowsiness, insomnia, dry mouth, loss of appetite, and confusion . The intensity of each symptom at the time of the encounter in the clinic was determined by each patient based on a 010 numerical scoring system where zero means the absence of respective symptom and 10 means the greatest severity a person could possibly imagine . The performance status was determined using the palliative performance scale (pps), which is a reliable and valid observer - rated tool for measuring performance status in pc patients . The pps consists of five domains (on a scale of 0%100%, with increments of 10%), namely ambulation, self - care, activity level / evidence of disease, intake, and conscious level . All interviews were conducted by one of the authors (oa) to avoid the potential of inter - rater variability . Data analysis was conducted using the software package sas version 9.2 (statistical analysis system, sas institute inc ., the student's t test for independent samples was used to compare means between two groups, such as those related to gender, age group, encounter type, and performance status . One - way analysis of variance was used for comparing the means among more than two groups such as in the case of grouping according to cancer type . A value of p less than 0.05 was considered significant . A total of 124 patients were interviewed, with a median age of 56 years and dominance of female gender (59%). One third of the patients were seen on follow - up basis and the rest were new to the palliative care outpatient clinic . All patients had advanced incurable cancer, with the majority (102; 82.3%) diagnosed with metastatic disease . For patients who were still receiving anticancer treatment, the most frequently encountered cancer types were female breast (27.4%), head and neck (15.3%), and genitourinary (12.9%). Only one patient denied any nonpain symptom while all other patients had from 1 to 10 nonpain symptoms per patient, with a mean number of symptoms of 5.1 2.2 . The majority of patients (93; 75%) had more than 3 nonpain symptoms each . The mean number of symptoms was higher in patients with pps of less than 70% (5.9 1.9) as compared with those who had better performance status (4.6 2.3) (p = 0.002). However, the mean number of symptoms did not show any statistically significant difference between groups of patients based on age, gender, cancer type, or the type of outpatient encounter . The most frequently reported symptoms were tiredness (79.8%), loss of appetite (71.8%), dry mouth (69.4%), and anxiety (60.5%), whereas the least prevalent symptoms were nausea and confusion (22.6% each) [table 2]. For symptomatic patients, the mean severity scores were highest for tiredness (5.1) and loss of appetite (5) and lowest for nausea and confusion (3.4 each) as shown in table 2 . Older patients (above the median age, 56 years) had lower mean anxiety score compared with younger patients, 1.9 and 3.2, respectively (p = 0.003). Females had higher mean severity scores than males in 4 symptoms, namely, tiredness (4.6 vs 3.5; p = 0.04), nausea / vomiting (1.0 vs 0.4; p = 0.02), anxiety (3.2 vs 1.7; p = 0.001), and loss of appetite (4.2 vs 2.7; p = 0.003). Patients with lower pps tended to have significantly higher severity scores for tiredness, shortness of breath, drowsiness, dry mouth, and loss of appetite, see table 2 . The mean severity scores of symptoms did not significantly differ between patients according to their cancer type or the nature of outpatient visit (new vs follow - up). However, when patients with primary or secondary lung or pleural disease were pooled in one group, they had higher mean severity scores of shortness of breath compared with other patients, 2.4 and 1.2, respectively (p = 0.01). Symptom prevalence and severity the participation of all approached patients in our study was not unexpected because the questions were all focused on the presence and severity of symptoms known to be fairly common among cancer patients . The median age of our study population agrees with the findings of a previous report on a larger group of advanced cancer patients in sa and appears generally younger than the median age of the palliative care population in western countries. [911] this might be explained by the fact that more than 97% of the saudi population represents those younger than 65 years . The finding that metastatic or locally advanced disease is the rule in the studied population may be explained by the trend of delayed diagnosis of cancer in sa as well as the common practice of delayed referrals to palliative care in kfshrc . Symptom prevalence studies on patients with advanced cancer have mostly targeted inpatients rather than outpatients . Furthermore, most reports have major variations in demographics of surveyed populations and in methodologies implemented, which obviously make comparisons between the findings of various reports less meaningful . That being said, our findings have shown higher prevalence of nonpain symptoms among patients with advanced cancer compared with what other researchers have reported . For instance, grond and colleagues have reported that cancer patients attending an outpatient pain clinic were having on average 3.3 nonpain symptoms the most prevalent of which were insomnia (59%) and loss of appetite (48%). According to several reports, it seems that the more symptoms are actively assessed the more symptoms will likely be reported. [1517] similar to our results, there is a common finding among various reports that tiredness (also reported as fatigue or weakness in some studies) is often on the top of the list of prevalent nonpain symptoms among patients with advanced cancer. [1620] potter and coauthors have reported nonpain symptoms in patients seen at an outpatient pc clinic in the uk, with the most prevalent symptoms being tiredness and nausea (18% each) and loss of appetite (17%). A recent report from lebanon has shown high prevalence of fatigue (88.4%), dyspnea (65.1%), depression (53.5%), and confusion (37.2%) among inpatients with advanced cancer . Alshemmari et al . From kuwait have studied cancer patients who were largely having advanced incurable disease and found the most common nonpain symptoms to be fatigue (80%), anorexia (67%), weight loss (49%), and dyspnea (42%). Overall, the severity of symptoms reported by our patients was generally more than what has been reported elsewhere on a group of cancer patients hospitalized for palliative reasons . Our findings have supported previous reports suggesting that poorer performance status may be associated with higher symptom burden . In addition, we have found that some symptoms might be associated with higher severity scores in patients with poor performance status . Have found, our data failed to show an association between symptom prevalence and age or gender of patients . However, younger patients in our study had higher severity scores of anxiety than older patients . Also, females reported higher severity scores as compared with males in few symptoms including anxiety . Our finding that patients with primary or secondary lung or pleural cancer were having severe shortness of breath supports what has been suggested by kirkova et al . That some symptoms may vary in prevalence or severity (or both) based on cancer site . In the pc outpatient clinic at kfshrc, it is common for patients relatives to attend follow - up clinics on behalf of patients for the sake of medication refill, hence the lower proportion of follow - up patients as compared with new patients in our study . However, one reason could be the fact that many patients reside outside riyadh and, like most saudis, have widely extended families with good chances of having relatives living in riyadh who would be happy to visit the clinic on behalf of the patients . Although air fares for health care - related travel are usually covered by the government, some patients may find it difficult to attend the clinic in person due to poor performance status . However, we are under the impression that a good number of patients who prefer to send relatives to attend the clinic on their behalf might be having symptoms that are fairly controlled . This could partially explain the high prevalence and severity of symptom burden in our sample as well as the finding that symptom prevalence and severity in our study was similar in patients new to the pc clinic and those returning to the clinic for follow - up . Limitations of our study include the relatively small number of participants and the cross - sectional nature of the study design . A longitudinal design with sequential assessments of a larger group of patients from different centers could have generated more generalizable data . A much longer list of nonpain symptoms could have been better representatives of symptom burden among this group of patients . Future multicenter research on the same lines may be focused on longitudinally assessing symptom burden in such patients, with emphasis on exploring the efficiency and effectiveness of symptom management strategies in pc outpatient settings . Despite the wide variation in reports that explored symptom pattern and prevalence in patients with advanced cancer, it is clear that they all agree upon the fact that patients with advanced cancer are polysymptomatic . The significant prevalence and severity of nonpain symptoms among new and follow - up cancer patients seen in a pc outpatient clinic emphasizes the need for comprehensive assessment and routinely audited symptom management plans.
Tuberculosis (tb) is common disease in developing and developed countries caused by various strains of mycobacteria, usually mycobacterium tuberculosis (m. tuberculosis)14). The most common initial infection site is the lung, but the brain can be infected by hematogenous transmission . Tb meningitis and tuberculoma are the two most common forms of tb of the central nervous system . Surgical treatment is only recommended when medical therapy fails, decompression is necessary, or the diagnosis is uncertain9,15). Transient worsening, appearance of new symptoms, or radiological manifestations of tb can develop after initiating of anti - tb medication4). These paradoxical responses are not uncommon in human immunodeficiency virus - negative patients and have been observed in 6 - 30% of patients with a tb infection6,7). These episodes of paradoxical deterioration are commonly associated with extrapulmonary tb and the central nervous system is the most common location for this presentation . However the lesion revealed indistinguishable radiological findings of a malignant intracranial tumor such as a high grade glioma after anti - tb treatment . We describe the radiological findings of the paradoxical response of a cerebral tuberculoma mimicking a malignant brain tumor . She underwent a chest computed tomography (ct), which showed minimal cardiac fluid collection with pleural effusion but without pleural enhancement or thickening . The pleural fluid was an exudate, with a white blood cell count of 907/mm and 30% lymphocytes . The quantiferon tb and immunoglobulin m for the ebstein - barr virus capsid antigen was positive . She refused anti - tb medication, and her symptoms improved after aspirin and steroid treatment . Brain magnetic resonance images (mri) showed 18 mm and 6 mm sized lesions on the pons and occipital lobe, respectively . The lesions were hypointense on t1-weighted images, and iso - intense with a central hyper - signal on t2-weighted images associated with perilesional edema and rim - enhancement after contrast administration (fig . The diffusion images showed diffusion restriction in the central area but no increased cerebral blood volume on mr perfusion images (fig . A follow - up chest ct showed randomly distributed miliary nodules and mixed small centrilobular nodules, suggesting pulmonary tb with miliary dissemination . Anti - tb medication was started with isoniazid, rifampicin, ethambutol, and pyridoxine . At the 1 month follow - up, the thickened, peripherally enhanced lesions were enlarged and associated with aggravated perilesional edema (fig . H mr spectroscopy revealed increased choline, lactate and lipid peak, and reduced n - acetyl aspartate and creatine, suggesting a high grade glioma rather than tuberculoma (fig . 2), and pcr for tb was positive . Her medication was changed to isoniazid, rifampicin and ethambutol . After 13 months of medication, the right hemiparesis improved and the lesion had decreased in size with decreased perilesional edema (fig . She continued the anti - tb medication to treat the remaining lung and brain lesions . Involvement of the central nervous system (cns) is the most severe form of tb11). A ring - enhancing lesion can be seen in intracranial infections or tumors such as tuberculomas, neurocysticercosis, bacterial cerebral abscesses, neurosarcoidosis, cerebral metastases, glioblastomas, and cns lymphomas8). A caseating tuberculoma with a solid center shows ring and central heterogeneous enhancement and is iso- to hypointense on t1- and t2-weighted mri . A caseating lesion with a liquid center shows ring enhancement and is hypointense on t1- and hyperintense on t2-weighted mri . Intense focal gyral enhancement can be seen, and a secondary cerebral infarction from obliterative end arteritis can be associated with intracranial tb16). A tuberculoma with liquid necrosis shows restricted diffusion with a low apparent diffusion coefficient, whereas those with solid necrosis do not . H mr spectroscopy of the lesion reveals lipid with increased choline and reduced n - acetyl aspartate and creatine12). Mr perfusion also shows increased cerebral blood volume on initial images, which gradually normalize with treatment, suggesting healing of the lesion12). The diagnosis is established based on the pathology results of a biopsy or detecting m. tuberculosis dna in a pcr study2). Treatment of a tuberculoma is based on anti - tb treatment regimens, which include two important antibiotic agents10). Isoniazid and rifampicin can be used for several months with combinations of other agents due to bacteria resistance . The initial treatment of extrapulmonary tb includes these two agents and the brain is not an exception . Expansion of an intracerebral tuberculoma or newly detected lesions can be seen on follow up images after anti - tb medication, which is called a paradoxical response or paradoxical progression5,17). The tuberculomas increase in size at 1 - 7 months after starting chemotherapy . The explanation for this paradoxical response during therapy remains unclear, but it may be related with local tissue reactivity17). The brain is known as' immunologically privileged', meaning that immune reactivity in brain is selective and modified . Foreign antigens including pathogens deposited in the brain parenchyma are not detected efficiently by the immune system in the cns . The experimental data showed that peripheral immune sensitization can result in the immune - mediated delayed - type hypersensitivity response, which can be used to explain' paradoxical reaction' of intracranial tuberculoma3). Once the active tb is under control with chemotherapy, enhanced delayed type hypersensitivity can cause activation and accumulation of lymphocytes and macrophages at the site of bacillary deposition or toxin production1). Therefore, many factors may combine to produce the paradoxical response, which might be related to a host immune response, virulence of the tubercle bacilli, the infection site, antigen load, or the effects of chemotherapy . These aggravated lesions can be misdiagnosed as treatment failure or other tumorous pathology . In our case, after 1 month of treatment, our patient developed worsening symptoms, radiological aggravation with tumoral spectrum on h mr spectroscopy, and increased blood volume on mr perfusion . These findings mimicked a high grade glioma even if this center of the lesion with liquid necrosis showed a diffusion restriction . Initial improvement followed by deterioration despite adequate diagnosis and treatment is strong evidence to suspect the paradoxical response on clinical grounds . The response can be effectively managed by continuing the anti - tb drugs, and eventual clinical improvement is observed in almost all reported cases . Systemic corticosteroids are probably indicated to manage symptoms and cerebral edema for most patients with a cerebral paradoxical response . However, careful consideration of corticosteroid administration must be given due to possible complicating factors such as the host's immune status or the presence of concomitant infections . Surgical intervention should be considered for patients with increased intracranial pressure or an uncertain diagnosis . Brain tuberculoma could be aggravated mimicking brain malignancy during administration of anti - tb medication . This paradoxical response can be effectively managed by continuing the anti - tb drugs with careful radiologic follow - up.
Left ventricular noncompaction (lvnc) is a rare primary congenital cardiomyopathy arising from intrauterine arrest of myocardial compaction . It results in persistent prominent ventricular trabeculations and deep intertrabecular recesses and is characterized morphologically by 2 layers i.e., thin compacted epicardial layer and thickened noncompacted endocardial layer.1) the prevalence of lvnc is approximately 0.05%-0.24% in the general population according to echocardiography, the diagnostic tool of lvnc.2) the etiology of lvnc is unidentified; however, possible linkage between the disease and various genetic background is recently suspected.3) diverse clinical manifestations previously reported range from no symptom to the development of heart failure, arrhythmias, systemic embolic events or sudden cardiac deaths.3) interestingly, several abnormal electrocardiograms (ecgs) were reported in lvnc patients but none had the feature of left atrial (la) standstill imitating atrioventricular (av) nodal block . We described a 29-year - old man who presented with a symptom of cerebrovascular accident (cva) and concomitant asymptomatic lvnc and la standstill . A 29-year - old man with the history of stroke was transferred from a local medical center to determine whether he should receive pacemaker insertion due to abnormal ecg findings . At first, he was admitted to a local clinic with dysarthria a month prior to the transfer, and was diagnosed as left middle cerebral artery territory infarction . His brain magnetic resonance imaging (mri) demonstrated multiple cortical infarcts implying embolic origin (fig . 1). Magnetic resonance angiography findings showed no evidence of pathologic stenosis or atheroma of the neck and head arteries . Asymptomatic bradycardia was shown on the local ecg with 2:1 av block reported on local holter monitoring (seer light, ge medical, usa). There was neither specific family history nor past history of specific diseases except the recent stoke . The initial ecg obtained after transfer showed bradycardia (55 beats / min), pr prolongation followed by av block characteristic of second degree av block, mobitz type 1 with left axis deviation and poor r wave progression (fig . The patient's vital signs were as follows: blood pressure, 120/80 mmhg; heart rate, 49 beats / min; respiratory rate, 20 breaths / min; temperature, 36.7. he had white blood cell count 802010/l (neutrophil 68.5%); hemoglobin, 15.1 g / dl; platelet count, 202000/l; and serum c - reactive protein, 0.42 mg / dl . Additionally, serum electrolyte, serum creatine kinase - myocardial band, serum troponin i level and n - terminal pro b - type natriuretic peptide were within normal range . Treadmill test was performed for the evaluation of a chronotrophic response; however, the patient's maximum heart rate observed during bruce protocol stage 3 was 117 beats / min without reaching the target heart rate of 163 beat / min . Transthoracic echocardiography demonstrated a loss of a wave mimicking atrial fibrillation with peak e wave velocity of 59.72 cm / sec and deceleration time of 183 msec . Global left ventricular (lv) ejection fraction was 67% with e / e' of 5.24 implying normal lv systolic function and filling pressure . End diastolic diameters of each ventricle were within normal ranges i.e. 51.80 mm on the left and 33.10 mm on the right ventricle . Contrast echocardiography showed flow communication between multiple intertrabecular recesses and lv cavity with noncompacted / compacted ratio of approximately 3:1 (fig . 3b). Cardiac mri demonstrated prominent trabeculations and deep intertrabecular recesses with thinner compact epicardial layer of the lv characteristic of lvnc (fig . 4). Mild spontaneous echo contrast in la appendage without definite thrombi and decreased mean la appendage flow velocity of 19.93 cm / sec were observed on transesophageal echocardiography with no definite atherosclerotic change from the aortic arch to thoracic aorta . Eletrophysiologic (ep) test was performed to evaluate the cause of arrhythmia and to determine pacemaker insertion . 5a); interestingly, although there was 1:1 av relationship during pacing from high right atrium (ra), there was no atrial signal on coronary sinus (cs) bipole with no correlation between atrial and ventricular signal while pacing from ra appendage that implied absence of electrical activity of la on ep study (fig . For further corroboration of the results, we carried out an additional ep study using 3 dimensional voltage mapping (fig . The map showed no voltage signal of the entire la, septum and body of ra, which explained the capture failure during pacing of ra septum and la . Based on the prior results, we decided to initiate warfarinzation keeping in mind permanent pacemaker insertion; however, we could not perform the procedure due to the patients' refusal . He was discharged from hospital and remained asymptomatic without further complication until the latest follow up visit . We reported a case of a young male patient with concomitant lv noncompaction and la standstill that has not been reported before . The etiology of cva in this case can be explained by 2 conditions: firstly, the noncompaction itself as a complication; and secondly, low velocity in the la appendage . Lvnc was first described as " spongy myocardium " in 1975 by dusek et al.4) and is characterized by morphological abnormality of prominent trabeculae representing " persistent sinusoids " and myocardial wall thickening; the new term " isolated non - compaction of lv myocardium " was introduced by chin et al.5) in 1990 emphasizing deep recesses communicating with the cavity of lv and absence of other cardiac deformities . Although it is defined as a primary cardiomyopathy by the american heart association, it still remains as a " unclassified " cardiomyopathy according to the world health organization.6) the etiologies of isolated lvnc are yet obscure, though several genetic mechanisms that might be involved in the arrest of myocardial development during embryogenesis were recently suggested.3) the various clinical manifestations of lvnc reportedly include congestive heart failure, systemic embolic events, and arrhythmias . The degree of symptoms usually depends on the grade of noncompaction and ventricular function.3) two representative methods used for the diagnosis of lvnc are echocardiography and cardiovascular magnetic resonance (cmr). Generally, the diagnosis of lvnc largely depends on 2 dimensional/3 dimensional echocardiography, with or without contrast echocardiography due to cost - effectiveness . We applied 3 different echocardiographic diagnostic criteria by chin et al.,5) jenni et al.,7) and stllberger et al.8) based on parameters of ventricular morphology, although there is no officially established diagnostic criterion; therefore, multimodal imaging approach inevitably improves diagnostic accuracy . In case of low echocardiographic image quality, cmr shows better signal to noise ratio, contrast to noise ratio, unlimited imaging planes and ability to use tissue characterization . There are 2 cmr criteria proposed by petersen et al.9) and jacquier et al.,10) either using the ratio of noncompact / compact myocardium depth (> 2.3) or trabecular mass (> 20%). Management of lvnc focuses on standard medical therapy for systolic and diastolic ventricular dysfunction and prevention of systemic embolic events.3) we applied warfarin for the prevention and treatment of cva . There is controversy regarding cardioverter - defibrillator (implantable cardioverter - defibrillator) implantation in patients with lvnc but should be considered as primary prevention in high risk patients; alternatively, cardiac transplantation can be considered in these patients.11) once lvnc is diagnosed, family screening of first degree relatives is recommended.3) the prognosis of lvnc is still controversial . Initial reports showed very poor prognosis in patients with lvnc; however, recent studies supported a better prognosis.6)12) one study showed 15% mortality among lvnc patients during a mean 30 month follow - up.12) previously reviewed studies and case reports indicate various ecg findings among the lvnc patients; for example, normal ecg, atrial fibrillation, bundle branch block, qt interval prolongation, left ventricular hypertrophy, av block, and repolarization abnormality.13) there were several cases of both atrial standstill in lvnc patients; however, our patient showed isolated la standstill that has not been reported previously in lvnc patients . Atrial standstill is an uncommon arrthymia characterized by the absence of electrical and mechanical activity in the atrium.14) on egc, atrial standstill is distinguished by the absence of p waves with bradycardia and narrow junctional escape rhythm.14) known etiologies of atrial standstill are electrolyte imbalance, familial cause, cardiomyopathies, valvular disease, muscular dystrophy and toxicity of antiarrhythmic agents.15) several types of atrial standstill were previously described; the more frequent transient type, the rare persistent type and total or partial types.15) recently, familial atrial standstill was reported in several studies showing tendency of sudden cardiac death with higher probability of cardiomyopathies at a young age . The genetic causes of familial type are still obscure; however, cardiac sodium channel gene scn5a was identified as a related gene mutation.16) the study patient showed features of isolated la standstill demonstrating electrical silence in the la that is isolated from partially electrically active ra . Isolated la standstill is extremely rare; it has previously been reported in severe mitral valvular stenosis, dilated cardiomyopathy or following la ablation.15) however, none of the possible factors was found in our patient, thus raising the question whether the abnormal ecg finding was correlated with lvnc or occurred independently . The ep study demonstrated complete electrical block between ra and la, showing no atrial conduction impulse in the cs bipole with no electrical activity in la, which supported la thrombus formation as the etiology of cva . In conclusion, both lvnc and la standstill have risks of embolic events . While the connection between these 2 conditions is still ambiguous, coagulation is a certainty . Here we reported a case of a young man who had lvnc combined with la standstill with cva sequel without any cardiac symptoms.
The deregulation of signaling pathways in tumors can lead to enhanced cancer cell growth, proliferation, survival, invasion, and metastasis or reduced apoptosis 1, 2 . Such pathways became the focus of the development of targeted cancer therapies during the last decades 3 - 5 . Kinases are of special interest within these systems, either as receptor molecules or downstream regulators of signaling cascades (see table 1 for an overview of targeted therapies). Examples of receptor kinases to be further discussed in this review are the human epidermal growth factor receptors 1 (egfr) and 2 (her2). Both kinases are targets for anticancer drugs and can be analyzed for their expression by clinically approved tests, such as immunohistochemistry (ihc) and fluorescence in situ hybridization (fish). Egfr overexpression due to gene amplification is often found in human cancers; in gliomas, this deregulation is often associated with structural rearrangements leading to in - frame deletions in the extracellular domain of the receptor 6 . Her2 overexpression as found in 25 to 30% of human breast cancers can be mediated either by transcriptional activation or gene amplification 7 - 10 . The her2 status of breast cancer patients does not only have a predictive value, but the receptor itself is also a target for the monoclonal anti - her2 antibody trastuzumab 8 . Recent evidence has demonstrated that besides being an important therapeutic target in breast cancer, her2 is a target for the treatment of metastasized gastric cancers 11, 12, and thus, the anticancer drug herceptin (trastuzumab) was approved for the treatment of advanced gastric carcinomas . Her2 is currently detected by immunohistochemistry (ihc) and fluorescence in situ hybridization (fish), and protocols for the detection of her2 in gastric cancers by ihc were recently suggested by rschoff et al . 13 . However, although her2 is well - established as a therapeutic target, recent evidence suggests that down - stream signaling molecules may be better predictors for a response to a her2 directed therapy than the receptor itself . This follows from the fact that the membrane - bound molecule alone does not necessarily lead to an activation of the signaling cascade . For that reason, identifying the activation status of cancer - related signaling cascades might provide a better insight into the mechanisms underlying the success and failure of targeted therapies, thus providing a useful approach to stratify patients for optimal personalized treatment regimens . To individualize cancer, biomarker identification has become even more important for the stratification of patients for special treatment regimens . Urokinase - type plasminogen activator (upa) and its inhibitor plasminogen activator inhibitor1 (pai-1) are prominent examples of such biomarkers . Both are used to provide a more detailed prognosis for nodal - negative breast cancer patients and have reached the highest level of evidence (loe i) for this purpose 14 . However the level of these two markers can just be determined in fresh - frozen tissue . This is problematic as formalin - fixed, paraffin - embedded (ffpe) tissue is the main source of patient material world wide . Therefore for clinical determination of diagnostic and therapeutic targets new methods are crucially needed to maximize the data that can be gleaned from this kind of tissue . The recently developed method to extract full - length, immunoreactive proteins from ffpe tissues (for review see 15) is a major progress for such approaches and provides the means to quantitate clinically relevant proteins like her2 and analyze cancer - related pathways by reverse phase protein microarray (rppa)16 . Because the field of targeted therapy is growing rapidly, it is important to emphasize the necessity of standardization for pre - analytical as well as analytical settings . In this review, we shed light on new developments for the detection of therapeutic protein targets and diagnostic biomarkers in clinical tissues and comment on suggestions for the standardization of tissue handling and analysis . Formalin is the standard fixative in clinical tissue collections worldwide and the method of formalin fixation was described in detail in a previous article of the authors 17 . The protein and nucleic acid cross - linking induced by formalin maintains the tissue in an excellent condition for the pathological examination of the morphological characteristics of diseased tissues 18 . With the new interest in macromolecules (dna, rna, proteins) as therapeutic targets or biomarkers, scientists began to analyze these entities first in frozen material because ffpe fixation required to preserve the tissue morphology had adverse effects on macromolecules, especially proteins . By adoption of antigen retrieval first described by ikeda et al . 19 it was possible to develop an assembly of protocols enabling extraction of proteins from ffpe tissues 18 - 26 . An overview of these methods is provided in a recent review by berg et al ., compared to other recent studies 27 - 30, using these protocols non - degraded, full - length, and immunoreactive proteins are obtained and may then be used for protein profiling for enhanced diagnosis . However one should keep in mind that all protocols require high sds concentrations, temperature and ph, which is problematic for some down - stream applications such as elisa assays . To assure the integrity of isolated proteins we always do a quantification of the total protein obtained by extraction (bradford - assay) and additionally check for correct protein masses by control western blots . Other authors suggest controlling for protein integrity by ms 26, 31 - 33 . In the scope of tumor marker research and personalized medicine further aspects of investigation became of major interest: pre - analytical workflow and tissue quality . For molecular diagnostics protein biomarkers need to be precisely measured in high - quality tissue samples . It is known that specimens undergo numerous processing steps from the collection of patient samples to the final diagnostic analysis . This aspect of the sample history is very important with regard to the identification of disease - related biomarkers . But so far there were no detailed investigations concerning for example the influence of temperature and time during transportation, fixation method and storage on the molecular integrity of tissue samples . In order of reproducibility of subsequent diagnosis the impact of pre - analytical parameters on biomolecular integrity and expression needs to be analyzed in detail . Furthermore we need guidelines for the standardized collection, handling, stabilization and storage of biosamples and quality assurance indicators for artificial, post collection changes of biological samples . Two large international initiatives are working toward this goal, one funded by the eu (www.spidia.eu) and one by the us (http://biospecimens.cancer.gov). In table 2 we highlight the most important problems occurring during the pre - analytical phase of sample preparation . The issue of the evaluation of adverse and variable effects of sample preparation is discussed in more detail in a recent review by becker and taylor 34 . Although morphological parameters, namely tumor size, grade and staging, are the most important parameters for diagnosis, they do not address the complexity and heterogeneity of individual tumors at the molecular level 35, 36 . However, since individualized cancer therapies are on gaining ground more detailed patient classifications are necessary to be able to identify the right therapy for each patient . Anyway, it is important to realize that transcript profiling does not accurately reflect the complexity of cellular protein networks (e.g. Protein - protein interactions, protein localization or posttranslational modifications) 37 . Additionally, several groups demonstrated that there is no constrained correlation between gene transcript levels and protein expression or the functional state of a protein . However especially the communication in such a protein network, which is responsible for activation and deactivation of involved proteins, is often altered in cancer and may then lead to aberrant cellular functions that result in proliferation, apoptosis, differentiation, survival, invasion and metastasis . That's why it is of great importance not only to look at the expression of genes or at the activation state of a single protein but to analyze whole protein signaling networks that then can provide fundamental information about the functional state of signaling pathways 42, 43 . One method which meets all demands for such a network monitoring is the reverse phase protein microarray (rppa). This array format allows the simultaneous analysis of multiple samples for the expression of several proteins under the same experimental conditions even from small numbers of tumor cells or small specimens such as biopsies 44, 45 . Like this changes in protein expression levels or phosphorylation states, before and after treatment, between disease and non - disease states and between responders and non - responders the procedure to generate such an array is simple . After protein extraction from any kind of material (e.g. Cells, ffpe tissue or fresh - frozen material), each sample is arrayed in triplicate on nitrocellulose - coated slides using several dilutions to ensure that each analyte / antibody combination can be analyzed in the linear dynamic range . After the spotting each slide may be detected with an antibody against the desired protein (for an overview of the rppa methodology, see figure 1). However one should keep in mind that for a successful implementation of rppa highly specific antibodies are needed as one can't distinguish between different molecular weights (as e.g. In western blot) but only one signal is obtained . Especially for phosphorylated proteins and other posttranslationally modified proteins this seems to be one of the major limiting factors 35, 46 . Another advantage of the rppa is the possibility to quantify the total amount of protein in the sample via the application of purified recombinant proteins one the same slide 15, 16 . This method enables to measure protein expression from ffpe tissues more precisely than it is possible by ihc (e.g., for her2) and implement the analysis of clinical markers, which until now were only measurable from fresh or frozen tissues (e.g. For upa / pai-1). This highly quantitative approach is advantageous for patient selection; however, the correlation between protein abundance and histology, as provided by ihc, is lost . Nevertheless, we successfully established a set of about 50 antibodies for use in rppa analysis by stringently selecting for specificity and sensitivity by western blot 15, 16 thus providing a starting point for the analysis of major cancer - related signaling pathways from ffpe tissues of cancer patients . Based on this expertise we recently developed an algorithm to validate antibodies for their simultaneous application to extract based and classical morphology based methods (schuster c, malinowsky k et al . We plan to combine the advantages of both approaches to get broad insight into all aspects of cancer formation and development . For the application of rppa generated data for such a purpose in clinical routine it is necessary to be able to compare the results of arrays generated in different hospitals and experimental settings . This can be achieved by reference samples on each slide, to which the signal may be normalized to . Such a control has to be renewable, reproducible in large - scale, successful over a broad range of end points, stable over a long period of time and as closely related to the test sample as possible 47 . To validate the reproducibility of the method, we performed experiments analyzing the variability between sample preparations, array slides and experimental setups (inter - sample as well as inter - assay comparisons) and found the method to be highly reliable 15 . In comparison to the widely used elisa approaches rppas are more sensitive and a two - site antibody sandwich method is not used, hence there won't be any experimental variability introduced due to labeling yield (or) epitope masking . They are used for the rapid and comprehensive analysis of new drug candidates found by in silico approaches or by binding screens for their biological functions 48 - 50, as well as for biomarker screenings 51 . Many recent studies have demonstrated that rppa technology is a very promising tool for signaling pathway profiling of human tissues and cell lines to produce valuable information for the development of new therapeutics or patient selection . Feinberg et al . Was the first to utilize a microspot technique to detect antigens in serum 52 . In 2001, another study used rppas to show the activation of pro - survival proteins and pathways during prostate cancer progression 45 . In our group, we provided insight into the role of upa / pai-1 in cancer related signaling 16 . Other prominent examples of cancer - specific signaling deregulation namely the activation of the phosphatidylinositol 3-kinase (pi3k) pathway in a significant number of ovarian and colon tumors 53, 54.and, alterations in the mitogen - activated protein kinase (mapk) pathway or the overexpression of her2,8, 53 - 57 were summarized in an earlier review by the authors 17 . These examples demonstrate the diverse signaling - based mechanisms underlying cancer progression and indicate that cancer types (e.g., breast cancer) can be subdivided e.g., into small, well - defined subgroups that express a typical protein profile . Some authors go even further, suggesting that the use of a targeted therapy should not be based on the identity of the cancer but rather on the deregulation of a certain pathway 58 . Such an approach would challenge the design of future clinical studies and the approval of new drugs for targeted therapy but could be beneficial for patients by providing highly specific therapies that are only based on the availability of specific targets regardless of the classical characterization of the cancer type . Such a specific treatment could greatly increase the quality of life for patients by minimizing adverse side effects of cancer treatment . In addition to classical biomarker screenings rppa is also an adequate tool to compare different types of samples regarding their protein expression profiles . One prominent example is the expression analysis of her2 from resected tumors versus core needle biopsies . Currently, core biopsies are routinely used for diagnosis of breast cancer and they are often the only sample for providing prognostic and predictive markers before treatment . After extraction of full - length proteins from ffpe tissues, berg et al . Used rppas to compare her2, estrogen receptor (eralpha), and progesterone receptor (pgr) expression levels in a series of 35 ffpe breast cancer surgical specimens and their corresponding core biopsies . We found a high concordance between protein expression in core biopsies and surgical specimens . In this study, the authors could show that her2, eralpha, and pgr expression can be assessed reliably on core biopsies of ffpe breast cancer tissues using rrpa . These results might further strengthen the implementation of rppa technology in routine clinical settings (berg et al . Protein microarray - based comparison of her2, estrogen receptor and progesterone receptor status in core biopsies and surgical specimens from ffpe breast cancer tissues . Gene expression profiles (e.g., genes regulating cell cycle, invasion or metastasis) have been proposed as potential biomarkers for the prognosis, prediction of treatment response and the identification of potentially new drugs 59 . In addition to the well - established mrna profiling, the analysis of small non - coding rnas called mirnas has become a focus in biomarker research . In recent years, evidence has suggested that whole groups or families of mirnas are deregulated in different diseases, especially cancer 60, 61 . The quantitative analysis of protein expression described in this review may enable the parallel identification of protein and rna profiles (mrna and mirna) using only one ffpe tissue sample . With this method, it is possible to simultaneously determine mutations in critical genes and relate them to expression profiles . Gene mutations frequently lead to a loss of gene function, although the mutated gene is still expressed at the rna and protein levels . A classical example for such a mutation is the cell adhesion molecule e - cadherin, in which a loss of e - cadherin function is found in many diffuse - type gastric cancers, although the protein is expressed at high levels in these tumors 62 . Another useful application of the combined dna and protein analysis is the utilization of egfr as a therapeutic target; although egfr expression is detectable on a protein level, many patients with colorectal cancers do not respond to cetuximab therapy 63 - 65 . The downstream target of egfr is mutated in many of the non - responders, therefore blocking the egfr does not inhibit proliferative signaling . Therefore, the parallel assessment of the level of egfr protein and the mutational status of the egfr gene is one important prerequisite for successful cetuximab treatment 63 - 65 . The remaining egfr - positive patients express the wild - type form of the protein and provide a good example for the need of additional biomarkers for the prediction of therapeutic response . Many of these patients do not respond to cetuximab therapy, probably due to further alterations in pathways downstream of egfr . In recent years, several additional candidate biomarkers of egfr resistance were suggested . Molecular aberrations occur in braf, pik3ca and pten, which are known downstream effectors of egfr 66 - 69 . Somatic mutations in these egfr effectors correlate with the reduced efficacy of cetuximab in patients with metastatic colorectal cancers 70, 71 . An activating mutation of braf, which lies downstream of the egfr signaling cascade, was found in a proportion of patients with wild - type egfr that do not respond to cetuximab treatment 66 . For that reason, braf mutational analysis is currently recommended by the national comprehensive cancer network (nccn) clinical guidelines for patients with wild type metastatic or recurrent colorectal cancers that are receiving cetuximab 72 . Recently, we succeeded in the parallel extraction of proteins and rna, including mirna, from the same ffpe tissue sample (malinowsky et al . ; unpublished data), demonstrating the proof of principle of our vision . While ffpe samples are a large challenge of the clinical application of the combined analysis of protein and rna, new formalin - free fixatives, e.g. Paxgene tissue fixation and stabilization reagents 73, hope 74 - 76, rcl2 77 - 79, finefix 80, metacarn 81 or umfix 82 show great potential to serve as novel multimodal fixatives for modern pathology, enabling extensive protein biomarker studies on clinical tissue samples . Regardless of the nature of the fixative, our approach allows the integration of analysis at all three levels of gene expression (dna, rna and protein), providing insight into the whole spectrum of potential biomarkers (figure 2). In the scope of new targeted cancer therapy approaches new technologies which meet the issues of fast and precise target detection and quantification are desperately needed . It has already been implemented in several biomarker studies and becomes even more powerful when combined with the huge sample reservoir provided by ffpe tissues . It is clear now that proteins can be extracted from ffpe tissues and reliably analyzed . We believe that applying these new techniques - probably in conjunction with novel tissue fixatives - will greatly facilitate the search for new biomarkers and therapeutic targets in the near future . More importantly, the great translational potential of the new approaches are apparent as the methodologies discussed can easily combine classic histopathology and modern multiplex protein profiling . However, in depth evaluation of antibodies and the use and design of proper controls for tissue quality are essential.
The fastq file for illumina genome analyzer iix (gaiix) run accession srr944107 (single - end reads) was downloaded from http://www.ebi.ac.uk/ena/data/view/srr944107, having sourced the accession code via http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse49302 . Briefly, hek293 t cells were suspended in 1% low - melt agarose prior to lysis . Free dna ends (sites of dsbs) were ligated to a double - stranded biotinylated adapter oligonucleotide before digestion with the restriction endonuclease sau3ai . Dsb site - containing termini were phase - purified using streptavidin paramagnetic particles, eluted via ecori restriction endonuclease digestion and then subjected to sau3ai site adapter ligation and pcr amplification . Pcr products were ligated to illumina adapters, allowing them to be represented in either orientation . Library fragments of ~ 200400 bp (insert plus adapter and pcr primer sequences) were band isolated from agarose gels and the purified libraries were sequenced in single - ended fashion using the illumina genome analyzer iix sequencing platform . We used our custom software image 1 to produce a modified representation of image 2 . Briefly, it filters reads based on the observation of expected arrangements of adapter sequences, with the stringent requirement that both adapters be evident in a given read . Reads exhibiting evidence of ligation artefacts or insufficient evidence of expected adapter sequences were removed . Accepted reads were processed to trim adapter sequences, and those with library inserts greater than or equal to 25 nucleotides in length were retained and transformed to orient the dsb site at the start . The concatenated sequences of image 3 plus human reference genome build image 4, represented as image 5, were indexed using bwa (version 0.7.5a) using the command: reads of the transformed fastq file were then mapped to image 7 using bwa, thus: samtools (version 1.3.1) was used to convert from sam file format to bam file format and to sort the resulting bam file with the following command: bedtools (version 2.17.0) was then employed to produce a bed file representing the mapping, including cigar string information and mapping orientation, with the following command: to reduce false positives resulting from mapping artefacts, we filtered out reads that overlapped with encode project blacklist regions and repeatmasker - derived repetitive regions as follows (image 14 represents a file created by sorting a concatenation of the hg19 co - ordinate - associated files image 15 and image 16): we then used our custom software image 19 (available at https://github.com/djpark1974/raft_hotspots_se) to further filter the data and to count the number of observations of dsbs at co - ordinates in image 20 (yielding image 21). Since we presented the dsb at the beginning of each read prior to mapping, we can determine the exact location of the dsb at the single nucleotide level for each read . Reads that mapped in either orientation were treated as likely to be erroneous if the cigar string showed evidence of clipping at either terminus . Additionally, we required reads to exhibit mapping qualities (mqs) of greater than 40 for them to be included in our dsb site counting . For increased specificity, we removed dsb sites located within 5 base pairs of a sau3ai consensus site (gatc), thus (image 22was derived via a custom python script): to enable detailed downstream analyses, we have supplemented the co - ordinate - dsb - count data with annotation derived from the encode project and blast alignment scores derived from aligning u13369.1 to the human genome . Encode annotations are recorded with the identity and proximity of respective encode elements and blast alignment scores provide the highest scoring sequence similarity match for a contiguous sequence spanning a given co - ordinate . 2 illustrates the frequency of dsbs at single nucleotide resolution sites across the hg19 reference human genome . The fastq file for illumina genome analyzer iix (gaiix) run accession srr944107 (single - end reads) was downloaded from http://www.ebi.ac.uk/ena/data/view/srr944107, having sourced the accession code via http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse49302 . Briefly, hek293 t cells were suspended in 1% low - melt agarose prior to lysis . Free dna ends (sites of dsbs) were ligated to a double - stranded biotinylated adapter oligonucleotide before digestion with the restriction endonuclease sau3ai . Dsb site - containing termini were phase - purified using streptavidin paramagnetic particles, eluted via ecori restriction endonuclease digestion and then subjected to sau3ai site adapter ligation and pcr amplification . Pcr products were ligated to illumina adapters, allowing them to be represented in either orientation . Library fragments of ~ 200400 bp (insert plus adapter and pcr primer sequences) were band isolated from agarose gels and the purified libraries were sequenced in single - ended fashion using the illumina genome analyzer iix sequencing platform . We used our custom software image 1 to produce a modified representation of image 2 . Briefly, it filters reads based on the observation of expected arrangements of adapter sequences, with the stringent requirement that both adapters be evident in a given read . Reads exhibiting evidence of ligation artefacts or insufficient evidence of expected adapter sequences were removed . Accepted reads were processed to trim adapter sequences, and those with library inserts greater than or equal to 25 nucleotides in length were retained and transformed to orient the dsb site at the start . The concatenated sequences of image 3 plus human reference genome build image 4, represented as image 5, were indexed using bwa (version 0.7.5a) using the command: reads of the transformed fastq file were then mapped to image 7 using bwa, thus: samtools (version 1.3.1) was used to convert from sam file format to bam file format and to sort the resulting bam file with the following command: bedtools (version 2.17.0) was then employed to produce a bed file representing the mapping, including cigar string information and mapping orientation, with the following command: to reduce false positives resulting from mapping artefacts, we filtered out reads that overlapped with encode project blacklist regions and repeatmasker - derived repetitive regions as follows (image 14 represents a file created by sorting a concatenation of the hg19 co - ordinate - associated files image 15 and image 16): we then used our custom software image 19 (available at https://github.com/djpark1974/raft_hotspots_se) to further filter the data and to count the number of observations of dsbs at co - ordinates in image 20 (yielding image 21). Since we presented the dsb at the beginning of each read prior to mapping, we can determine the exact location of the dsb at the single nucleotide level for each read . Reads that mapped in either orientation were treated as likely to be erroneous if the cigar string showed evidence of clipping at either terminus . Additionally, we required reads to exhibit mapping qualities (mqs) of greater than 40 for them to be included in our dsb site counting . For increased specificity, we removed dsb sites located within 5 base pairs of a sau3ai consensus site (gatc), thus (image 22was derived via a custom python script): to enable detailed downstream analyses, we have supplemented the co - ordinate - dsb - count data with annotation derived from the encode project and blast alignment scores derived from aligning u13369.1 to the human genome . Encode annotations are recorded with the identity and proximity of respective encode elements and blast alignment scores provide the highest scoring sequence similarity match for a contiguous sequence spanning a given co - ordinate . 2 illustrates the frequency of dsbs at single nucleotide resolution sites across the hg19 reference human genome . Here, we present the relative frequencies of dsbs across the human reference genome for hek293 t cells, at single nucleotide resolution . Since dna strand breakage and genomic rearrangements are highly relevant to cancer and other diseases,,, it is probable that our new data will have utility for the development of clinically important diagnostic tests . The highest ranking dsb regions reported by tchurikov et al . For the srr944107.fastq dataset predominantly relate to regions that would be likely to present problems to short read mapping software, such as satellite sequences . In an attempt to reduce mapping - related artefacts, we have elected to remove regions known to result in low - confidence mapping from our analysis . Top ranking single nucleotide - resolved dsb sites resulting from our analysis relate to regions listed previously as enriched for dsbs, albeit to a lesser extent than reported for numerous low - complexity (and low confidence) sequence regions . The raft protocol from which our data are derived is theoretically enriched for blunt - ended forum domain termini, previously shown to be associated with transcriptional control,, . They will be biased towards termini that occur within a particular range of genomic distances from a sau3ai restriction endonuclease site . Future protocols that make use of multiple restriction endonucleases for cleavage following the initial ligation step, as alternatives to (and as well as) sau3ai, should mitigate this to a large extent . We have applied high - stringency thresholding on mapping quality as part of our algorithm and discarded library elements that could not be uniquely assigned to a single genomic location with high confidence and, as such, repetitive genomic elements harbouring dsb sites will not be represented . It should be noted that the data we present relate to human hek293 t cells . Other cell - types will likely exhibit differences in their raft - detectable dsb profiles due to variations in higher - order chromosomal architecture and dna cleavage - inducing enzyme activity . These differences will be elucidated with the expansion of studies to a range of cell and tissue types . We have supplemented the profiling of the relative frequency of dsb sites in hek293 t cells with encode - derived annotation, including regional information pertaining to important transcription factor binding sites and other marks of gene regulation, regions of dnasei hypersensitivity and repetitive elements . Further, we provide annotation in the form of sequence similarity scores, derived from blast analysis, for sites that occur in regions with high similarity to human ribosomal dna, since such sequences are known to include hot spots for dsbs and present particular mapping challenges due to their representation at high copy number at multiple sites in the genome . This information should assist with the selection of suitable targets for diagnostic test design, allowing the user optionally to avoid sites that present excessive mapping difficulties or to focus on regions associated with particular genomic marks, for example . The refined characterisation of the propensity for particular types of dsbs, such as those identified by the raft procedure, across the human genome will likely allow more efficient assessment of genomic . This should be highly relevant to clinical management approaches such as risk stratification for particular types of cancer and treatment response prediction . As such, the use of these data has the potential to be beneficial to the reduction of disease associated mortality and morbidity.
Collagen is a major connective tissue protein that plays an important role in the extracellular matrix in animals . As such atelocollagen is a type of soluble collagen produced from tropocollagen, the collagen molecule that makes up collagen fibrils, via the elimination of the telopeptide moieties, which are considered to account for most of collagen's antigenicity [1, 2]. Thus, atelocollagen is considered to have little immunogenicity, which makes it a safe biomaterial . For example, a minipellet atelocollagen formulation has been demonstrated to sustain the release and maintain stable blood concentrations of protein drugs for more than 1 week . Many kinds of protein drugs such as interferon-, interleukin-2, nerve growth factor, and bone morphogenetic protein, and so forth, have been administered using this drug delivery system, and interferon- and interleukin-2 showed strong antitumor activities in animal models when administered in this manner [3, 4]. In the past decade, as well as being used as a solid substrate, dissolved atelocollagen has been used as a drug delivery vehicle for nucleic acid - based medicines for gene conversion, inflammatory disease [8, 9], and tumor therapy . Atelocollagen can be used to deliver most kinds of nucleic acid - based medicines including plasmid dna, antisense oligodeoxynucleotides (odn) [1113], short interference rna (sirna) [1420], and micro rna (mirna) [2123]. It is also capable of delivering oligonucleotides to subcutaneous xenografts and metastatic tumors after its local and/or systemic administration . Many studies, including some involving in vivo tumor models, have evidenced the contribution of atelocollagen to the enhancement of drugs' antitumor activities, and some of them described the mechanisms . For example, nucleic acids delivered by atelocollagen are protected against degradation by host nucleases [8, 14, 24], and it has also been shown to improve the delivery efficiency of oligonucleotides to tumors [15, 16]. However, the biological functions of atelocollagen and the mechanism by which it enhances delivery efficiency are still not fully understood . It is essential to reveal the biological characteristics of atelocollagen in order to be able to fully exploit its drug delivery potential . While we were studying the basic properties of atelocollagen, we discovered another of its functions: it increases endothelial permeability . Here, we describe the results of a study of the effects of atelocollagen on intercellular sealing function . We measured transendothelial electrical residence (ter) in order to estimate intercellular barrier function and performed an immunohistochemical examination to see whether any cellular morphological changes were induced . Rhodamine red - conjugated atelocollagen was prepared in accordance with the manufacturer's instructions (fluoreporter rhodamine red - x protein labeling kit; life technologies japan, tokyo, japan). The oligodeoxynucleotides (odn) and double stranded rna (dsrna) were synthesized by eurogentec (seraing, belgium). The sequences of the oligonucleotides are listed in table 1 [20, 25, 26]. Each atelocollagen - oligonucleotide formulation (ac formulation) was prepared by gently mixing aqueous atelocollagen with a solution containing a defined concentration of oligonucleotides . The final oligonucleotide concentration was usually 5 m and that of atelocollagen was 0.1% w / v unless otherwise stated in the text, tables, and/or figures . Normal human dermal microvascular endothelial cells (hmvec) were purchased from eidia (tokyo, japan). These cells were cultured in egm (endothelial growth media; eidia, tokyo, japan) until they reached confluence on 12 mm transwell filters with a 0.4 m pore size (corning glass works; corning japan, tokyo, japan) coated with rat tail collagen . Porcine brain microvascular endothelial cells (bmvec) were purified and maintained according to the method described in a previous study . Ter was determined using an evom voltohmmeter and an endohm-12 chamber (world precision instruments, sarasota, fl) at 37c . Cell growth was monitored by measuring ter . Once stable intercellular seals had formed; that is, at confluence, the medium in the inner chamber was exchanged for 400 microliters of culture medium containing 30% v / v of ac formulation . One hour after treatment with the ac formulation, which was performed as described in section 2.2, 100 l of 0.36% w / v texas red - conjugated dextran (mw: 40 kda; life technologies japan, tokyo, japan) were added to the inner chamber . One hour later, the dextran concentration of the medium in the outer chamber was analyzed by measuring its fluorescence . To investigate the enhancement of paracellular transport by atelocollagen, solute transportation was compared among the ac formulation, bovine serum albumin, and dextran . Once stable intercellular seals had formed, the medium in the inner chamber was exchanged for 400 microliters of culture medium containing 30% v / v of the ac formulation, which had been produced using 0.1 or 0.3% w / v rhodamine red - conjugated atelocollagen (i.e., approximately 0.03 or 0.1% w / v atelocollagen was added; molecular weight (mw): 300 kda); 0.1% w / v of fluorescein conjugated dextran (mw: 70 kda; life technologies japan, tokyo, japan); 0.1% w / v of alexa fluor 594 conjugated bovine serum albumin (bsa; mw: 66 kda; life technologies japan, tokyo, japan). The solute concentrations of the outer chambers were analyzed by measuring their fluorescence at 1 and 2 hours after the medium exchange . After treatment for 1 hr with the ac formulation, oligonucleotide alone, atelocollagen alone, or phosphate - buffered saline (pbs) as a control, hmvec cells were fixed with 1% paraformaldehyde for 10 min and then treated with 0.2% triton x-100 for 10 min . After preincubation with 5% skimmed milk, they were incubated for 1 hr at room temperature with rabbit or mouse antibodies against vascular endothelial (ve)-cadherin (bd biosciences, san diego, ca), zonula occludens-1 (zo-1) (zymed laboratories, san francisco, ca), claudin-5 (zymed laboratories, san francisco, ca), and -tubulin (amersham, poole, uk). Then, the samples were incubated for 1 hr with appropriate secondary antibodies labeled with alexa fluor-488 or alexa fluor-596 (life technologies japan, tokyo, japan). Actin filaments were labeled with alexa fluor-546 phalloidin (life technologies japan, tokyo, japan). The expression of each protein was examined using a laser scanning confocal microscope (mrc 1024; bio - rad, hercules, ca). Western blotting was performed according to the method described in a previous report . For western blotting of the total cell lysates, the dishes were washed with pbs, and then 300 l of sample buffer (1 mm nahco3 and 2 mm phenylmethylsulfonyl fluoride) was added to 60 mm culture dishes . The cells were scraped and collected in microcentrifuge tubes and then sonicated for 10 sec . The protein concentrations of the samples were determined using a bca (bicinchoninic acid) protein assay reagent kit (pierce chemical, rockford, il). For each sample, aliquots of protein (15 g per lane) were separated by electrophoresis in 4/20% sodium dodecyl sulfate polyacrylamide gels (cosmo bio, tokyo, japan) (sds page). After being electrophoretically transferred to nitrocellulose membranes (immobilon; millipore, billerica, ma), the membranes were saturated with blocking buffer (trisbuffered saline [tbs] supplemented with 0.1% tween 20 and 4% skimmed milk) for 30 min at room temperature and incubated with antiactin, anti - zo-1, anti - ve - cadherin (bd biosciences, san diego, ca), anticlaudin-5 (zymed laboratories, san francisco, ca), anti - p38 mitogen - activated protein kinase (map kinase or mapk) (santa cruz biotechnology, santa cruz, ca), antiphospho - p38 mapk (cell signaling, beverly, ma), anti - p42/44 mapk (promega, madison, wi), antiphospho - p42/44 mapk (cell signaling, beverly, ma), anti - rho - a, and anti - cdc42 (santa cruz biotechnology, santa cruz, ca) antibodies (1:1000) for 1 h at room temperature . The membranes were then incubated with horseradish peroxidase - conjugated anti - rabbit or mouse igg (dako a / s, copenhagen, denmark) at room temperature for 1 h. the immunoreactive bands were detected using an ecl western blotting analysis system (ge healthcare, little chalfont, uk). Paracellular flux is dependent on the function of tight junctions [30, 31]. We assessed the effects of an ac formulation on the ter of hmvec to evaluate their tight junction function . As shown in figure 1 and table 1(a), the odn containing ac formulation caused a time - dependent reduction in ter, while ter was hardly affected by treatment with odn or atelocollagen alone . As for the type of oligonucleotide in the formulation, phosphorothioate odn produced a more significant reduction in ter than phosphodiester odn, which only produced slight alterations . Various formulations containing different ratios of odn and atelocollagen were examined in order to understand which parameters have the greatest effect on the change in ter . As a result, we found that the ter change was dependent on the size of the odn and the composition of the formulation, but not the odn sequence, as shown in tables 1(b), 1(c), and 1(d). Specifically, odn composed of 15 or more bases were effective and those containing around 30 bases were the most effective, but 10-base - long odn were not effective . The change in tight junction function was also dependent on the concentrations of odn and atelocollagen in the formulation . To verify that the ac formulation increased paracellular flux, the amount of texas red - labeled dextran (molecular weight: 40 kd) transported across an endothelial cell layer was examined . As shown in figure 2(a), dextran transport was increased approximately twofold in the cell cultures incubated with the ac formulation . Next, the paracellular transport of atelocollagen was analyzed and compared with those of bsa and dextran . Only very small amounts of bsa and dextran penetrated the cell sheet during the 2-hour study period; on the other hand, much more atelocollagen passed through, even though the molecular weight of atelocollagen is 4 - 5 times higher than those of bsa and dextran (figure 2(b)). An examination using bmvec was performed to determine whether the effect of the ac formulation was specific to hmvec . As a result, we found that the ter value of the bmvec was also reduced by the ac formulation (and only the ac formulation), as shown in figure 3 . Bmvec forms the blood - brain barrier (bbb), where intercellular sealing function is strictly maintained . These results showed that the ac formulation is able to affect the paracellular flux of endothelial barriers . It is well known that increased endothelial permeability is associated with impaired intercellular contact [3235]. We carried out an immunohistochemical analysis of the cells treated with the ac formulation to clarify how their intercellular sealing was affected . As shown in figure 4(a), treatment with the ac formulation markedly reduced the degree of intercellular contact, as shown by intercellular gap formation, actin stress fiber formation, cellular contraction, and a lack of ve - cadherin . Adequate expression of claudin-5, one of the key components of the endothelial barrier, was noted at the cell periphery . However, zo-1 protein expression was absent from the intercellular gaps . On the contrary, western blotting revealed that treatment with the ac formulation did not affect the expression of these proteins (figure 4(b)). Although the ter value remained low as long as the ac formulation was present in the culture medium, the treatment did not cause toxicity . The cells survived well for at least 24 hrs, and both the ter and morphology of the cells could be recovered by removing the formulation (data not shown). No such morphological changes were induced by treatment with odn or atelocollagen alone (figure 4(a)). Microtubules play an important role in regulating actin formation and hence, endothelial barrier function [36, 37]. As shown in figure 5, -tubulin formed a fine network in the blank control . However, treatment with the ac formulation caused the peripheral fine structure of the -tubulin network to be lost . Many studies have shown that increased endothelial permeability and impaired intercellular contact can be induced by signal transduction, mainly that of rho a [32, 38] and p38 map kinase [36, 3942]. Thus, we investigated the effects of the ac formulation on signal transduction . As a result, no differences were found in the expression levels of rho - a, cdc42, or p42/44 map kinases or their phosphorylated forms . Regarding p38 map kinase, although no changes were noted among the control, oligonucleotide alone, or atelocollagen alone groups, the levels of phosphorylated p38 map kinase were markedly increased in the cells treated with the ac formulation (figure 6), which indicates that the impact of the ac formulation on tissue permeability is associated with the activation of p38 map kinase . Collagen plays an important role in the extracellular matrix by supporting cells so that they can form tissues and organs . Atelocollagen is produced from type i collagen and is widely used in its solid state as a biomaterial for medical and surgical products because of its biocompatibility and workability . However, the kinetics, dynamics, and biological functions of atelocollagen after its injection into the living body are still poorly understood, and it is essential to elucidate the characteristics of atelocollagen in order to fully exploit its potential . Here, we demonstrated a novel biological function of atelocollagen . When endothelial cell sheets were treated with atelocollagen or oligonucleotides alone, the intercellular structure of the sheet was not changed . However, when the atelocollagen and oligonucleotides were administered together, intercellular gaps formed and consequently the paracellular flux of the sheet was elevated . The abovementioned changes were elicited via the activation of p38 map kinase, a signal - transduction - related molecule . In addition, the changes observed in this study are similar to those triggered by thrombin, histamine, tnf- [34, 36], and vegf (vascular endothelial growth factor), and so forth . As shown in section 3.1, the degree to which endothelial function was affected was dependent on the molecular structure of the oligonucleotides including their size and chemical modifications, suggesting that the three - dimensional structure of the oligonucleotide and atelocollagen complex stimulates a signal transduction pathway that acts as a permeability modulator, although the specific pathway that it stimulates remains unknown . To date, no severe systemic edema or side effects of the ac formulation have been noted, even after the intravenous administration of atelocollagen as an oligonucleotide drug carrier . These findings indicate that atelocollagen could be used as a permeability enhancer at local treatment sites without the adverse systemic effects that cytokines and chemokines sometimes provoke . Since tight junction modulators are regarded as practical drug delivery enhancer candidates [4446], the function of atelocollagen demonstrated in the present study should be thoroughly investigated . The unique biological functions of atelocollagen have led to the development of unique antitumor therapies and products, such as surgical products; formulations that sustain the release of antitumor proteins [24]; treatments that enhance the antitumor activities of various molecules including antisense odn [1113], sirna [1420, 24], and mirna [2123]. Obtaining more information about atelocollagen would allow us to develop the next generation of atelocollagen - mediated drug delivery systems.
In order to characterize the radial undulations from a simulated vesicle, we start by recasting the positions of the bilayer beads into an equi - angular discrete surface representation using a, -grid (colatitude and longitude respectively), where [0,] and [0, 2], with = 0 at the northern pole . The angular resolution is defined as1 where s is the arc - length where the bilayer fluctuations transition between a continuum mode to molecular (s 4 nm), r0 is the average vesicle radius, and ns is the number of sampling points per s with ns 2 to satisfy nyquist sampling theorem . To render the surface, we define the origin at the com of the vesicle (lipids + membrane inclusions) and then transform the system into spherical coordinates (xi, yi, zi ri,i, i, where ri,i, i corresponds to radius, colatitude, and longitude for each bead i, respectively). The direct use of an equi - angular, -grid to bin bead positions introduces discontinuities at the poles . We mitigate these discontinuities by implementing an arc - length low - pass filter with filter cutoff qarc = 2/s . Our previous work with undulation analysis on flat patch systems identified a cutoff wavenumber, q0 = 1.5 nm, at which the long - wavelength undulations transition into molecular structure fluctuations . We use this q0 as a guide in defining an appropriate qarc for the vesicle systems . Figure 1a shows a snapshot from an equilibrated, 34 nm dmpc vesicle, and figure 1b presents a cutaway view, emphasizing the vesicle s diameter relative to its thickness . We parse all lipids into the inner and outer monolayer using the average vector between the acyl - chain terminal beads and the phosphate bead, where the radial component defines which monolayer the lipid belongs to (i.e., r <0, inner monolayer; r> 0, outer monolayer). Using an arc - length filter with qarc = 2.5 nm, we define the local, -membrane surface for both inner and outer monolayers, rin(,) and rout(,), respectively . Figure 1c schematizes the first stages of our method, with the inner (red) and outer (blue) monolayer beads shown for numerous, -positons . On the left hemisphere of figure 1c, regions of rin(,) and rout(,) are presented for illustration . The average of the two surfaces define the undulating radial surface as2 a. snapshot of a 34 nm dmpc - lipid vesicle . C. an arc - length filter is used to define a surface for both inner and outer monolayers (blue and red selection beads respectively). D. rund(,) is determined as the average of the inner and outer monolayers (color - map indicates fluctuations about the average radius, r0, (units in nm). Figure 1d shows rund(,) calculated from one frame of the dmpc trajectory . The color - map displays the deviations about the average vesicle radius, r0 (units in nm), which is typically smaller than the ideal sphere radius r0 . From rund(,), we then define the normalized radial fluctuations as3 next, spectral decomposition of rund(, spherical harmonics are standing waves on a sphere . Given that rund(,) is a discrete surface defined on a spherical manifold, we can represent it as a linear combination of spherical harmonics with degree (l) and order (m), corresponding to the number of waves in the, -dimensions, respectively . Spha decomposes rund(,) into a series of standing waves on a sphere with degree l and order m. helfrich s formulism for spha expands the normalized radial fluctuations in spherical harmonics as4where alm are the spherical harmonic coefficients of degree l and order m (l 0,1,lmax and m l,...,l) with lmax defined by the number of colatitude grid points.ylm are the spherical harmonic basis functions5where plm() are the fully normalized associated legendre polynomials with the normalization factor, nlm, defined as6 because f(,) is a real - valued function alm = al, m , the complex conjugate of alm . We thereby redefine alm such that7allowing us to write8where now m 0,...,l . We generate the matrix p with dimensions (2n lmax) n for a given, -distribution, where n is the number of colatitude parallels, such that for each li 0,...,lmax, the corresponding 2 m + 1 rows are defined as9from which the transformation matrix y follows as10 from the matrix y we can write the spherical harmonic forward transform as11where alm1 is a recasting of the spherical harmonic coefficients alm with dimension n 1 corresponding to the row construction of y, and f,1 is a matrix of positions with dimension (2n * lmax) 1 . Whereas the forward transform is exact, the inverse transform (i.e., spha) is overdetermined . Using the factorize package in matlab, we compute the pseudoinverse of y using qr - factorization and apply a least - squares approximation to determine alm1 as12 implementation of the spha algorithm results in error propagation from numerous sources (i.e., truncation error, round - off error, and least - squares approximation). We evaluated the magnitude of these effects by calculating the root - mean - squared - difference (rmsd) between rund(,) and the transformed rund(,), where13 rmsd calculated for the dmpc system is 3.5 10 nm and for the dmpc + cholesterol system is 1.8 10 nm . These errors are 2 orders of magnitude lower than the magnitude of the radial fluctuations, o (0.5 10 nm), allaying concerns regarding the inherent propagating errors . Membrane structure ((z) and al) for flat - patch membrane systems was determined using our previously published method . Briefly the urs was determined using the phosphate atoms and subsequently filtered with a 1.5 nm cutoff . The filtered surface was then used to reference every atom to the local bilayer midplane . These distances were subsequently binned and volume normalized to extract the number density profile . The standard method for determining the radial membrane number density profile, rbin(r), references every bead / atom in the system relative to the com of the vesicle in spherical coordinates, averaging across all, -angles . We have previously shown that the membrane structure profile is smoothed out in systems where long - wavelength undulations develop . By isolating the long wavelength undulations and referencing every bead / atom to the local undulating reference surface following the same principles for our method developed for flat - patch systems, we apply a low - pass ideal filter to the spherical harmonic coefficients with an order cutoff lcut = qarcr0 0.5 such that14where lm1 are the filtered coefficients . For the dmpc vesicle lcut = 26, whereas for the slightly larger dmpc + cholesterol vesicle, lcut = 29 . We next apply an the inverse spherical harmonic transform to resolve a filtered radial - undulation surface rund(,) as15 with the filtered surface we resolve the vesicle s local membrane structure, uc(r), by referencing every bead / atom i relative to it is position on the surface, such that16where rund(,)nearest corresponds to the closest equal - angular grid point on rund(,). The grid definition samples the corresponding filter wavelength at least 2 times (satisfying nyquist sampling theorem). As a default we employ 4-samples per wavelength (at qarc = 2.5 nm the arc - length resolution is 0.4 nm), providing a close approximation to the unique rund, i(,). The resulting ri(,) is then binned with a bin - width of dr = 0.1 nm and subsequently normalized by the differential volume shell17with r0the radius of the ideal sphere with equal volume as rund(,) defined as18and adjusted for every bin index b by the corresponding distance from the surface . In addition to number density profiles, we can identify the vesicle s area per lipid, al, both as a whole and independently for both monolayers . Instead of defining r0 with all vesicle beads / atoms, we parse them independently as inner and outer monolayers . Then using eq 18, we obtain two additional ideal sphere radii (three in total), r0,vesicle, r0,inner, and r0,outer . The corresponding al for each ideal radius is simply192021where nl, inner and nl, outer refer to the number of lipids in the respective monolayer . From alm1, we obtain the undulation power spectrum by binning the modulus of the spherical harmonic coefficients across degree l. the resulting profile can be interpreted according to the helfrich continuum model for undulations on a sphere with vanishing spontaneous curvature as22where t is temperature and l 2,...,lmax . In the flat - patch spectral method, the undulations power spectrum, \|u(q)\|, determined by direct fourier transformation of the bilayer selection atoms can be modeled as23where n is the number of selection atoms, a is the projected area per lipid, and s(q) is the in - plane structure factor . S(q) is subtracted from n\|u(q)\| to provide a broader range of modes within the q regime . Kc is then determined by fitting the low - q, long - wavelength modes . Using a filter to define the urs attenuates the spectral intensity of the undulations . This attenuation can span a broad frequency bandwidth, bleeding through into the desired frequencies of the signal . We explored a range of arc - length filter cutoffs to characterize the filter s frequency response, with the goal of identifying a cutoff where signal attenuation is limited to frequencies above the crossover wavenumber, q0 = 1.5 nm . Figure 3 presents the power spectra for the dmpc vesicle system using four different arc - length filters with wavenumber cutoff, qcut, ranging from 0.5 nm to 2.5 nm . Undulation spectra for a range of arc - length cutoff wavenumbers, (qcut = 0.5 nm: blue; 1.5 nm: green; 2.0 nm: red; and 2.5 nm: cyan) for the dmpc vesicle system . The frequency response of the arc - length filter is far from ideal, with significant bleed - through extending below the desired cutoff wavenumber . This is most noticeable when comparing the crossover wavenumber q0 = 1.5 nm (black dashed - line)the wavenumber where continuum undulations transition into molecular fluctuations to the 1.5 nm filter cutoff (green triangles). There is significant loss of undulation intensity for degrees 1225, all before the 1.5 nm crossover wavenumber . This loss of intensity skews the kc - fit, resulting in a larger kc . Furthermore, increasing the filter cutoff increases the number of degrees that comprise the linear region below q0, thereby improving the fit to eq 22 . Bending with qcut = 2.5 nm, all 26 degrees (spanning the full range, q0 1.5 nm) can be used to determine kc . All simulations were run using the martini force field and using the gromacs v4.5.3 program . Isobaric (npt) ensemble at constant pressure and temperature (1 bar and 303 k, respectively) using either a 25 fs time step for pure lipid or 10 fs time step for cholesterol systems . Pressure coupling was isotropic for the vesicle systems and semi - isotropic for flat - patch, with independent xy- and z - barostats, resulting in a tensionless bilayer . Initial equilibration for vesicle systems included 100 000 steps of steepest descent minimization followed by 500 ns of dynamics using the velocity - rescaling thermostat and a berendson barostat (flat - patch systems underwent 10 000 steps steepest descent minimization and 100 ns velocity - rescaling dynamics). Hoover thermostat and the parrinello rahman barostat with a time constant of 2.5 and 250 ps, respectively . Pressure coupling was applied isotropically for vesicle simulations and semi - isotropically for flat - patch systems . Vesicle production simulations were 2.5 s (10 s scaled simulation time), flat - patch simulations were 5 s (20 s scaled time) sampled every 1 ns . The starting configurations for the dmpc vesicle (11 126-dmpc, 1 123 315-coarse grained water + antifreeze, high - water, 1 113 742-cg water + antifreeze low - water) and dmpc + cholesterol (12 771-dmpc + 5473-cholesterol, 1 313 698-cg water + antifreeze) were constructed by randomly seeding two opposing monolayers with the appropriate number of lipids (dmpc and/or cholesterol) based on the area per lipid of each species, the surface area of the monolayer shell, and the mole fraction of the mixture . A second dmpc vesicle was simulated using the 3 s frame as a starting configuration with 10% of the internal water beads removed to explore effects of system construction on structure and bending rigidity . Flat - patch bilayer systems were constructed with 3200-dmpc lipids and 2240-dmpc + 960-cholesterol, randomly seeded in flat monolayers with 70,400 coarse grained water + antifreeze particles . During the early stages of equilibration, rapid pore formation occurred throughout the vesicles . These pores coalesced and closed quickly within 70 ns providing an opportunity for both lipid flip - flop and water exchange across the vesicle to equilibrate the system . For the low - water system, the vesicle collapsed into an ellipsoidal shape within 100 ns, and maintained an ellipsoidal character throughout the 3 s production run . Trajectories were manipulated and processed using both the gromacs v.4.5.3 simulation package and the mdanalysis python library . Further data analysis and figure rendering was performed using matlab (v.r2012a) with use of the factorize library . Having established the framework to define rund(,) for vesicle simulations, it now becomes possible to extract the underlying membrane structure from the fluctuating vesicle s trajectory . Figure 4 presents the number density profiles for the dmpc vesicle, rbin(r) and uc(r), and the dmpc flat - patch, uc(z), systems . Vesicle profiles are illustrated as dashed lines, flat - patch profiles as filled distributions, with the component groups color - coded as described in the caption . Negative radial positions correspond to the inner monolayer, positive values correspond to the outer monolayer . Comparisons of component number densities for dmpc vesicle and flat - patch systems . Flat - patch profiles are filled - distributions with headgroup (black / gray), carbonyl - glycerol (red / pink), and acyl - chain (blue / cyan). (a), (b) rbin for the high - water and low - water dmpc system, respectively . (c), (d) uc for high - water and low - water dmpc system . The same flat - patch profile has been included in all panels . Figure 4a compares the high - water vesicle profile, rbin(r), to the flat - patch profile, uc(z). The uc(z) profile was translated by the average vesicle radius, r0, to allow direct comparison to the vesicle profiles . As expected, using a global reference frame with a fluctuating bilayer results in a broadening of the component distributions (rmsd between rbin(r) and uc(z) is 3.08 nm). This broadening results in a loss of structural resolution in the vesicle profile, most noticeably in the acyl - chain distributions, where the typically pronounced terminal methyl trough is absent . Figure 4b compares rbin(r) from the ellipsoidal, low - water dmpc system to the flat - patch profile . As fluctuations increase, the low - water rbin(r) displays significant distortion (rmsd is 7.12 nm) across all lipid - component distributions, highlighting the problem with using a global com reference frame to define membrane structure . Figure 4c, d present comparisons of our new method, uc(r), to the flat - patch profile for both the high- and low - water dmpc systems, respectively . In both cases, the resulting number density more closely resembles the flat - patch result (rmsd between uc(r) and uc(z) is 1.66 nm for low - water dmpc and 2.34 nm for high - water). Each component distribution is tightened up relative to rbin(r), and key structural features (e.g., the terminal methyl trough) are properly characterized . In the high - water system (figure 4c), the headgroup distributions are centered at the appropriate position (i.e., the bilayer thickness matches that of the flat - patch). However, the amplitude and width of the headgroup distributions do not fully agree with the flat - patch result: the amplitudes are too high and the widths to narrow . This small discrepancy suggests that the lipids are under tension, either due to water - density or lipid density asymmetry across the bilayer . Comparison of the low - water profiles (figure 4b, d) demonstrates the dramatic improvement in calculated membrane structure when undulations are isolated and characterized during structure determination . Even with a dramatic ellipsoidal geometry, the uc(r) method extracts the underlying membrane structure . The low - water profile amplitudes agree with the flat - patch profile, more so than the high - water result . However, the membrane appears thicker and the component distributions are slightly broader than the flat - patch result . Comparing figure 4c with 4d (uc(r) for the spherical and ellipsoidal dmpc vesicle systems) as expected, varying the number of waters inside the vesicle can lead to different membrane tensions and corresponding structures . With our new algorithm, this type of comparison can serve as a guide in the initial construction of vesicle simulations, evaluating the membrane structure for imbalances in water density or lipid density across the bilayer . Analysis of the dmpc + cholesterol system highlights additional complexities that can develop due to the different system geometries . Supplemental figure 1 presents rbin(r) and uc(r) for the dmpc + cholesterol system . The undulation correction still sharpens the component distributions, albeit to a lesser extent than the pure dmpc system . Cholesterol s facile ability to flip - flop during the time - scale of the simulation results in an asymmetric cholesterol distribution across the bilayer in the vesicle geometry . The development of an asymmetric cholesterol distribution is not surprising for the vesicle as the spontaneous curvature difference between inner and outer monolayers will induce an asymmetric partitioning of lipids with non - neutral spontaneous curvature . Close inspection of all vesicle number density profiles highlight an asymmetry (particularly in the headgroup distributions) where the inner monolayer has a higher density relative to the outer monolayer (figure 4 and supplemental figure 1). We cannot know for certain whether this asymmetry is due to a redistribution of lipid components or an artifact from a poorly equilibrated vesicle . Vesicle structure does not change across the last 1.5 s for both the dmpc and dmpc + cholesterol systems . However, the differences in structure between high - water and low - water dmpc vesicle systems highlight the effect of water density imbalances across the bilayer . Furthermore, in all simulations, lipid flip - flop was only observed for cholesterol and never for dmpc . Significantly longer simulation times (> 100s of s) of standard md would be necessary to allow for both water equilibration and sufficient lipid flip - flop events to fully equilibrate the vesicle . Our focus here has been on the development of a method to calculate structure from vesicle simulations and not to perfectly refine these particular simulations . Nevertheless, the problem of vesicle construction and equilibration is an important challenge that must be answered to facilitate further study of these more complex systems . Risselada et al . Employed an artificial pore allow for both water and lipid exchange across the vesicle prior to the production md simulation . For simple lipid compositions, this approach is preferable over an iterative refinement of the lipid and water distributions (altering them by hand on the basis of structural profile imbalance) and far more computationally efficient than extending the simulation until sufficient lipid flip - flop is observed . Figure 5 presents the area per lipid (al) time - series for the dmpc vesicle systems . We compare values determined along the urs for the full vesicle (all, blue) and both inner and outer monolayers (green and red, respectively) with those of the flat - patch al (cyan). A summary of the average al is presented in table 1 . In the high - water dmpc system (figure 5a) there are three distinct differences between the vesicle and flat - patch result: (1) the inner and outer monolayer have different al, with inner monolayer lipids occupying more lateral area than those in the outer leaflet; (2) all measures of al are greater than the flat - patch al; and (3) the fluctuations in al are significantly decreased in the high - water vesicle system versus the flat - patch . The imbalance between inner and outer monolayers correlates with the structural asymmetry in the headgroup uc(r). (a) al trajectory for the high - water dmpc vesicle system with total vesicle (blue), inner monolayer (green), outer monolayer (red), and flat - patch system (cyan). We presume that this reflects that the inner monolayer is under greater tension than the outer, constraining the inner headgroups in the radial dimension and resulting in a tighter position distribution (recall that the thickness is the same, figure 3b). There are two potential causes of this tension, either an imbalance in the water density or the lipid density across the vesicle . The area per unit cell, auc = 2 asurface / ndmpc, for the dmpc + cholesterol system shows a similar trend as the dmpc vesicle (see supplemental figure 2). Figure 5b illustrates the same al analysis for the ellipsoidal, low - water dmpc system . It is immediately evident that the water content dramatically affects the vesicle s structure and dynamics . The ideal sphere al is increased for all three al metrics, even though the average vesicle radius is smaller . There is a partial recovery in the al symmetry across the monolayers (i.e., a reduction in al = al, out the residual discrepancy between al, out and al, in likely stems from a lipid density imbalance across monolayers, which is still evident in figure 4d . Although the structure from the low - water system more closely matches the flat - patch profile, it is the al of the high - water system that is in better agreement . The number density profile calculation isolates the increased fluctuations in the low - water system, whereas the al does not . Figure 6 presents the power spectra and subsequent model fits for dmpc high - water (black) and dmpc + cholesterol (red) in both vesicle (figure 6a - c) and flat - patch (figure 6d) geometries . In each case the spectra was determined over the final 1.5 s of the trajectory . As expected, in both the flat - patch and vesicle simulations cholesterol reduces the magnitude of the undulation intensity, reflecting the sterol s well - know rigidifying effect.kc values were determined by fitting the linear regime across low degrees corresponding to q0 1.5 nm to the appropriate helfrich continuum model for vesicle or flat - patch geometries (l 26 for dmpc and l 29 for the slightly larger dmpc + cholesterol vesicle). As shown in table 2, we observe a similar cholesterol - induced increase in kc in the vesicle- and flat - patch geometry (the latter of which was obtained via our the previously published method). Power spectra for both vesicle systems (a) with corresponding kc fit for dmpc (b) and dmpc + cholesterol (c). Undulation power spectrum and kc - fits for flat - patch systems . For all panels, dmpc (black) and dmpc + cholesterol (red). The * denotes values from brandt et al . ; experimental values (* *) from pan et al .. the high - water (hw) dmpc vesicle kc is in better agreement with our previously published kc from a large flat patch, where a 30 000 lipid dmpc coarse - grained flat - patch system had kc = 1.5 10 j. to ensure the use of an equi - angular grid did not introduce any artifacts into the vesicle analysis, we repeated the spha on the high - water dmpc vesicle in a 90 rotated reference frame . The results were identical to the nonrotated system (see supplemental figure 3). The increased kc from our smaller 3200 lipid dmpc flat - patch highlights potential system - size effects with spectral methods to extract kc . The low - water (lw) system shows a reduced kc, relative to the high - water kc, agreeing with the increased al fluctuations (see figure 5b, table 2, and supplemental figure 4). As we discuss below, other factors may also contribute to the differences in kc across the two system geometries, including hydration levels . For example, in the low - water system, we observed a dramatic increase in fluctuations and concomitant decrease in kc . The increase in rigidity for the dmpc + cholesterol system, determined as the ratio of kc between dmpc + cholesterol and dmpc, shows excellent agreement for the high - water (hw) case, 1.8 for flat - patch versus 1.9 for vesicles . For both cases (pure lipid and + cholesterol), the vesicle s kc is smaller than the corresponding flat - patch (1.3 10 j versus 2.1 10 j for dmpc and 2.4 10 j versus 3.8 10 j for dmpc + cholesterol). In both the flat - patch and vesicle systems, the calculated kc value for dmpc is an order of magnitude greater than that determined experimentally (0.58 10 j, determined from diffuse x - ray scattering on oriented bilayer stacks). However, given that our goal is to establish a reliable and robust (i.e., force - field independent) algorithm, we consider the agreement between the established flat - patch method and our new vesicle method as the relevant measure of success, rather than close agreement between our result and experiment . That said, we do note that there is much better agreement between the kc calculated from simulation and the experimental values for both dmpc low - water (6.1 10 j versus 5.8 10 j) and the dmpc + cholesterol systems (2.4 10 j versus 2.73 10 j, respectively). Achieving better agreement with experiment in these values remains a challenge for force - field development, though it remains to be seen whether any force field (even fully atomistic ones) will be able to accurately capture kc across all lipid types . Because of the sensitivity of the spectrum to the arc - length filter cutoff (see figure 3), it was important to evaluate the sensitivity of the kc fit to the loss of undulation intensity . We did so by varying the number of degrees used in the data - fitting, testing both the 1.5 nm and 2.5 nm filters . Supplemental figure 5 illustrates the sensitivity of kc fits and time course of kc for both dmpc and dmpc + cholesterol vesicle systems . The increased linear region for the 2.5 nm spectra results in a much broader span of converged kc fits for both vesicle systems (supplemental figure 5b). This added robustness confirms our choice of filter parameters, specifically the 2.5 nm arc - length filter (for rendering the initial surface) and the 1.5 nm ideal filter (to define the urs and truncate the sph coefficients for the kc - fit at the transition from continuum to molecular length scales). The evolution of kc shows very small changes across the duration of the simulations for both dmpc and dmpc + cholesterol systems (supplemental figure 5c). We have developed a method to determine membrane structure (number density and area per lipid) and bending rigidity from md simulations of lipid vesicles . Comparisons of number density profiles determined from vesicle simulations using either a global or a local reference frame highlight the broadening effect introduced by local bilayer fluctuations, similar to what was previously observed in large flat - patch simulations . Using a urs removes the fluctuation - induced broadening effect and recovers the underlying structure profile, even under extreme vesicle deformations (e.g., elliptical vesicles). Our new spha method can be applied to any md simulation where the topography is such that radial lines intersect the topography only once (e.g., vesicles, bubbles, micelles). In systems where the topography is more tortuous (e.g., where radial lines intersect the topography multiple times) the spha method breaks down . Nevertheless, the local structure profile can still be resolved via the urs, as long as the method used to define the urs is modified to accommodate the more complex topography . Our choice of using an equi - angular grid in order to define the urs as well as in implementing the spha was done to simplify the calculation of the sph coefficients via matrix transformation (eq 12). Alternative surface definitions (e.g., geodesic gridding) are equally viable; however, they may introduce increased complexity and computational cost in determining the transformation matrix . Any predefined, surface reference frame requires either a real - space filter (e.g., arc - length filter) or a direct spherical harmonic transformation that requires treating every membrane bead as a radial delta function to obtain the urs . Although the latter is the direct corollary to our flat - patch direct fourier method, the increased computational cost of the explicit sph transform makes that approach prohibitive . The spherical harmonics analysis of the urs allows us to determine the al for the equivalent ideal sphere that samples the same area as the undulating bilayer . We define a unique al for the vesicle as well as both monolayers . These three al metrics describe structural imbalances that exist due to initial vesicle construction (e.g., water or lipid imbalances across the bilayer). The convergence of these three metrics may serve as a simple readout for vesicle equilibration . This paper lays the groundwork for an iterative process to improve vesicle construction . With flat - patch membranes, numerous studies have been successful in matching experimental structure profiles by tuning the al through altering the periodic box dimensions . The vesicle membrane geometry raises a new and more difficult challenge: obtaining al, structure, and bending rigidity correct by varying the lipid distribution in the two monolayers and the water density inside the vesicle . Our algorithm can extract these structural and mechanical properties to guide the construction and equilibration of these complex vesicle systems . Comparison of these structural and mechanical results with experimental measurables is the ultimate goal . Although we are currently limited by the accuracy of the simulation force fields, changes in structure that correlate with changes in rigidity may provide insight into the interpretation of experimental results.
Recent research indicates that at least one - third of us adults report regularly getting less than the 79 hours of sleep per night recommended by the national sleep foundation . Insufficient sleep, variably defined, has been associated with health - risk behaviors, such as smoking [2, 3], alcohol use [2, 3], and with adverse health outcomes such as obesity, and frequent mental distress (fmd) (14 days / past 30 days in which respondents report their mental health was not good). Noting that the average sleep duration among us adults has decreased during the past 50 years along with a concomitant increase in the prevalence of obesity, wheaton et al . Found a strong positive relationship between perceived insufficient sleep and body mass index (bmi) among community - dwelling adults . Given the linkage between insufficient sleep and increased bmi, the association between insufficient sleep and diabetes is not surprising . Consistent with these findings, sleep restriction (defined as 5 hours / night for one week) was found to be associated with a significant decrease in insulin sensitivity . Notably, the prevalence of several factors associated with insufficient sleep has been reported to vary between different race / ethnicities in large epidemiologic studies . Specifically, an analysis of 20062008 bmi data from the behavioral risk factor surveillance survey (brfss) revealed prevalences of obesity of 23.7% among non - hispanic whites (nhw), 35.7% among non - hispanic blacks although ai / an were not specifically delineated in the latter survey, rates of obesity in ai / an adults have been reported to be 1.5 times greater than among nhw adults . Similarly, aggregated prevalences (19992004) of diagnosed diabetes reported from the national health and nutrition examination survey (nhanes) were 5.9% among nhw, 11.1% among nhb, and 10.9% among mexican americans . An analysis of data from both the brfss and the indian health service across 19942002 revealed that the age - adjusted prevalence of diabetes among ai / an adults was more than twice that of us adults overall . Despite the relatively high prevalence of factors associated with insufficient sleep in ai / an, few epidemiologic investigations of insufficient sleep have been conducted in this population . In investigations delineating race / ethnicity disparities, short sleep duration (average 6 hours/24-hour period) among workers was reported significantly more frequently by nhb (38.9%), nh other (35.3%), and nh asian (33.2%) than among nhw (28.6%) or hispanic (28.8%). In related research using nhanes 20052008 data to assess sleep - related difficulties (e.g., concentrating, remembering, driving, and working), nhb reported a greater prevalence of sleep - related difficulties in driving or taking public transportation (14.8%) than the other racial / ethnic populations studied (nhw, mexican americans, and others). Similarly, reporting 30 days of insufficient sleep or rest was significantly more prevalent among nhb (13.3%) than nhw (11.2%) 2008 brfss respondents . It is worthy of note that because of small sample sizes of ai / an in national surveillance systems, ai / an are often categorized as others, thus rendering it difficult to ascertain the prevalence of sleep sufficiency and risk factors in this population . As many risk factors associated with frequent insufficient sleep appear to be of increased prevalence in ai / an, we sought to determine the prevalence of insufficient sleep in a community - based sample of this population . Specifically, to examine if ai / an are more likely to experience insufficient sleep relative to nhw, as has already been reported among nhb, we aggregate data from the 2009 and 2010 brfss . Furthermore, we assess the impact of recognized sleep correlates (i.e., socioeconomic indicators, lifestyle behaviors, obesity, age, and fmd) on potential race / ethnicity disparities in insufficient sleep . Data were obtained from the recent brfss, a large, random - digit - dialed telephone survey conducted in all 50 states, the district of columbia, and us territories . The 2009 - 2010 brfss collected data on health - related behaviors, including sleep, smoking, physical inactivity, binge drinking, obesity, and frequent mental distress among the us civilian population aged 18 years living in households with landline telephones . As previously noted, we combined 2009 and 2010 brfss data in order to yield an adequate sample size of ai / an . Before aggregating these data, we observed no difference between 2009 and 2010 in frequent insufficient sleep, defined as 14 days / past 30 days in which respondents reported that they did not get enough rest or sleep . Additionally, the two years shared a similar median response rate to the question assessing frequent insufficient sleep (52.5% and 54.6%, respectively) (brfss 2009 and 2010 summary quality report, version 1, revised on 2/18/2011 for 2009 and on 5/2/2011 for 2011). A detailed description of the brfss survey design, data collection techniques, and the full - text questionnaire can be found at http://www.cdc.gov/brfss . Data were obtained from 810,168 respondents (96.9%) who self - identified as nhw, (n = 671,448), nhb (n = 67,685), hispanic (n = 59,528), or ai / an (n = 11,507) after excluding respondents who had missing data on the insufficient sleep question (n = 15,322) and other variables of interest (n = 25,636). All respondents were asked, during the past 30 days, for about how many days have you felt you did not get enough rest or sleep? We defined frequent insufficient sleep as 14 days, as this cutoff has been shown to have a strong relationship with the prevalence of chronic disease and health risk behaviors . Sociodemographic characteristics included sex, age in years (1824, 2534, 3544, 4554, 5564 or 65), years of education (<12, 12, or> 12), and employment status (employed for wage / self - employed, unemployed, retired, unable to work, or homemaker / student). Health - related lifestyle behaviors included smoking status (current smoker, former smoker, or never smoked), binge drinking (for men, 5 alcoholic beverages on one occasion in the previous 30 days; for women, 4 alcoholic beverages on one occasion in the previous 30 days), and physical inactivity (respondent indicated no to the question, during the past month, other than your regular job, did you participate in any physical activities or exercising such as running, calisthenics, golf, gardening, or walking for exercise?). Frequent mental distress (fmd) was defined as a response of 14 days to the question, now thinking about your mental health, which includes stress, depression, and problems with emotions, for how many days during the past 30 days was your mental health not good? . Assessment of obesity was based on the body mass index (bmi, kg / m), calculated from respondents' self - reported height in inches and weight in pounds (obese: bmi 30 kg / m versus not obese: bmi <30.0 kg / m). First, the racial / ethnic - specific distributions of the selected characteristics and frequent insufficient sleep were obtained in order to examine unadjusted race / ethnicity disparities . Next, the prevalence of frequent insufficient sleep by race / ethnicity and groups defined by obesity, smoking, binge drinking, physical activity, and fmd status was calculated to demonstrate race / ethnicity - specific associations between these covariates and frequent insufficient sleep . We used multivariate logistic regression modeling to assess the race / ethnicity disparity in frequent insufficient sleep in separate models controlling first for age, then in a second model with the addition of sex, education, and employment, to which obesity was added to create a third model . A fourth model added smoking status, physical inactivity, and binge drinking to the third model, with a fifth model adding fmd . All analyses were conducted using sas - callable sudaan to account for the complex sampling design . Ai / an had a higher proportion of male respondents than other racial / ethnicity groups . Relative to nhw, we found that nhb, hispanics, and ai / an tended to be younger, to report <12 years of education and were more likely to be unemployed or to report being unable to work (p 0.01). In addition, nhb, ai / an, and hispanics were more likely to be obese relative to nhw (p 0.01) and, concomitantly, to report they were physically inactive (p 0.001). Ai / an were more likely to report being current (33.2%) or former (25.7%) smokers than any of the other race / ethnicity groups examined . Binge drinking was similar in all race / ethnicity groups except among nhb who were less likely to report binge drinking . Table 1 also indicates that the unadjusted prevalence of fmd was significantly greater (p 0.001) among ai / an than any other race / ethnicity group . Most notably, we found significantly higher unadjusted prevalences (95% confidence interval [95% ci]) of frequent insufficient sleep among ai / an (34.2% [32.136.4]) and nhb (29.4% [28.630.1]) and a significantly lower prevalence among hispanics (25.4% [24.726.1]) compared to nhw (27.4% [27.127.6]). Table 2 lists the race / ethnicity - specific prevalences of frequent insufficient sleep by selected characteristics . Obese respondents had a statistically higher prevalence of frequent insufficient sleep than nonobese respondents among all race / ethnicities, excepting ai / an . Regardless of race / ethnicity, current smokers were more likely to report frequent insufficient sleep than former smokers or never smokers (p 0.01). A higher prevalence of frequent insufficient sleep among binge drinkers compared to nonbinge drinkers reached statistical significance only among nhw and nhb (p 0.01). A positive relationship between physical inactivity and frequent insufficient sleep was observed regardless of race / ethnicity (p 0.01). Persons reporting fmd were significantly more likely to report frequent insufficient sleep than those without fmd in all race / ethnicity groups (p 0.001). Results from our adjusted multivariate logistic regression models comparing the likelihood of frequent insufficient sleep among nhb, hispanics, and ai / an relative to frequent insufficient sleep in nhw are shown in table 3 . After age adjustment (model 1) the disparity in frequent insufficient sleep between nhw and nhb (pr = 1.02; 95% ci: 0.991.04) was no longer statistically significant . However, both nhb (pr = 0.95; 95% ci: 0.920.98) and hispanics (pr = 0.84; 95% ci: 0.810.86) remained significantly less likely (p 0.001) to report frequent insufficient sleep than nhw, even after controlling for all the covariates in the model (model 5). Table 3 also reveals a significant age - adjusted excess prevalence of frequent insufficient sleep in ai / an relative to nhw in model 1, (pr = 1.20; 95% ci: 1.121.27). This was modestly weakened by the addition of sex, education, and employment (pr = 1.12; 95% ci: 1.041.19) as indicated by model 2 . This relationship was further attenuated in ai / an by the separate addition of obesity in model 3 (pr = 1.10; 95% ci: 1.031.18), and then by the addition of smoking, physical inactivity, and binge drinking (pr = 1.08; 95% ci: 1.011.16) in model 4 . Finally, the disparity in frequent insufficient sleep was no longer significant after adding fmd in model 5 (pr = 1.05; 95% ci: 0.991.13). This is the first investigation to document the excess prevalence of frequent insufficient sleep among ai / an relative to other race / ethnicity groups . Over 34% of ai / an reported frequent insufficient sleep 14 days in the past month in contrast to 29% of nhb, 27% of nhw, and 25% of hispanics . The disparity in frequent insufficient sleep prevalence between ai / an and nhw remained even after we adjusted for recognized sleep correlates that included sociodemographic characteristics, lifestyle behaviors, and obesity . However, we also found that this disparity was attributable, in part, to the relatively high rate of fmd in the ai / an population . Notably, previous sleep research in this population has largely been limited to the identification of factors associated with the increased prevalence of sudden infant death syndrome [1618] and has documented the prevalence of sleep disturbance among ai / an veterans . Other research on sleep health in ai / an suggests possible implications of the results of the present investigation . Specifically, noting that cardiovascular disease (cvd) is the leading cause of death among ai / an, sabanayagam et al . Assessed both symptoms of insomnia including short sleep duration and self - reported cvd among 449 ai / an aged 55 years of age in the native elder care study . Notably, these investigators found that relative to participants reporting a 7-hour sleep duration, the multivariable odds ratio for cvd of those with a sleep duration 5 hours was 2.89 (95% ci: 1.177.16), after adjusting for demographic, lifestyle, and clinical factors . These results suggest that the excess insufficient sleep documented in the present investigation could manifest itself in the increased risk for cvd - related morbidity and mortality in the ai / an population . In a review of the literature metabolic cost, and triggers a cascade of metabolic, neuroendocrine, and behavioral changes designed to increase food consumption and decrease energy expenditure, thereby giving rise to obesity . The excess insufficient sleep observed in this study may thus underlie findings indicating that ai / an are at increased risk of obesity relative to other race / ethnicity groups [2325]. Furthermore, as diabetes is associated with obesity [2629], our findings may describe a heretofore unrecognized risk factor underlying the increased rate of diabetes among ai / an [3032]. First, our measure of frequent insufficient sleep was based on subjective self - report and is not corroborated by actigraphy or polysomnography . Second, the wording of the insufficient sleep question includes both the terms rest and sleep, thereby being potentially subject to varying interpretations among respondents . Third, our sample is limited to only households with landline telephones and to respondents residing in noninstitutionalized settings; thus, our findings may not be generalizable to the overall us population . Our finding of excess insufficient sleep among ai / an thus appears to confirm the importance of sleep health to chronic disease prevention and health promotion in this population . Specifically, assessment of sleep sufficiency appears particularly germane to healthcare providers serving this population, as well as to public health messaging targeting sleep health (including sleep correlates) in ai / an communities . Available interventions designed to promote awareness of the importance of sufficient sleep include a summary for patients describing the influence of both diet and sufficient sleep on efforts to lose weight, as well as cognitive behavioral therapy for insomnia [34, 35], with the latter improving both subjective and objective sleep quality, as measured by polysomnography . Finally, as fmd appears associated with frequent insufficient sleep among ai / an, mental health assessment and intervention emerge as important components in the evaluation of insufficient sleep in this population.
Because steroid hormones play an important role in a wide range of physiological processes, the potential to disturb endocrine effects is a major concern in the development of novel pharmaceutical drugs such as etomidate and aminoglutethimide . The adrenal gland is the most common target for toxicity in the endocrine system in vivo, because steroid hormones are primarily synthesized through enzymatic reactions in the adrenal cortex [25]. Indeed, in these studies based on chemically induced endocrine lesions observed in vivo, the most frequent site of reported effects was the adrenal gland . Therefore, the prediction of human adrenal toxicity based on the mechanism of on- or off - target actions in the early stages of drug development is important . The nci - h295r human adrenocortical carcinoma cell line has been used to elucidate mechanisms of adrenal steroidogenic disrupting compounds [1, 6]. The h295r cell line was established by gazder and his collaborators in 1990, which expresses all key steroidogenic enzymes and steroidogenesis - related proteins [79]. H295r cells have the physiological characteristics of zonally undifferentiated human fetal adrenal cells and the ability to produce steroid hormones found in the adult adrenal cortex [1, 7, 9]. In vitro bioassays using the h295r human cell line have been able to evaluate the effects of chemicals on steroid hormone production [1015], steroidogenic enzyme activities [11, 16, 17], and the expression of steroidogenic genes [11, 18]. In transcriptome studies, the mechanisms of action of many steroidogenic disrupting compounds have been qualitatively assessed in terms of adrenal toxicity . Furthermore, measuring a few specific steroid hormones may not be a useful approach to study the mechanisms of steroidogenic disrupting effects in complex pathways such as adrenal steroidogenesis . To systematically understand how exogenous compounds affect adrenal steroidogenesis, simultaneous determination of all detectable steroid hormones and integrative analysis of these complex data would be important . As an exploratory approach to analyze complex data, toxclust developed by zhang and colleagues in 2009 is able to visualize concentration - dependent response relationships in the characteristics of chemically induced toxicological effects . However, this exploratory approach is unable to provide a quantitative understanding of the mechanism of action of adrenal toxicants or reveal systematic information about the effect of each enzymatic reaction, interactions, and feedback in the adrenal steroidogenesis pathway . Systems biology based on computational models of biological processes and the comprehensive measurement of biological molecules is the most powerful approach to quantitatively understand the influence of each factor in complex biological pathways . In recent studies by our collaborators, a computational model of adrenal steroidogenesis has been developed in nci - h295r cells, including the steroidogenic disrupting effects of metyrapone to inhibit enzymatic reactions of cyp11b1 [21, 22]. The model reproduces the dynamics of adrenal steroidogenesis in nci - h295r cells and the influence of metyrapone . A current computational model of adrenal steroidogenesis was incorporated with a reaction of oxysterol synthesis as a bypass to consume cellular cholesterol . In addition, all reactions in this model are described by a kinetic equation of the first - order reaction . It is difficult to quantitatively evaluate the influence of each protein in the complicated system of adrenal steroidogenesis using the reported models, because it is simple and any biochemical and cellular biological information is not sufficient . For example, to clearly understand the cause of the change from the differentially dynamic patterns of steroid hormones, it is necessary to consider the substrate inhibition of steroidogenic enzyme because most of steroidogenic enzymes recognize multiple steroids as the enzymatic substrate . However, the substrate inhibition of steroidogenic enzyme cannot be described by the mathematical model based on kinetic equations of first - order reaction that does not consider michaelis constant km expressing the affinity of the substrate . To quantitatively estimate the mechanism of steroidogenic disrupting compounds from comprehensive experimental data of adrenal steroidogenesis in nci - h295r cells, the reported model should be improved according to the following two points . First, the kinetic equation of enzymatic reactions should be exchanged from the first - order equation to a steady - state kinetic equation based on the mechanism of the enzymatic reaction . Because a mathematical model organized by first - order equations operates in a simple structure - dependent manner, it does not show complex behavior based on molecular interactions, feedback, or regulation . Second, intracellular localization processes of cholesterol should be incorporated as a considerable mechanism . Because intracellular cholesterol molecules are stored as cholesterol esters or widely distributed as membrane components, only a few cholesterol molecules localized on the mitochondrial inner membrane are available for the adrenal steroidogenesis pathway [23, 24] moreover, cholesterol - trafficking processes from the outer to inner mitochondrial membranes, which are regulated by steroidogenic acute regulatory (star) protein, are one of the rate - limiting steps in adrenal steroidogenesis . By overcoming these limitations in the reported steroidogenesis model, systems analysis of adrenal steroidogenesis in h295r cells may be able to quantitatively estimate the mechanism of action of steroidogenic disrupting compounds . In the present study, to quantitatively estimate the toxicological mechanism of endocrine - active compounds in adrenal steroidogenesis and to predict human adrenal toxicity of novel pharmaceutical drugs in the drug discovery phase, we developed a novel computational model of steroidogenesis in nci - h295r cells . It includes cholesterol transport into intracellular regions from the extracellular space, the cholesterol translocation system in intracellular regions, including oxysterol synthesis, the metabolic pathway of adrenal steroidogenesis, and transport of steroid hormones . Global sensitivity analysis of this adrenal steroidogenesis model is able to evaluate the influence of each steroidogenic enzyme and related protein for each steroid hormone observed in an in vitro steroidogenesis assay of nci - h295r cells . Furthermore, the mechanisms of action of steroidogenesis disrupting compounds for steroidogenic enzymes can be estimated by the optimization method to solve the reverse problem from the concentration changes of 12 steroid hormones measured by liquid chromatography / mass spectrometry in the steroidogenesis assay of nci - h295r cells in vitro . Using this developed model of adrenal steroidogenesis and the analytical approach, the in vitro steroidogenesis assay of nci - h295r cells can assess the human adrenal toxicity of a novel pharmaceutical drug based on quantitative understanding of its toxicological mechanism in adrenal steroidogenesis . Nci - h295r human adrenocortical carcinoma cells were purchased from the american type culture collection (cat #crl-2128, manassas, va) and cultured at 37c in a humidified atmosphere with 5% co2 . The cells were maintained in a 1: 1 mixture of dulbecco's modified eagle's medium (dmem, gibco, life technologies, carlsbad, ca) and f-12 medium (mp biomedicals inc ., irvine, ca) supplemented with 15 mm hepes (dojindo laboratories, kumamoto, japan), 0.00625 mg / ml insulin (sigma - aldrich, inc ., st . Louis, mo), 0.00625 mg / ml transferrin (sigma - aldrich inc ., st . Louis, mo), 30 nm sodium selenite (wako pure chemical industries ltd ., osaka, japan), 1.25 mg / ml bovine serum albumin (bsa, sigma - aldrich inc ., st . Louis, mo), 0.00535 mg / ml linoleic acid (sigma - aldrich inc ., st . Louis, mo), 2.5% nu serum (becton, dickinson and company, franklin lakes, nj), 100 u / ml penicillin (meiji seika pharma, tokyo, japan), and 100 mg nci - h295r cells were stimulated with adrenocorticotrophic hormone (acth), forskolin, and angiotensin ii to initiate steroidogenesis . Changes in steroid concentrations over time were measured after stimulation in both cells and culture medium to construct a simulation model . The cells were seeded at 6 10 cells / well in 6-well plates . After 3 days of culture, the culture medium was changed to stimulation medium consisting of dmem / f-12 (1: 1) medium supplemented with 0.00625 mg / ml insulin, 0.00625 mg / ml transferrin, 30 nm sodium selenite, 1.25 mg / ml bsa, 0.00535 mg / ml linoleic acid, 10% fetal bovine serum (gibco, life technologies, carlsbad, ca), 100 u / ml penicillin, 100 mg / l streptomycin, 50 nm acth (sigma - aldrich inc ., louis, mo), 20 m forskolin (sigma - aldrich inc ., st . Louis, mo), and 100 nm angiotensin ii (calbiochem, merck millipore, darmstadt, germany). Culture media and cells were collected at 0, 8, 24, 48, and 72 h after stimulation . The cells were collected in 100 l distilled water and sonicated to produce a cell lysate . The cultures were conducted in four wells / time point (n = 4). The concentrations of 12 steroids, pregnenolone (preg), 17-hydroxypregnenolone (hpreg), dehydroepiandrosterone (dhea), progesterone (prog), 17-hydroxyprogesterone (hprog), androstenedione (dione), testosterone (testo), 11-deoxycorticosterone (dcortico), 11-deoxycortisol (dcort), corticosterone (cortico), cortisol (cort), and aldosterone (aldo), in the medium and cell lysate were measured by lc / ms . Concentrations of estrone (e1) and 17-estradiol (e2) were measured by enzyme - linked immunosorbent assays (wako pure chemical industries ltd . In addition, the concentration of cholesterol was measured using a commercial kit (wako pure chemical industries ltd ., osaka, japan) based on the cholesterol oxidase method . A lc - vp series (shimadzu, kyoto, japan) consisting of an sil - htc autosampler, lc-10advp pump, cto-10acvp column oven, and dgu-14am degasser was used to set the reverse - phase liquid chromatographic conditions . The column was a cadenza cd - c18 column (100 2 mm i.d ., 3 m, imtakt corp ., kyoto, japan) used at 45c . The mobile phase included water / acetonitrile / formic acid 95/5/0.05 (v / v / v, solvent a) and water / acetonitrile / formic acid 35/65/0.05 (v / v / v, solvent b). The gradient elution programs were 0% b (0 - 1 min with an isocratic gradient), 040% b (1 - 2 min with a linear gradient), 40% b (27 min with an isocratic gradient), 40100% b (712 min with a linear gradient), 100% b (1214 min with an isocratic gradient), 1000% b (14 - 15 min with a linear gradient), and 0% b (15 - 16 min with an isocratic gradient) at a flow rate of 0.3 ml / min . The autosampler tray was cooled to 45c and the injection volume was 5 l . A triple quadrupole mass spectrometer api4000 (applied biosystems / mds sciex, concord, canada) coupled with an electrospray ionization source was operated in the positive ion mode . The optimized ion source conditions were as follows: collision gas, 6 psi; curtain gas, 40 psi; ion source gas 1, 50 psi; ion source gas 2, 80 psi; ion source voltage, 5500 v; ion source temperature, 600c . Nitrogen was used as the collision gas in the multiple reaction monitoring (mrm) mode . The conditions of declustering potential, collision energy, and collision cell exit potential were optimized by every steroid . The transitions in mrm were as follows: preg m / z 317 299, hpreg m / z 315 297, dhea m / z 289 271, prog m / z 315 109, hprog m / z 331 109, dione m / z 287 97, dcort m / z 331 123, dcortico m / z 347 161, cortico m / z 347 100, cort m / z 363 309, aldo m / z 361 343, and testo m / z 289 109 . Mass spectroscopic data were acquired and quantified using the analyst 1.4.2 software package (applied biosystems / mds sciex, concord, canada). Cell volume was estimated from the number of cells in the well and the average diameter of the cells . Cells were detached from the well using 0.025% trypsin (mp biomedicals, inc ., irvine, ca) in a 0.02% edta solution (dojindo laboratories, kumamoto, japan) at the start of preculture, start of stimulation, and at 24, 48, and 72 h after stimulation . The numbers and diameters of the cells were measured by a cell counter vi - cell xr 2.01 (beckman coulter, krefeld, germany) after trypan blue staining . Parameters of the cell volume and number of cells were estimated to fit experimental time - course data using exponential curves . Nci - h295r cells were exposed to seven well - characterized inhibitors of steroidogenesis, and then the concentrations of the steroids in the culture medium were measured to estimate the enzyme inhibition to evaluate the performance of the simulation model . The adrenal steroidogenic inhibitors included aminoglutethimide (agt, bachem ag, bubendorf, switzerland), o, p-ddd (ddd, sigma - aldrich inc ., st . Louis, mo), spironolactone (sp, sigma - aldrich inc ., st . Louis, mo), metyrapone (mp, sigma - aldrich inc ., st . Louis, mo), ketoconazole (kc, wako pure chemical industries, ltd ., osaka, japan), miconazole (mc, wako pure chemical industries, ltd ., osaka, japan), and daidzein (dz, sigma - aldrich inc ., st . The cells were also exposed to four adrenal toxicants whose adrenal toxicity is not mediated through steroidogenesis inhibition . The toxicants were acrylonitrile (an, wako pure chemical industries, ltd ., osaka, japan), salinomycin (sm, sigma - aldrich, inc ., st . Louis, mo), thioguanine (tg, tokyo chemical industry co., ltd ., tokyo, japan), and fumaronitrile (fn, wako pure chemical industries, ltd ., osaka, japan) and added to the culture medium at 1: 1000 dilutions . Nci - h295r cells were cultured for 3 days in 6-well plates and then stimulated with the above - mentioned compounds . Upon the start of stimulation, various concentrations of test chemicals were added to the cultures . After a further 3 days of culture with the chemicals, the concentrations of 12 steroids (preg, hpreg, dhea, prog, hprog, dione, dcortico, dcort, cortico, cort, aldo, and testo) in the culture medium were measured by lc / ms / ms . The cytotoxicity assay was conducted in 96-well plates using atp content in cells as an endpoint (celltiter - glo luminescent cell viability assay, promega). The test concentrations of adrenal steroidogenesis inhibitors and other compounds are shown in table 1 . Comparisons were performed by the two - sample welch's t - test with bonferroni multiple testing correction for each steroid hormone species . Steroid hormones secreted from human adrenal corticocarcinoma nci - h295r cells are synthesized from cholesterol through the c21-steroid hormone biosynthesis pathway . A mathematical model of adrenal steroidogenesis in nci - h295r cells was constructed with cholesterol transport and the intracellular localization pathway, the oxysterol synthesis pathway as a bypass of steroidogenesis, the c21-steroid hormone biosynthesis pathway as the main steroidogenesis pathway, passive transport of steroid hormones, and cell proliferation (figure 1). In this model, two compartments, the intracellular space and culture medium, were incorporated as the available region . Equations and parameters of the cell proliferation and diffusional transport of steroid hormones have been proposed by previous studies [21, 22]. Cholesterol transport and the intracellular localization pathway including the oxysterol bypass were integrated using a part of the acth - stimulated cortisol secretion model described by dempsher and colleagues . The c21-steroid hormone biosynthesis pathway includes 14 steroid hormones, preg, hpreg, dhea, prog, hprog, dione, testo, dcortico, dcort, cortico, cort, aldo, e1, and e2, and 17 enzymatic reactions catalyzed by nine steroidogenic enzymes, cholesterol side chain cleavage enzyme (cyp11a1), 17-hydroxylase (cyp17h), c17,20-lyase (cyp17l), 3-hydroxysteroid dehydrogenase (hsd3b2), 21-hydroxylase (cyp21a2), 11-hydroxylase (cyp11b1), 18-hydroxylase (cyp11b2), 17-hydroxysteroid dehydrogenase (hsd17b3), and aromatase (cyp19a1). In this mathematical model of adrenal steroidogenesis in nci - h295r cells, the flux velocities of molecular transportation and enzymatic reaction rates of steroidogenic enzymes were defined based on the first - order reaction and rapid - equilibrium enzyme kinetics, respectively . All equations in the mathematical model of adrenal steroidogenesis of nci - h295r cells were described in a supplementary document (see supplementary material available online at http://dx.doi.org/10.1155/2016/4041827). The rate constants and the maximum activities were estimated by fitting to experimental time - course data of the concentrations of cholesterol and all steroids . Initial values of cholesterol and the 14 steroid concentrations were used in each experimentally measured value, and every steroid concentration was assumed to rapidly reach the equilibrium state between the culture medium and intracellular space . All fixed values of static parameters and initial values of variable parameters in this model were described in tables s1 and s2 in a supplementary document, respectively . This computational model of adrenal steroidogenesis in nci - h295r cells was developed on the simbio platform which is a general environment of biological dynamic simulation and computational model development . Odes were solved by the fourth - order runge - kutta method with a variable time step . The time step (dt) was adjusted to refer to the maximum absolute value of flux velocities or enzymatic reaction rates at each time point, and the range of the time step was from 1 10 to 10 . To confirm whether the range of the time step was suitable, the numerical error ratio was calculated by certain fixed time steps in the range of the time step, which was under 1 10 in every time step . The duration time of computational simulation of adrenal steroidogenesis in nci - h295r cells was set at 72 h. to reconstruct experimental time - course patterns of the concentrations of cholesterol and the 14 steroids in the culture medium and intracellular space, we optimized every rate constant and maximum velocity of the steroidogenic enzymes . This parameter optimization problem was solved by the levenberg - marquardt method which is one of the nonlinear least squares methods [4951]. The objective function of optimization was used as the following normalized least squares distance (nlsd):(1)nlsd=h i jxh, i, jexpxh, i, jsim2xh, imax2,where h is the compartment (culture medium or intracellular space), i is the molecular species (cholesterol and the 14 steroids), j is the time point (0, 8, 24, 48, and 72 h), xh, i, j is the experimentally measured concentration of molecule i in compartment h at time point j, xh, i, j is the simulated concentration of molecule i in compartment h at time point j, and xh, i is the maximum concentration of molecule i in compartment h over all time points . Data points under the lower quantitation limit were excluded from the evaluation by the objective function . Effects of every static model parameter for parameter optimization were calculated from differences of fitting the objective function using sensitivity analysis . Metabolic steroid profiling and differential patterns of the adrenal steroid hormones by chemical perturbation were reconstructed to optimize the relative activities of the steroidogenic enzymes . The input data for the quantitative mechanistic analysis of adrenal toxic compounds was a fold change (ratio) of the measured 12 steroid concentrations induced by drug exposure for 72 h. the two - step optimization method of the real - coded genetic algorithm (rcga) was adopted as a global optimization method in the quantitative mechanistic analysis of adrenal toxic compounds . The operations of the crossover and generation alteration model in rcga were used for the real - coded ensemble crossover (rex) and just generation gap (jgg) [5255]. As the initial parameters of rcga, maximum generation, population size, selection size of parent individuals, population size of child individuals, and termination criteria were 1000, 100, 6, 25, and under 0.1 of nlsd, respectively . The search space for the relative activities of the steroidogenic enzymes was from 1/100 to 100 . To evaluate the fitness of each individual, the sum of squared residuals for fold changes of measured 12 steroid concentrations was used as the objective function . Nonlinear least squares optimization by the levenberg - marquardt method was used as a local search [4951]. As the estimated mechanisms of actions of the adrenal toxic compounds, the relative activities of eight steroidogenic enzymes (cyp11a1, cyp17h, cyp17l, hsd3b2, cyp21a2, cyp11b1, cyp11b2, and hsd17b3) were optimized by the above - mentioned 2-step optimization method . The property of every kinetic parameter in this computational model of steroidogenesis in nci - h295r cells was evaluated by dynamic sensitivity analysis . The sensitivity (sx, y) of kinetic parameter x for variable parameter y was defined by the following equation:(2)sx, yt=yt / ytx / x, where variable parameter y was the concentration of a steroid hormone in the cytosolic space of nci - h295r cells . The perturbation for kinetic parameters was + 10% (x / x = 0.1). All steroid hormones in the culture medium were significantly increased after 72 h of stimulation with 50 nm acth, 20 m forskolin, and 100 nm angiotensin ii (figure 2(a)). Mass balances in steroidogenesis of nci - h295r cells under nontreatment and control (stimulated) conditions are shown in figures 2(b) and 2(c), respectively . Under stimulation, the dynamics of net mass in these experiments were unchanged, and accumulated cholesterol was converted to adrenal steroids . Viabilities of cells treated with each compound were expressed as a relative value to the atp level of the control . Effects of an, sm tg, fn, agt, ddd, sp, mp, kc, mc, and dz on cell viability were determined to be valid under 80% of the relative atp level at 7 days after treatment . An, sm, tg, and fn showed cytotoxicity at over 100, 1, 10, and 10 m, respectively . Agt, mp, and dz did not affect cell viability at up to 100 m . Ddd, sp, kc, and mc induced less than 80% of cell viability at over 100, 50, 100, and 25 m, respectively . After nci - h295r cells were exposed to each test compound during three days, the concentrations of 12 steroid hormones in the culture medium were simultaneously measured by lc / ms / ms . All effects of the compounds on adrenal steroidogenesis were evaluated at the concentration without any overt cytotoxicity . The differential metabolic steroid profiles of 11 adrenal toxic compounds were classified and visualized by using hierarchical clustering analysis (figure 3). Four adrenal toxicants without steroidogenic inhibition, an, sm, tg, and fn, did not change the medium concentrations of all steroid hormones by more than 2-fold . Above - mentioned 4 compounds at every condition and 7 adrenal steroidogenic inhibitors at the low exposure concentration were gathered into a big cluster as nonchange group . The 7 steroidogenic inhibitors at the maximum exposure concentration showed the characteristic steroid profiles each, but 100 m dz and 10 m sp were classified as a cluster . Agt drastically decreased the medium concentrations of preg, hpreg, dhea, prog, dcortico, cortico, and aldo at 100 m . Ddd dose - dependently decreased the medium concentrations of prog, dcortico, cortico, cort, and aldo at> 10 m and decreased preg, hpreg, dhea, prog, hprog, dione, and dcort at the maximum exposure concentration of 25 m . Sp increased preg, hpreg, and dhea and decreased prog, dione, dcortico, dcort, cortico, aldo, and testo at 10 m . Mp dose - dependently decreased cortico, cort, and aldo and decreased dhea, hprog, dione, and testo at the maximum exposure concentration of 100 m . Kc drastically decreased the medium concentrations of preg, hpreg, dhea, hprog, dione, dcortico, dcort, cortico, corto, aldo, and testo at 10 m . Mc increased the medium concentrations of prog and decreased dione, dcort, cort, and testo at 10 m . Dz increased preg, hpreg, and dhea and decreased dione, dcortico, dcort, cortico, cort, aldo, and testo at 100 m . The mathematical model of adrenal steroidogenesis in nci - h295r cells was optimized for several kinetic parameters of cholesterol transport, intracellular localization, the oxysterol pathway, and maximum velocity of steroidogenic enzymes to fit the experimental time - course data . The optimized mathematical model was reconstructed with the experimental dynamic patterns of cholesterol and the 14 steroid hormones in the intracellular space and culture medium . Optimized kinetic parameters were calculated sensitivities for the nlsd value as the fitting score and are shown in table s1 in a supplementary document . The highly sensitive parameters for fitting the nlsd score were the extracted nine kinetic parameters, kcholesterol transport, kf, kf, kb, vmax, km, vmaxa, vmaxa, and vmaxa, which had higher than 3.0 fitting sensitivity . Effects of adrenal toxic compounds on steroidogenic enzymes were quantitatively predicted from the change in the ratio of the measured medium concentrations of the 12 steroid hormones at 72 h after drug exposure using the mathematical model of adrenal steroidogenesis in nci - h295r cells . The estimated effects of 11 adrenal toxic compounds on eight steroidogenic enzymes are shown in figure 5 . The adrenal toxic compounds without steroidogenic inhibition, such as vasculotoxic agents (an, sm, tg, and fn), were not estimated for the target steroidogenic enzymes under noncytotoxic conditions . Every fitness values were under 0.05 of nlsd values used as the fitting objective function (figures 5(a)5(d)). Other steroidogenic inhibitors (agt, ddd, sp, mp, kc, mc, and dz) are described in detail below . The mechanism of action of agt in adrenal steroidogenesis was estimated by inhibition of cyp11a1, hsd3b2, cyp21a2, and cyp11b1 at 100 m (estimated inhibitions were 77.0%, 78.0%, 81.1%, and 59.8%, resp .) (figure 5(e)). Agt has been reported to inhibit cyp11a1, cyp21a2, cyp11b1, and cyp11b2 [6, 2730]. In particular, cyp11a1 appeared to be inhibited strongly by agt . In our study, hsd3b2 inhibition of agt was shown by mechanistic analysis based on systems biology approaches as a novel mechanism of action of agt . However, the concentration of aldo in the culture medium decreased to 3.8% of the normal stimulated condition . Inhibition of agt by cyp11b2 has been shown using sheep adrenal homogenates as well as a human adrenal homogenate from a patient with cushing's syndrome . The activity of 18-hydroxylase induced by cyp11b2 was determined as the conversion of corticosterone to 18-hydroxycorticosterone in the previous study . The cause of the discrepancy regarding the effect of agt on cyp11b2 was suggested to be substrate inhibition, because the intracellular concentration of cortico was increased by over 10 times of that in the culture medium to reach 50 m . Another possibility was poor quantitative reliability of the experimental data, because the aldo concentration was under the lower limit of quantification at 100 m agt . Hecker and colleagues reported that 3 m agt decreases preg and prog concentrations and increases the testo concentration . One possibility is that the concentration of testo was already enhanced by about 3.3-fold through stimulation with acth, forskolin, and angiotensin ii . The mechanism of action of ddd in adrenal steroidogenesis was estimated by dose - dependent inhibition of cyp11a1, hsd3b2, cyp21a2, and cyp11b1 (estimated inhibitions at 25 m were 87.0%, 86.9%, 76.9%, and 84.9%, resp .) Ddd has been reported to inhibit cyp11a1, hsd3b2, cyp21a2, cyp11b1, and cyp11b2 [29, 31, 32]. Inhibition of ddd by cyp11b2 was not estimated in our study . However, the concentration of aldo in the culture medium decreased to 3% of that in the normal stimulated condition . Inhibition of ddd by cyp11b2 has been shown using mitochondrial and microsomal fractions prepared by standard centrifugation procedures from a bovine adrenal cortex homogenate . The cause of the discrepancy regarding the inhibition of ddd by cyp11b2 could not be explained by same effect in the case of agt . The mechanism of action of sp in adrenal steroidogenesis was estimated by inhibition of hsd3b2, cyp21a2, and hsd17b3 (estimated inhibitions at 10 m were 70.2%, 59.5%, and 59.3%, resp .) Sp has been reported to inhibit cyp17h, cyp17l, cyp11b1, and cyp11b2 [6, 3335]. The inhibitory effect of sp on the hsd3b2 enzyme was a novel mechanism of action . Sp has also been reported to exert some off - target effects by binding to androgen, glucocorticoid, and progesterone receptors [5658]. Sp has been shown to inhibit the production of aldo and cort from preg induced by angiotensin ii in h295r cells, but the specific mr antagonist eplerenone did not show the inhibitory effects . Therefore, hsd3b2 inhibition by sp is not mediated via mr, and the action might be direct inhibition of hsd3b2 enzymes or a part of known off - target effects mediated through other nuclear hormone receptors . Regarding cyp17h and cyp17l, our results were consistent with previous reports [33, 34]. The fact that there were no inhibitions of cyp17h or cyp17l in our study suggests that sp might not be deacetylated to 7-thiospironolactone in nci - h295r cells . It has been shown that 30 m sp inhibits cyp11b1 and cyp11b2 in human and bovine adrenal mitochondria . The cause of cyp11b1 and cyp11b2 inhibition by sp could not be determined in our study, which might be due to the lower maximum exposure concentration of sp than that in the previous report . We could not examine sp concentrations over 10 m because these concentrations were cytotoxic in nci - h295r cells . The mechanism of action of mp in adrenal steroidogenesis was estimated by dose - dependent inhibition of cyp11b1 (estimated inhibitions at 1, 10, and 100 m were 57.1%, 82.7%, and 98.2%, resp .) Mp has been reported to inhibit cyp11b1 as its major effect and cyp11a1 and cyp11b2 as a weak effect [6, 29, 3639]. The results were able to show that mp is a selective inhibitor of cyp11b1 in the previous report . However, the estimated effect of mp at a high concentration, 100 m as the maximum exposure concentration, was unclear . According to the previous report, selectivity of mp for cyp11b1/cyp11b2 is about five times . In addition, 20 m mp has a slight inhibition effect on cyp11a1 in h295r cells . The mechanism of action of kc in adrenal steroidogenesis was estimated by inhibition of cyp11a1, cyp17h, cyp17l, hsd3b2, cyp21a2, cyp11b1, and cyp11b2 (estimated inhibitions at 10 m were 92.6%, 94.3%, 51.8%, 83.0%, 88.2%, 97.4%, and 79.8%, resp .) Kc has been reported to inhibit cyp11a1, cyp17h, cyp17l, hsd3b2, cyp21a2, and cyp11b1 [6, 29, 4043]. Kc inhibits cyp11a1, cyp17h, cyp21a2, and cyp11b1 in nci - h295r cells at 10 m and cyp17h, cyp17l, cyp21a2, and cyp11b1 in human adrenal mitochondria and leydig cell microsomes at 25 m [60, 61]. However, kc has shown only weak inhibition of hsd3b2 and hsd17b3 in leydig cells at the millimolar level [60, 61]. Regarding cyp11b2 and hsd17b3, we considered that these estimated inhibitions of kc did have sufficient reliability in terms of quantitative prediction precision, because aldo and testo concentrations were less than the lower limit of quantification at 10 m kc . The mechanism of action of mc in adrenal steroidogenesis was estimated by inhibition of cyp17h, cyp17l, cyp11b1, and hsd17b3 (estimated inhibitions at 10 m were 69.1%, 53.0%, 76.4%, and 57.1%, resp .) (figure 5(j)). Mc has been reported to inhibit not only cyp17h and cyp17l but also cyp11a1, cyp21a2, and cyp11b1 [41, 44, 45]. The results in the previous reports were able to estimate that mc is a cyp17 inhibitor . However, cyp11a1 inhibition by mc, probably instead of a reduction in star expression, was not clearly detected in our study using nci - h295r cells, because there were no decreases in the concentrations of preg and prog in the culture medium . Indirect inhibition of cyp11a1 via the peripheral - type benzodiazepine receptor has been reported in mouse adrenocortical y-1 cells treated with mc in the absence of stimuli by measuring preg production . On the other hand, reductions of star protein expression and/or transport activity without affecting total steroid synthesis or cyp11a1 and hsd3b2 enzyme expression or activities have been reported in (bu)2camp - stimulated ma-10 leydig tumor cells treated with mc by measuring prog production . Therefore, the effect of mc on the initial reaction in adrenal steroidogenesis from cholesterol should be different according to the cell type and stimulation condition . Inhibition of cyp21a2 and cyp11b1 by mc has been reported as decreases in the consumption of prog and dcortico, respectively . Inhibition of cyp11b1 was estimated by the action of mc in this study, but that of cyp21a2 was not detected . In the previous experimental report, inhibitory sites by mc might have been reflected by inhibition of cyp17h activity, because cyp21a2 activity was measured as a decrease in labeled prog . The mechanism of action of dz in adrenal steroidogenesis was estimated by inhibition of cyp11a1, hsd3b2, cyp21a2, cyp11b1, and hsd17b3 (estimated inhibitions at 100 m were 58.6%, 94.1%, 96.5%, 87.2%, and 98.1%, resp .) (figure 5(k)). These estimated effects of dz on cyp11b1 and hsd17b3 were unclear, because the concentrations of aldo and testo were less than the lower limit of quantification at 100 m . In addition, these enzymes act downstream of the strong action points of dz, such as hsd3b2 and cyp21a2 . To comprehensively understand the dynamics of adrenal steroidogenesis, dynamic sensitivities were calculated for steroid concentrations secreted by nci - h295r cells using our constructed mathematical model of steroidogenesis . The results of dynamic sensitivity analysis at 72 h of duration and 6 h of interval time are presented as a heat - map in figure 6 . The top 10 parameters of the total area under the curve of dynamic sensitivity for cholesterol and the 14 steroids in culture medium were vmaxa, vmax, vmaxa, kf, kb, km, vmaxa, kcholesterol transport, kf, and vmaxb in order from the top . Cholesterol uptake (kcholesterol transport), star protein (kf), and cyp11a1 (vmax), which are determining factors of the capacity for steroidogenesis, promoted the production of mineralocorticoids (dcortico, cortico, and aldo) and restrained the synthesis of glucocorticoids (dcort and cort) and sex steroids (dione, testo, and e1) because of the accumulation of intermediate molecules in steroidogenesis (preg, hpreg, dhea, prog, and hprog) only by self - activation . The dynamic patterns of the intermediate molecules in steroidogenesis were mainly dependent on the activity of cyp17h and hsd3b2 with preg as the substrate of these enzymes, in which the dynamic sensitivities of vmaxa for hpreg, and hprog and vmaxa for prog, hprog, and dcortico reversed the direction of sensitivity at 4966 h after stimulation . The dynamic sensitivities of the maximum activities of hsd3b2 for preg (vmaxa) and cyp21a2 for prog (vmaxa) were related to all steroids at 72 h. almost all model parameters had positive sensitivity for downstream steroids in the adrenal steroidogenic pathway and negative sensitivity for direct - binding steroids as substrates of the steroidogenic enzyme . The sensitivity of vmax in all steroidogenic enzymes was relatively higher than km for the same steroid substrate . To clearly show the property of the metabolic shift between mineralocorticoid and glucocorticoid biosynthesis, we performed two - dimensional parameter scanning of the enzymatic activities of cyp17h and hsd3b2 (figure 7). Nci - h295r cells lost the ability to produce all steroid hormones when enzymatic activities of cyp17h and hsd3b2 were changed by over 60% and 30%, respectively . Activation of cyp17h and/or hsd3b2 induced the metabolic shift that enhanced the glucocorticoid biosynthesis and deviated from the mineralocorticoid biosynthesis . On the other hand, inhibition of cyp17h and/or hsd3b2 induced the metabolic shift that enhanced the mineralocorticoid biosynthesis and deviated from the glucocorticoid biosynthesis . Moreover, the enzymatic activity of hsd3b2 regulated the metabolic balance of sex steroids and the precursors on adrenal steroidogenesis of nci - h295r cells . E1, testo, and dione were produced by nci - h295r cells when activating the enzymatic activity of hsd3b2 . Conversely, e2 and dhea were produced by nci - h295r cells when suppressing the enzymatic activity of hsd3b2 . The biosynthesis of downstream steroids in adrenal steroidogenesis pathway, such as mineralocorticoids and glucocorticoids, was almost completely terminated when the enzymatic activity of hsd3b2 was decreased by over 80% . Our systematic analysis using the mathematical model of adrenal steroidogenesis in nci - h295r cells revealed that the enzymatic activity of 3-hsd controls the dynamics of adrenal steroidogenesis . The activity of the star protein controls the net capacity of steroidogenesis in steroidogenic cells, which is the transport of cholesterol from the outer to inner mitochondrial membranes . Both the expression levels of star protein and mrna are rapidly elevated in response to stimulation by tropic hormones such as acth [62, 63]. Another important factor in adrenal steroidogenesisis is the cholesterol side chain cleavage enzyme cyp11a1, the first rate - limiting and hormonally regulated step in the synthesis of all steroid hormones, which is conversion of cholesterol to pregnenolone in mitochondria . According to our results of global sensitivity analysis (supplementary table 1 and figure 6(d)), in addition to cyp11a1 and star proteins, 3-hsd was one of the key regulators in adrenal steroidogenesis of nci - h295r cells . And also, this result suggests that a significant regulatory mechanism in steroidogenesis pathway is very reasonable . Star, cyp11a1, and 3-hsd (isoforms 1 or 2 in humans) proteins generally respond to the same hormones that stimulate steroid production through common pathways such as camp signaling in adrenal glands and testes [65, 66]. Moreover, our data also support recent experimental evidence from clinical and in vivo studies, suggesting that the enzymatic activity of 3-hsd plays an important role in the regulation of mineralocorticoid synthesis in adrenal steroidogenesis and contributes to hypertension caused by abnormal overproduction of aldosterone [6770]. Circadian clock - deficient cry - null mice show salt - sensitive hypertension due to abnormally high synthesis of aldosterone, which is caused by constitutively high expression of hsd3b6 mrna and protein in the adrenal cortex [67, 68]. Recent clinical observations have revealed predominant expression of hsd3b2 mrna and protein in tumor cells of aldosterone - producing adenoma (apa), and hsd3b1 mrna significantly correlated with cyp11b2 mrna levels and plasma aldosterone concentrations in apa patients [69, 70]. However, the relationship is unclear and disputed in a small - scale clinical study indicating that genetic variation in hsd3b1 affects blood pressure and hypertension in apa patients . The results of our simulation study suggest that 3-hsd protein (human genes are hsd3b1 and hsd3b2) is one of the determination factors for the dynamic property of adrenal steroidogenesis . Our results support the clinical evidence of doi and colleagues, and we believe that the hsd3b1 enzyme has a promising potential as novel drug target for endocrine hypertension . The metabolic properties of adrenal steroidogenesis in nci - h295r cells were revealed by dynamic sensitivity analysis using the mathematical model (figure 6). Mineralocorticoids, such as dcortico, cortico, and aldo, and intermediate steroids upstream of the adrenal steroidogenesis pathway, such as preg, hpreg, dhea, prog, and hprog, were accelerated by reactions of cholesterol import (kcholesterol transport), star protein (kf and kf), and cyp11a1 (vmax). On the other hand, glucocorticoids, such as dcort and cort, and sex hormones, such as dione, testo, and e1, were suppressed by these model parameters . Therefore, enhancement of the net adrenal steroidogenesis capacity, which supplies preg precursor to the pathway, causes a production shift from glucocorticoids to mineralocorticoids by substrate inhibitions of cyp17h, hsd3b2, and cyp21a2 caused by accumulation of initial intermediate steroids such as preg and prog . Sensitivities of cyp17h (vmaxa) and hsd3b2 (vmaxa) for the products were dynamically changed and these parameters determined the metabolic balance of downstream steroids in the adrenal steroidogenesis pathway . According to these results of dynamic sensitivity analysis of star, cyp11a1, cyp17h, and hsd3b2, we suggest that the enhancement of cyp17h and hsd3b2 activity during acth stimulation was important to shift the steroidogenic output away from aldo biosynthesis towards cort biosynthesis, as well as adrenal androgen production . This suggestion partially supports a comparative animal study in which molecular and cellular variations in cyp17h activity dramatically affect acute cortisol production, resulting in distinct physiological and behavioral responses . Results of two - dimensional parameter scanning of the enzymatic activities of cyp17h and hsd3b2 quantitatively showed the detail of the metabolic relationship between mineralocorticoid and glucocorticoid biosynthesis (figure 7). Particularly, the results showed that the balance of these enzymatic activities was very important for the typical function of nci - h295r cells, namely, the ability to produce all steroid hormones . Nci - h295r cells lost this function when enzymatic activities of cyp17h and hsd3b2 were changed by over 60% and 30%, respectively . In addition, they became mineralocorticoid (aldo) secreting cells when the enzymatic activity of cyp17h or hsd3b2 was inhibited by over 50% or glucocorticoid (dcort and cort) secreting cells when these enzymes were activated by over 50% . In particular, this analysis also showed that hsd3b2 was a key player in the adrenal steroidogenesis of nci - h295r cells, because hsd3b2 inhibition by over 80% almost completely inhibited the biosynthesis of downstream steroids . The ratio of cyp17a1 to hsd3b2 mrna expression levels has been related to several endocrine diseases with a low level in apas and high level in cortisol - producing adenomas . Furthermore, the expression levels or enzymatic activities of cyp17a1 and hsd3b1 have been related to androgen production in polycystic ovary syndrome [75, 76]. These clinical studies support our simulation results indicating that the balance of enzymatic activity of cyp17h and hsd3b2 determines the shift in steroidogenic output to mineralocorticoids, glucocorticoids, or androgens . According to our results obtained using the mathematical model of steroidogenesis in nci - h295r cells, such as sensitivity analysis, comprehensive analysis based on systems biology is available to quantitatively estimate the mechanism of action of steroidogenic disrupting compounds from differential profiling of adrenal steroid hormones, because dynamic patterns of steroid hormones in adrenal steroidogenesis pathway are highly complex . Our proposed method of quantitative mechanistic analysis of steroidogenic inhibitors was able to predict known action sites in the adrenal steroidogenesis pathway at only one time point (72 h after drug exposure). Moreover, according to the results of sensitivity analysis (figure 6), vmax of all steroidogenic enzymes was more sensitive than the km, because the intracellular concentrations of steroid hormones were almost maintained at sufficiently high levels compared with km values of steroidogenic enzymes . These results suggested that estimation of the mechanism of action of drugs is more effective and detectable when using the influences of vmax as the searching parameters such as our proposed method . Our data showed that the proposed method based on a systems biology model is a very powerful tool for exploratory screening of steroidogenic disrupting compounds . Rcga as a solver of parameter estimation problems in systems biology has been applied to biological network identification of gene regulatory networks and metabolic pathways and optimization of biological processes using experimentally observed time - course data [7782]. In this study, rcga was useful to estimate the mechanism of action of novel pharmaceutical drug candidates for adrenal steroidogenesis as a new application of rcga in systems biology . We had two issues when applying rcga to the quantitative mechanistic analysis of drug actions . These issues were the vast calculation cost and multimodality of quasi - optimum solutions in solving the optimization problem, because the mathematical model in systems biology consists of many equations and parameters . A proposed optimization strategy using rcga based on rex / jgg was a highly stable and efficient calculation method for a better quasi - optimum solution than the unimodal normal distribution crossover (undx)/minimum generation gap (mgg) method that is well applied in the engineering field . In addition, we expanded the rcga optimization program based on rex / jgg to a hybrid method of ga and then applied a local search as recommended by harada and kobayashi [28, 83]. A final optimal solution was obtained with a good convergence property . Because these problems are general in systems biology studies, we suggest that the proposed hybrid method based on rex / jgg is a very useful tool for quantitative mechanistic analysis of novel pharmaceutical drugs, not limited to steroidogenic disrupting compounds . The novel mathematical model of adrenal steroidogenesis was constructed in this study, including cholesterol transport and distribution, the c21-steroid hormone pathway, steroid transport, and cell proliferation, which could reproduce adrenal steroidogenesis in nci - h295r cells . According to the results of dynamic sensitivity analysis using the new model, hsd3b2 plays the most important role in the metabolic balance of adrenal steroidogenesis in nci - h295r cells . Moreover, to quantitatively estimate mechanisms of action of adrenal toxic compounds, we analyzed differential metabolic profiles of 12 steroid hormones at 3 days after exposure to 11 adrenal toxic compounds, by using the new mathematical model and a hybrid optimization method of the rcga and a local search (nonlinear least squares). We could estimate which steroidogenic enzymes were affected by these compounds using the hybrid optimization method . Vasculotoxic agents were estimated to have no effect according to the results obtained by our method . In terms of adrenal steroidogenic inhibitors, the predicted action sites were approximately matched to the target enzymes as reported in the literature . Thus, our computer - aided method based on a systems biology approach may be useful to analyze the mechanism of action of endocrine - disrupting compounds and to assess the human adrenal toxicity of novel pharmaceutical drugs based on steroid hormone profiling.
Chronic low - grade inflammation (inflammaging) in the elder population is considered a risk factor for the development of aging - related diseases and frailty . Geriatric frailty is associated with increased inflammatory activity as increased levels of several biomarkers like tnf - a, interleukin-6 (il-6), cytokine antagonists and acute phase proteins are identified in the serum of frail older individuals . C - reactive protein (crp) is playing a key role in several disease processes, and elevated serum crp levels have been identified to accompany increased vulnerability for disease and mortality in older patients . The aim of this review was to search for the relation between the commonly used inflammatory marker crp and the risk of incidence of physical frailty in older individuals as extracted from the published literature . We conducted a pubmed search using the combinations of the terms crp, c - reactive protein and frailty, frail, elders or elderly . We also reviewed the bibliographies of all extracted manuscripts attempting to identify additional relevant publications . The database search, as well as the review of the references of the relevant publications, resulted in a total of 29 studies conducted between 2004 and 2016 which discussed and investigated the relationship between crp and frailty in elderly individuals . The studies are presented along with their main findings in table 1 [1 - 29]. Frailty is defined as the syndrome characterized by a reduced ability of individuals to re - establish homeostasis in response to stress . The phenotype of physical frailty consists of weight loss, slowness, weakness, exhaustion, and a low physical activity level . The causes of frailty seem to be complex, as genetic, biological, physical, social and environmental factors are involved in its pathogenesis . The mechanisms of the syndrome are multifactorial, as inadequate nutrition, endocrine, and immune system dysfunctions are involved in its development . Additionally, disadvantaged socioeconomic conditions and low cultural levels seem to be significantly related to this clinical entity [30, 31]. The clinical syndrome of frailty in the elderly consists of several pathologies and is characterized by low physical activity, weakness, exhaustion, and a global impairment of physiological reserves of several organ systems . The incidence of geriatric frailty is around 20 - 30% of the population over 75 years and increases with advancing age . Age and chronic disease - related activation of inflammation, as also neuroendocrine dysregulation and metabolic changes lead to physiological and clinical frailty . The fact that frailty in the elderly is related to long - term adverse health - related outcomes like disability, dependency, hospitalization and mortality is of great importance . The chronic activation of the inflammatory response, called inflammaging, is considered a key component for the development of frailty in older individuals . Low - grade chronic inflammation and oxidative stress, mediated partly from the superoxide anion overproduction by nadph oxidase, consists part of the underlying pathology . Several biomarkers, like erythrocyte sedimentation rate, crp, white blood cell and lymphocyte counts, iron, albumin, cholesterol and other, are associated with a higher severity grade of the multidimensional prognostic index (mpi) and mortality . Many studies show that the clinical phenotype of frailty is associated with pathologic levels of laboratory markers suggesting as possible pathogenetic mechanisms hormonal dysregulation, immuno - aging, pro - coagulation and pro - inflammatory abnormalities . A useful approach to assess frailty in clinical settings is the use of biomarkers, thus making feasible and accurate the assessment of frailty by clinicians . Standard clinical tests and observations associated with inflammation are hypoalbuminemia, erythropoietin resistance, decreased iron saturation accompanied by high ferritin, physical frailty, low serum creatinine, reduced total and ldl - cholesterol, and increased crp . Frailty in the elderly is correlated with higher serum levels of inflammatory biomarkers like il-6, crp and tnf - a, which are inversely correlated with poor physical activity, muscle weakness and increased disability [1 - 3, 7 - 9, 17, 21 - 23, 25, 26, 29, 34 - 37]. Elevated crp levels are also associated with increased risk of mortality in frail older subjects [2, 5, 19], but they are not always a significant mortality predictor [15, 27]. Wassel et al in a prospective study with 1,353 participants in 2010 concluded that higher serum crp levels predicted a reduced survival time only among males . Serum crp levels were also associated with incident frailty in more specific subgroups, such as poor functional status and more advanced clinical stage in older patients with cancer and impaired physical performance in obese older adults . However, reiner et al in a 2009 prospective study concluded that there is little evidence associating crp levels and frailty among postmenopausal women . Among the studies reviewed, two of them showed significant correlations between incident frailty, cognitive functions, and serum crp concentrations only among women over 60 years old but not among men [14, 18]. Studies investigating specific crp gene polymorphisms and possible associations with frailty among older individuals were also identified . Statistically significant correlations were found between hand grip, as a frailty index, and three out of the five single nucleotide polymorphisms (snps) in the crp gene as well as the haplotype c - c - c - c - c, studied by lin et al in 2014 in a study involving 472 elderly subjects . Another crp polymorphism found to be linked with increased odds of frailty was the crp1846g> a . Two other common crp gene polymorphisms, rs1205 and rs3093059, were found to be significantly associated with serum crp levels but not with frailty in an elderly chinese population . In many specific populations, like patients with heart failure, studies show that there are specific relations between inflammatory markers and physical function . A higher frailty phenotype score was correlated with lower 25-hydroxy vitamin d and higher high sensitivity crp serum levels in patients with cardiac failure . The acute phase reactant crp increases with age and increased plasma levels of it have been identified as a biological component of frailty, defining elevated vulnerability for diseases and mortality with aging . Inflammatory markers seem to play a major role and are closely related to frailty as this is confirmed in the very old (85 +) by the newcastle 85 + study, previously established in younger old populations . Literature review shows that even mild increases in crp plasma levels are associated with an increased risk of sarcopenia, cardiovascular diseases, disability and cognitive decline in individuals over 65 years old . Also, in a study by yano et al, it was shown that after assessment of plasma pentraxin-3 and high sensitive crp for the early detection of cognitive decline in the elderlies, both parameters were significantly associated with the cognitive function calculated by the mini - mental state examination score . Among the studies presented and summarized in table 1, the cardiovascular health study (chs) by walston et al showed a significant relation of elevated crp levels with frailty after excluding cardiovascular disease and diabetes and adjusting for basic demographic characteristics, and the published data from the longitudinal aging study amsterdam (lasa) [28, 42]. Although a large number of studies show an association between serum crp levels and the presence of frailty in older patients, few others do not support these findings, concluding that no significant associations are found [12, 16]. The assessment of inflammatory markers in the setting of frailty in elderlies may represent a useful screening test and a potential target for further intervention . Of course, having a high concentration of more than one inflammatory marker may be more strongly predictive of incident frailty than a high concentration of only one . Measurement of serum crp levels is a widely accepted screening test that handles in daily clinical practice . Clinicians are initially using it as a tool to diagnose infections or clinical conditions closely associated with underlying inflammatory mechanisms . As the aging immune system is characterized by a low - grade, chronic systemic inflammatory state, markedly elevated inflammatory molecules, such as crp could be evaluated in an emergency or long - term basis as part of the assessment in the specific elderly population . Crp may play the role of a useful biomarker for the detection of frailty in elderly individuals as most of the published studies until now are showing a close relation with this clinical entity . Clinicians assessing older patients should always consider in their differential diagnosis that elevated levels of serum crp could be part of an underlying chronic inflammatory process related to the syndrome of frailty in the elderly . A large number of studies, referring to different ethnic populations are mentioning the correlation of increased serum crp levels with increased incidence of frailty in elder individuals . Studies presenting sex - specific differences between frailty and serum crp levels were also identified [14, 18, 24]. For extremely old populations like the centenarians, there is still a lack of large studies assessing further the role of crp in frailty . Also, the fact that there are few studies not replicating these findings, as well as studies implicating other biomarkers, inflammatory or not, with the frailty syndrome underlies the need for a better understanding of its pathophysiology . To understand better these pathophysiological pathways leading to the onset of frailty and disability in the elderly, finally, a question should always be raised in clinicians whether elevated serum crp levels should be taken into account as an index of acute inflammation or as part of the inflammaging (chronic inflammation) process . Frailty in the elderly appears in close association with chronic inflammation, and most of the published studies show a correlation between crp and this clinical entity in older individuals . Further research is needed to investigate the frailty - related pathologies as frailty seems to have substantial health and economic implications, and the screening of frailty using inflammatory markers should be a future debate . As crp has already been integrated part of the routinely measured biochemical panel for the investigation of many clinical entities, a potential role of such biomarkers and whether they can serve as indexes of vulnerability to age - related diseases is warranted.
Osteopoikilosis, spotted bone disease or osteopathia condensans is a rare asymptomatic bone dysplasia of unknown etiology.1 it is characterized by an abnormality in bone maturation process and often found incidentally on radiologic examination . Albers - schonberg was the first to describe this uncommon sclerosing bone dysplasia in 1915.2 incidence in both sexes is identical and it can occur at any age . Overall incidence of opk has been claimed to be one in every 50,000 subjects.3 radiologic signs of opk are homogeneous, small, well - defined symmetric round and ovoid radio opacities lesions . The commonly involved sites are metaphyses and epiphyses of long bones, scapulae, pelvis, carpi and tarsi.4 pain is not a prominent feature of opk, but in some patients, pain could be a presenting symptom of the disorder . A 46 year old female patient was referred to our pain clinic, complaining of pelvic pain starting 8 years ago . Her pain was constant, but worsened when climbing stairs and walking for long distances . The quality of pain was vague and its severity was rated as 56 on the visual analogue scale . She did not report any history of past or recent trauma or systemic disease and was not taking any medications . Inspection of pelvis showed bilateral symmetry of muscle bulk and there were no scares or bruising . Palpatory examination revealed slight tenderness over the great trochanters and symphysis pubis, however no trigger points were located . On physical exam, neurologic examination revealed no abnormality and provocative tests such as thomas test, patrick s test, femoral stretch test and straight leg raising were normal . All findings for complete blood count, erythrocyte sedimentation rate, c reactive protein, rheumatoid factor, serum electrolytes, alkaline phosphatase, calcium, phosphate and thyroid function tests were normal . Pelvic x - ray showed multiple oval shaped radiodensities of various degrees on the upper heads of femur, acetabulum and symphysis pubis (figs . 1, 2): osteopoikilotic lesions has also found in the foot x ray of the patient . In order to rule out metastases, abdomino pelvic ultrasonography was also normal . In light of these characteristic lesions and findings without cancer red flags, a diagnosis of osteopoikilosis was made . She was found to be pain free and asymptomatic in a follow up visit after the first month . Two months later, she returned to our clinic along with her 11-year - old son and sixteen year old daughter . Plain radiographs revealed the same characteristic ovoid sclerotic lesions of opk in distal end of tibia, fibula (fig 3, 4) and foot . Further work up showed no other abnormalities except for an exostosis in medial aspect of tibia in her son s x ray . This current report presents a 46 year old female patient and her son and daughter diagnosed with opk . An epidemiologic study on 53 patients affected with opk indicated that the frequent sites for opk, were phalanges (100%), carpal bones (97.4%), metacarpals (92.5%), foot phalanges (87.2%), metatarsals (84.4%), tarsal bones (84.6%), pelvis (74.4%), femur (74.4%), radius (66.7%), ulna (66.7%), sacrum (58.9%), humerus (28.2%), tibia (20.5%), and fibula (2.8%).5 in our cases, we found multiple oval shaped radiodensities of various degrees on the upper heads of femur, acetabulum and symphysis pubis . Characteristic radiologic signs of opk consist of numerous 210 mm ovals or round shaped densities, symmetrically distributed within epiphyses and metaphyses of long bones . These lesions appear as dense radiopaque spots.6 in the current study, pelvic x - ray showed multiple oval shaped radiodensities of various degrees on the upper heads of femur, acetabulum and symphysis pubis . Pain is not a dominant feature of opk but in 15%20% of patients, slight joint pain and effusion have been reported.4 the main complaint in our patients, which caused them to seek medical help, was persistent pain in affected areas . It is presumed that joint pain is an incidental finding in the course of opk . Increased localized bone metabolism at the location of the lesion, irritation of joint capsule attachment by sclerotic areas and increased intraosseous pressure due to venous stasis at the areas of lesion could produce joint pain.7 in nearly 25% of patients, opk is associated with buschke ollendrof syndrom.8 however, we could not find any dermatologic conditions as those in buschke therefore, the major differential diagnosis should be considered for osteoblastic metastases, mastocytosis and tuberoussclerosis.9 asymmetry, variation in size, axial skeleton involvement, osseous destruction and periostal reaction differentiates osteoblastic metastases from opk . In addition, scintigraphy can help distinguish opk from osteoblastic bone metastases but abnormal bone scan does not exclude opk.10 in our case, there were no hot spots indicating increased uptake of radiotracer in bone scan . Because of the familial nature of the disorder, when the patient came back with two offspring (her son and daughter), they were also evaluated and although the risk of malignant transformation with opk is very rare, some complications such as osteosarcoma,11 giant cell tumors12 and chondrosarcoma13 have been reported in the literature . Therefore, we regularly followed up with the patient and other affected members of her family . In conclusion, opk is an asymptomatic natural variation with benign nature but in patients with characteristic radiologic findings, the patients may be presented with pain . Thus, well - timed follow - up visits of the patients are recommended to survey other conditions which may require treatment.
Adverse drug reactions such as dermatologic events and hypersensitivity are common health conditions, which account for 3%6% of all hospital admissions and occur in 10%15% of hospitalized patients.1 adverse drug reactions are side effects of many medical treatments for chronic diseases . Postmarketing monitoring of drug safety using health and administrative registries and/or databases is essential.2 therefore, assessing the validity of diagnoses for adverse events in the registries is important to ensure high quality of data used for pharmacovigilance studies . National health registries in nordic countries have long been recognized as well suited for pharmacoepidemiologic research3 and have increasingly relied on study drug safety, especially in relation to treatments of chronic conditions for which drugs are dispensed through outpatient pharmacies . However, the validity of diagnoses of common drug reactions such as dermatologic events and hypersensitivity found in the nordic patient registries is not known.4,5 in this study, we estimated positive predictive values (ppvs) of case ascertainment algorithms for dermatologic events and hypersensitivity in the national hospital - based patient registries of denmark and sweden among women with postmenopausal osteoporosis (pmo), a disease where several new drugs have been introduced in the past 2 decades . We conducted a population - based validation study in denmark and sweden between january 1, 2005, and december 31, 2014, in a setting of universal health care access and routine recording of health events.6 the source population consisted of postmenopausal women (age 55 years or older). The danish source population was restricted to the central denmark region (size of the postmenopausal women population on january 1, 2011, was 189,319).7 aarhus university hospital is the largest hospital serving the area . In sweden, the source population comprised postmenopausal women residing in areas served by the 5 largest stockholm - area hospitals, karolinska university hospital, solna and huddinge, danderyd hospital, the south hospital, and st gran hospital . The size of the postmenopausal women population residing in the stockholm county on december 31, 2010, was 290,179.8 the study population included potential cases of dermatologic events and hypersensitivity among women with pmo seen in the hospitals in the selected geographic areas of denmark and sweden . The definition of pmo was based on an algorithm that included diagnostic codes indicating osteoporosis diagnosis, diagnosis of osteoporotic (fragility) fracture, or use of osteoporosis medications, among women 55 years of age or older . This cohort was initially assembled for an ongoing pharmacovigilance study, which has been described in detail elsewhere.9 to be included in the cohort, a woman had to meet at least 1 component of the osteoporosis algorithm on or after her 55th birthday (table s1). The index date was the date on which a woman first satisfied the inclusion criteria . Women with a diagnosis of paget s disease or a diagnosis of any malignancy (except nonmelanoma skin cancer) during the previous 12 months before the index date were excluded (table s2). Potential cases were identified from the danish national patient registry10 and the swedish patient register11 using the international classification of diseases, 10th revision (icd-10) diagnostic codes . Potential cases were defined as icd-10 codes indicative of hypersensitivity or a dermatologic event recorded as the primary diagnosis during a hospitalization or an emergency room / unplanned visit . The icd-10 codes used to identify the potential cases are listed in tables s3 and s4 . A priori, we planned to sample 100 potential cases of dermatologic events (50 for denmark and 50 for sweden) and 100 potential cases of hypersensitivity (50 for denmark and 50 for sweden). In denmark, we included all patients with dermatologic events (n=42) and hypersensitivity (n=32) from the period 20052012 and from aarhus university hospital sited in the central region of denmark . In addition, we randomly sampled 8 and 18 patients, respectively, from other hospitals in the central region in order to achieve a sample pool of 50 patients for each of the events . However, there were limitations on access to the medical records necessary for the review, and hence we decided to increase the sample pools to 60 by samples of patients treated at aarhus university hospital in 2013 and 2014 . The number of selected potential cases remained 50 each for dermatologic events and hypersensitivity in sweden . The sampled potential cases were confirmed through review of medical records (paper or electronic). We attempted to obtain all available medical records, which were reviewed and adjudicated by physicians . The adjudicating physicians confirmed the case status in 3 categories, according to predefined clinical criteria: 1) definite case, 2) definite noncase, or 3) insufficient information . In addition, we verified that each event (dermatologic reaction and hypersensitivity) was the primary diagnosis that led to a hospitalization or an emergency / unplanned visit . If neither definite case nor definite noncase status could be assigned by adjudicators, the case was categorized as having insufficient information and excluded from the analysis . From the available registry data, we compiled descriptive data on patients age, country, case year, department, and type of hospital visit (planned vs unplanned [emergency]). Unplanned hospital visits served as a proxy for emergency room visits as specific codes for the latter are lacking in the swedish patient registers . The ppvs were calculated as the proportion of potential cases that could be classified as definite cases by medical chart review . Because bullous or erythematous conditions as well as anaphylactic hypersensitivity are more severe conditions, which are frequently of interest in pharmacoepidemiology studies on drug safety, we conducted additional analyses on these specific subtypes . All ppvs were reported with 95% confidence intervals (cis) calculated according to the wilson score interval method.12 country - specific stratified analyses were conducted separately for dermatologic events and hypersensitivity leading to hospitalization or emergency room visit . Patient sampling and statistical analyses were performed using sas version 9.2, sas institute inc ., cary, nc, usa . In denmark, the study was approved by the danish data protection agency (record number 2010 - 41 - 5171) and by the data protection board of the danish central region (record number 1 - 16 - 02 - 1 - 08). In sweden, the stockholm county regional ethics review board approved the study (record number 2010/1617 - 31/3). According to danish and swedish law, informed consent from patients is not required for registry - based studies . We conducted a population - based validation study in denmark and sweden between january 1, 2005, and december 31, 2014, in a setting of universal health care access and routine recording of health events.6 the source population consisted of postmenopausal women (age 55 years or older). The danish source population was restricted to the central denmark region (size of the postmenopausal women population on january 1, 2011, was 189,319).7 aarhus university hospital is the largest hospital serving the area . In sweden, the source population comprised postmenopausal women residing in areas served by the 5 largest stockholm - area hospitals, karolinska university hospital, solna and huddinge, danderyd hospital, the south hospital, and st gran hospital . The size of the postmenopausal women population residing in the stockholm county on december 31, 2010, was 290,179.8 the study population included potential cases of dermatologic events and hypersensitivity among women with pmo seen in the hospitals in the selected geographic areas of denmark and sweden . The definition of pmo was based on an algorithm that included diagnostic codes indicating osteoporosis diagnosis, diagnosis of osteoporotic (fragility) fracture, or use of osteoporosis medications, among women 55 years of age or older . This cohort was initially assembled for an ongoing pharmacovigilance study, which has been described in detail elsewhere.9 to be included in the cohort, a woman had to meet at least 1 component of the osteoporosis algorithm on or after her 55th birthday (table s1). The index date was the date on which a woman first satisfied the inclusion criteria . Women with a diagnosis of paget s disease or a diagnosis of any malignancy (except nonmelanoma skin cancer) during the previous 12 months before the index date were excluded (table s2). Potential cases were identified from the danish national patient registry10 and the swedish patient register11 using the international classification of diseases, 10th revision (icd-10) diagnostic codes . Potential cases were defined as icd-10 codes indicative of hypersensitivity or a dermatologic event recorded as the primary diagnosis during a hospitalization or an emergency room / unplanned visit . The icd-10 codes used to identify the potential cases are listed in tables s3 and s4 . A priori, we planned to sample 100 potential cases of dermatologic events (50 for denmark and 50 for sweden) and 100 potential cases of hypersensitivity (50 for denmark and 50 for sweden). In denmark, we included all patients with dermatologic events (n=42) and hypersensitivity (n=32) from the period 20052012 and from aarhus university hospital sited in the central region of denmark . In addition, we randomly sampled 8 and 18 patients, respectively, from other hospitals in the central region in order to achieve a sample pool of 50 patients for each of the events . However, there were limitations on access to the medical records necessary for the review, and hence we decided to increase the sample pools to 60 by samples of patients treated at aarhus university hospital in 2013 and 2014 . The number of selected potential cases remained 50 each for dermatologic events and hypersensitivity in sweden . The sampled potential cases were confirmed through review of medical records (paper or electronic). We attempted to obtain all available medical records, which were reviewed and adjudicated by physicians . The adjudicating physicians confirmed the case status in 3 categories, according to predefined clinical criteria: 1) definite case, 2) definite noncase, or 3) insufficient information . In addition, we verified that each event (dermatologic reaction and hypersensitivity) was the primary diagnosis that led to a hospitalization or an emergency / unplanned visit . If neither definite case nor definite noncase status could be assigned by adjudicators, the case was categorized as having insufficient information and excluded from the analysis . From the available registry data, we compiled descriptive data on patients age, country, case year, department, and type of hospital visit (planned vs unplanned [emergency]). Unplanned hospital visits served as a proxy for emergency room visits as specific codes for the latter are lacking in the swedish patient registers . The ppvs were calculated as the proportion of potential cases that could be classified as definite cases by medical chart review . Because bullous or erythematous conditions as well as anaphylactic hypersensitivity are more severe conditions, which are frequently of interest in pharmacoepidemiology studies on drug safety, we conducted additional analyses on these specific subtypes . All ppvs were reported with 95% confidence intervals (cis) calculated according to the wilson score interval method.12 country - specific stratified analyses were conducted separately for dermatologic events and hypersensitivity leading to hospitalization or emergency room visit . Patient sampling and statistical analyses were performed using sas version 9.2, sas institute inc ., cary, nc, usa . In denmark, the study was approved by the danish data protection agency (record number 2010 - 41 - 5171) and by the data protection board of the danish central region (record number 1 - 16 - 02 - 1 - 08). In sweden, the stockholm county regional ethics review board approved the study (record number 2010/1617 - 31/3). According to danish and swedish law, informed consent from patients is not required for registry - based studies . Overall, 221 potential cases were identified; of these, 32 patients had missing information in the medical record or the medical record file could not be found (figure 1). Medical records were available for 189 patients (86%), including 100 potential cases of dermatologic events (mean age 77 years) and 89 potential cases for hypersensitivity (mean age 76 years; tables 1 and 2). Of these, 8 potential cases (8%) with dermatologic events and 6 potential cases with hypersensitivity (7%) had insufficient information for medical adjudication and were not included in the ppv calculations (table 3). Information verifying a dermatologic event leading to hospitalization or emergency room visit was found in 85 of 92 medical records, yielding a ppv of 92.4% (95% ci, 85.1%96.3%; table 3). The ppv was substantially lower for bullous dermatoses (52.5% [95% ci 37.5%67.1]) as well as for erythematous pathological subtype (12.5% [95% ci 2.2%47.1%]; table 3). Hypersensitivity leading to hospitalization or emergency room visits was confirmed in 49 of 83 medical records, yielding a ppv of 59.0% (95% ci: 48.3%69.0%). The ppv was 100.0% (95% ci, 67.6%100%) for hypersensitivity when restricting ppv calculation to codes for anaphylaxis . The overall ppvs of dermatologic events and hypersensitivity were comparable in denmark and sweden (table 4). Among women with pmo in denmark and sweden, we adjudicated hospital - based primary diagnoses of dermatologic events and hypersensitivity leading to hospitalization or emergency room visit coded in national patient registries . The higher ppv for dermatologic events than for hypersensitivity may reflect differences in the complexity of the disease definition and that 50% of the potential dermatologic event cases originated from specialist departments of dermatology, whereas> 90% of the potential hypersensitivity cases originated from departments of internal medicine and emergency rooms . The term hypersensitivity was often found to be used for normal physiological reactions of overdoses, for example, a high international normalized ratio (inr) as a result of too much warfarin . Most likely this mirrors the lack of time to find a more correct code . To the best of our knowledge, our study is the first to estimate the ppv of icd-10 codes for identifying patients with dermatologic events and hypersensitivity in the danish and swedish national patient registries . Only 2 studies have assessed the validity of icd-10 codes for skin diseases in the danish national patient registry with medical records as reference . A study of 38 cancer patients estimated a ppv almost as high as ours, 79% (95% ci, 64%90%) for skin infections when using an abstraction form and physician assessment in the same manner as done in our study, but the ppv was lower when confirmed by evidence - based criteria (45%, 95% ci, 30%60%), requiring, for example, at least 7 days of antibiotics.5 another study of 589 stroke patients validated several medical complications, among them the diagnosis of decubitus using a standardized abstraction form, but without physician assessment . With only 8 cases in the danish national patient registry, where 4 were verified, the ppv was found to be 50% (95% ci, 16%84%) for decubitus.4 skin disease codes in the swedish national patient registry have only been evaluated in 1 previous study.13 among children younger than 17 years, who were prescribed glucocorticoid or immunosuppressant medication recorded in the swedish prescribed drug register, the ppvs were 82% for unspecific dermatitis (n=199) and 45% for the eczema (n=108), with medical record review conducted by physicians as reference standard.13 in 2012, the american food and drug administration (fda) conducted a systematic review of us studies validating algorithms for anaphylaxis and related conditions . They concluded that more research is needed; more validation studies to test anaphylaxis algorithms need to be conducted . They found only 6 studies, with varying ppvs (38%72%) depending on cohorts and icd codes used.14 taken together, the literature in the area is limited and methods to validate differ . The danish and swedish national patient registries offer a variety of possibilities for pharmacoepidemiologic studies.10 based on our findings, the national patient registries can be used to study cohorts of patients with dermatologic events as well as postmarketing monitoring of dermatologic events and hypersensitivity of treatments . However, the low ppv of our algorithm for identifying hypersensitivity leading to hospitalization or emergency room visits is not adequate for monitoring in pharmacovigilance studies if absolute risk is the outcome of interest . In comparative safety studies, however, relative risks should be unbiased if specificity of the diagnosis is high and misclassification is nondifferential across exposure categories.15 this study was conducted among women with pmo in selected areas and hospitals of denmark and sweden . Scandinavian countries are welfare states with universal income - independent access to health care and uniform health care delivery . Hence, results of this validation study are likely generalizable to the overall postmenopausal female population in each country, although variation across geography and hospital size cannot be ruled out . By design, this study assessed ppvs and could not assess sensitivity because of a lack of an independent sample of confirmed cases . Our study showed that primary diagnoses of dermatologic events recorded at hospitalization or emergency room / unplanned visit have high ppvs in the danish and swedish national patient registries . In contrast, the ppv for hypersensitivity leading to hospitalization or emergency room visit was substantially lower . Thus, depending on study aims, the danish and swedish national patient registries may be useful for studying dermatologic events, but may not be adequate for studying hypersensitivity events.
Primary plasma cell leukemia (ppcl) is a very aggressive form of plasma cell disorder . Ppcl is very rare, reported in only 4 - 6% of patients with multiple myeloma (mm). Ppcl is defined as the presence of more than 20% plasma cells in the peripheral blood and an absolute plasma cell count greater than 2 10/l, without previous evidence of mm . Consistent with the high tumor burden, extramedullary involvement, such as hepatosplenomegaly, lymphadenopathy, soft - tissue plasmacytomas, or leptomeningeal infiltration, is more frequent in ppcl than in mm . Despite the development of new agents, such as proteasome inhibitors or immunomodulatory drugs (imids), for plasma cell disorders, the median overall survival of ppcl although ppcl is difficult to treat, high - dose melphalan followed by autologous stem cell transplantation (auto - sct) and/or allogeneic stem cell transplantation (allo - sct) seems to improve outcomes in younger selected patients . However, patients with ppcl who had central nervous system (cns) relapse after auto- and/or allo - sct were refractory to various treatments, and had an extremely poor prognosis . We herein present a ppcl patient with cns relapse after allo - sct who underwent concurrent intrathecal therapy (it) and radiotherapy (rt) followed by maintenance therapy with pomalidomide and low - dose dexamethasone (pd). A 46-year - old man without a previous significant medical history was referred to our hospital with complaints of anorexia and generalized fatigue . Laboratory findings revealed significant abnormalities, including a white blood cell (wbc) count of 19.710/l with 45% plasma cells, a hemoglobin level of 11.6 g / dl, a platelet count of 7.010/l, a creatinine level of 7.63 mg / dl, and a ldh level of 301 iu / l (table 1). On a peripheral blood smear, the plasma cells were medium - sized, with round nuclei, basophilic cytoplasm, and occasional cytoplasmic vacuoles (figure 1a). G / l, and the serum free light chain (flc) k/ ratio was 161.11 (table 1). In this case, a m - component of the igg - k type was identified by serum protein electrophoresis . Moreover, flow cytometry on the peripheral blood showed that these plasma cells were positive for cd38, cd49e, cd54, cd138 and cytoplasmic kappa light chain . T(11;14)(q13;q32) in 9 metaphase cells (figure 1b) and 46,xy in 11 metaphase cells, and a fluorescent in situ hybridization analysis revealed ccnd1/igh gene fusion rearrangement . The patient was admitted to an intensive care unit (icu) for continuous hemodiafiltration (chdf). On the same day, we started induction therapy with lenalidomide (15 mg / day orally on days 1 - 14), bortezomib (1.3 mg / m subcutaneously on days 1, 4, 8, and 11), and low - dose dexamethasone (20 mg / day orally on days 1, 2, 4, 5, 8, 9, 11, and 12), also known as rvd induction therapy, each for a 21-day cycle . Eight days after starting the induction therapy, the circulating plasma cells in the peripheral blood disappeared, and he was transferred from the icu after withdrawal of chdf . After a total of 3 cycles of chemotherapy, the laboratory abnormalities markedly subsided, consistent with a very good partial response (vgpr), because serum immunofixation was positive . He and his family consented to undergo allo - sct . At our outpatient department, because of grade 2 peripheral neuropathy, he continued induction therapy with lenalidomide (25 mg / day orally on days 1 - 14), bortezomib (1.3 mg / m subcutaneously on days 1 and 8), and dexamethasone (20 mg / day orally on days 1 and 8) in each 21-day cycle . After a total of 6 courses, allo - sct was performed with the bone marrow from an unrelated donor (hla - a one allele mismatch). The conditioning regimen consisted of fludarabine 120 mg / m (30mg / m on day -5, -4, -3, and -2), melphalan 180 mg / m (90mg / m on day-4 and -3), and rabbit anti - thymocyte globulin 2.5 mg / m (1.25mg / m on day -2 and -1). For the prevention of graft - versus - host - disease (gvhd), tacrolimus was started from day -1, and methotrexate was given on days 1, 3, and 6 . Complete donor chimerism was detected in the bone marrow on day 29 . During this admission, however, serum immunofixation was positive despite normal igg and flc levels; therefore, he was still considered to have a vgpr . Two months after discharge, the patient developed cervical, mediastinal, and axillary lymphadenopathy . He was therefore diagnosed with ebv - associated lymphoproliferative disease and was immediately treated with 2 cycles of rituximab monotherapy, resulting in a complete response . Tacrolimus was discontinued on day 180, and there was no evidence of chronic gvhd . However, 6 months after allo - sct, he had marked cytomegalovirus (cmv) antigenemia . The protein level in the cerebrospinal fluid was 53 mg / dl, and the glucose level was 55 mg / dl; 17 monocytes per microliter were detected . Cytological examination revealed that some of these cells resembled plasma cells (figure 2a). Magnetic resonance imaging (mri) with gadolinium (gd) enhancement revealed a small, enhanced nodule in the lateral medulla oblongata (figure 2b, arrowhead). Enhanced computed tomography (ct) showed no extramedullary tumors . Moreover, we still observed complete donor chimerism, without leukemic plasma cells, in his bone marrow . The patient underwent 4 courses of weekly it, consisting of methotrexate 15 mg, cytarabine 20 mg, and prednisolone 40 mg in combination with local cranial rt (5 fractions of 2 gy for a total dose of 10 gy). After this combined treatment, his neurological symptoms resolved and the plasma cells in the cerebrospinal fluid disappeared . To prevent cns recurrence, we administered pomalidomide 4 mg / day on days 121, with 20 mg of dexamethasone weekly for each 28-day cycle . Subsequently, a reduced dose of 2 mg was administered for 2 cycles, but grade 4 neutropenia occurred again . He resumed pomalidomide at 1 mg and continued the low dose without severe hematological abnormalities . Twelve months after the cns relapse, both enhanced ct and brain mri with gd enhancement revealed no extramedullary tumors . In our outpatient follow - up, he maintained vgpr without major complications for more than 18 months after the diagnosis of cns relapse . The prognosis of plasma cell disorder has improved following the introduction of new agents and the development of sct, but a cure remains elusive . The long - term survival rate of ppcl is low due to the aggressive nature of the disease that comprises high tumor burden and extramedullary involvement . In the us, upfront allo - sct is still one of the treatment options for young ppcl patients without major complications . In our study, we performed upfront allo - sct with a myeloablative - conditioning regimen for our young patients without major complications . The center for international blood and marrow transplant research (cibmtr) conducted a retrospective sct study in ppcl, and demonstrated high survival rates in an autologous group compared with those in an allogeneic group . In the cibmtr study, non - relapse mortality in the allogeneic group was much higher at 3 years (42% vs. 5%), despite this group possessing a lower relapse rate (39% vs. 61%). Our patient survived for more than 2 years after allo - sct, although he unfortunately suffered from cns relapse on day 350 . Consolidation and maintenance therapy after auto- and/or allo - sct in plasma cell disorder is still controversial . However, some clinical trials have recently shown that lenalidomide maintenance after auto - sct for mm significantly prolonged progression - free survival (pfs) and overall survival . Moreover, a prospective clinical trial by the intergroupe francophone du mylome group showed that a bortezomibbased regimen followed by auto - sct and subsequent maintenance therapy with lenalidomide, bortezomib, and dexamethasone for ppcl significantly improved pfs compared with those who received a reduced intensity conditioning allograft . In the future, auto - sct and maintenance therapy combined with novel agents may be a treatment option for young ppcl patients . The prognosis of plasma cell disorders with cns involvement is extremely poor . In a retrospective study on cns myeloma conducted in 12 greek institutions, there was no significant difference in median survival of cns myeloma (called post cns - mm) between novel drug - treated patients and those given other treatments (4 months vs. 2 months). By multivariable analysis, extramedullary lesions, prior to treatment with novel drugs, and high ldh levels at myeloma diagnosis were statistically independent predictors of post cns - mm survival . These findings corroborate our observations that our patient with cns relapse of ppcl had an extremely short - term outcome . It has been reported that 19% of post cns - mm patients met the criteria for ppcl . Furthermore, some of these ppcl patients developed isolated cns relapse during systemic remission after auto - sct . In a prospective study by royer et al ., 2 ppcl patients experienced neuromeningeal relapse after autologous- and allo - sct, respectively, and both patients soon died despite intrathecal chemotherapy . In the present case, although vgpr was maintained by rvd induction therapy followed by allo - sct, isolated cns relapse occurred within 1 year . Several drugs used in multiple myeloma, including bortezomib and lenalidomide, do not efficiently cross the blood of note, bbb penetration of lenalidomide was 11% in a rhesus monkey model, whereas that of thalidomide was 42% . In a murine model, pomalidomide displayed 39% bbb penetration and high anti - tumor activity in the cns . To the best of our knowledge, the anti - tumor effect of thalidomide is inferior to that of pomalidomide . Additionally, pomalidomide is more effective in ameliorating extramedullary involvement . In our case, concurrent it and rt followed by pd was initiated after the diagnosis of isolated cns involvement after allo - sct . In addition, maintenance therapy by pd might prolong survival to prevent systemic recurrence, including cns involvement, for more than 18 months . The role of pomalidomide for ppcl patients with cns involvement should be further explored in clinical trials . Our ppcl patient received rvd induction therapy followed by up - front allo - sct . However, concurrent it and rt followed by pomalidomide - based maintenance therapy well controlled his cns recurrence and prolonged his survival . To find an optimal treatment for this rare but aggressive ppcl, prospective clinical trials for this disorder should take into account the importance of myeloma drug penetration across the bbb in order to enhance the treatment of cns recurrence
In this issue of the jem, two articles describe a potential role for two bacterial toxins in the modulation of the immune system by interfering with t cell signaling . Using an in vitro jurkat t cell system, gerke et al . (8) report that yersinia pseudotuberculosis inhibits t cell activation and that the inhibitory activity was strictly dependent on the yersinia outer protein h (yoph), a potent tyrosine phosphatase that is delivered to host cells by a bacterially encoded type iii protein secretion system . Previous studies by a number of laboratories have identified several tyrosine phosphorylated proteins as apparent targets for the tyrosine phosphatase activity of yoph in cell culture systems . These include p130cas, focal adhesion kinase, and paxillin in hela cells, and fyb / slap130, p130cas, and skap55 in macrophages (912). In general, these are proteins involved in integrin signaling, which is consistent with the proposed role of yoph as an antiphagocytic molecule . More recent studies have shown that yoph can also inhibit t cell signaling in - vitro and that this activity could be correlated with the presence of yoph and the dephosphorylation of the tyrosine kinase lck (13). (8) have now extended those studies and added the t cell signaling adaptor proteins lat and slp-76 to the list of potential yoph substrates . These adaptor proteins are essential for the transduction of signals from the t cell receptor and its associated tyrosine kinases, and their tyrosine phosphorylation is required for their activities . (8) argue that tyrosine dephosphorylation of these adaptor proteins by yoph is responsible for the observed inhibition of t cell receptor signaling after yersinia infection of cultured jurkat t cells . They also present data that argue that both of these proteins are high affinity substrates of yoph, presumably requiring small amounts of translocated toxin to be targeted for dephosphorylation . In the second article, rossi paccani et al . (14) report that anthrax toxin also disrupts t cell signaling but by a completely different mechanism . Anthrax toxin, which is produced by bacillus anthracis, is composed of two enzymatic or a subunits known as edema factor (ef) and lethal factor (lf) that alternatively associate with a b subunit, known as protective antigen, which mediates their delivery into target cells (15). Ef is a potent calcium / calmodulin - dependent adenylate cyclase, which causes a massive increase of cyclic amp in intoxicated cells and global disruption of cell signaling . Lf is a zinc - dependent protease that specifically disrupts the mitogen - activated protein (map) kinase signaling pathways by cleaving the activating map kinase kinases mek1/2 and mkk3, mkk4, mkk6, and mkk7 (1619). (14) showed that treatment of peripheral blood lymphocytes with protective antigen in combination with either lf or ef effectively blocked t cell signaling as measured by the expression levels of the surface activation markers cd69 and cd25, the production of cytokines, and cell proliferation . Given the ubiquitous presence of the anthrax toxin receptors and the essential role for map kinases and camp in t cell signaling, the results presented by rossi paccani et al . The broader issue raised by these two and other studies with other bacterial toxins that disrupt immune cell function in vitro, however, is the in vivo relevance of the findings . Do these pathogens specifically interfere with t cell signaling during the course of an infection? Unfortunately, in vitro studies, although useful to provide testable hypotheses, cannot provide answers to these important questions (8) make a strong case for slp-76 and lat as high affinity substrates for this tyrosine phosphatase and hence of potential relevance during infection when presumably the pathogen would deliver small quantities of this toxin . Indeed, it is often the case that under vitro experimental conditions, cells are exposed to toxin quantities not usually delivered during actual infections . Nevertheless, it is not known how much yoph is delivered by yersinia during infection, and there is no evidence demonstrating that yersinia hampers t cell function during infection . Yersinia infections are usually acute (20), arguing against the need for these pathogens to counteract the host - specific adaptive immune responses during infection . However, although rare, chronic infections are sometimes observed (21, 22), and in those cases, the ability to interfere with t cell function might be useful to the pathogen . Whether this function of yoph has been specifically selected by evolution or is simply a by - product of another of its demonstrated functions, such as disruption of macrophages during acute infection, remains to be established . Anthrax toxin possesses even a more difficult challenge because its biochemical activity (such as inhibition of map kinase signaling) can potentially affect a large number of cellular processes . Therefore, the ability of anthrax toxin to inhibit map kinase signaling can result in the in vitro inhibition of any of the rather large number of intracellular signaling pathways that involve these kinases, regardless of their relevance during infection . For example, if added to cultured neurons, anthrax toxin would certainly prevent neurite outgrowth, a process strictly dependent on map kinase signaling (23). However, it would be hard to argue that this is a relevant function for the toxin during infection . By the same token, anthrax toxin predictably inhibited in vitro t cell signaling, a process strictly dependent on map kinase signaling . However, more experiments will be required to establish whether this activity of the toxin is important during infection . If untreated, b. anthracis infections are usually hyper acute (24); therefore, inhibiting t cell signaling may not provide a significant advantage to the pathogen . On occasions, b. anthracis can be associated with sub - acute infections (such as cutaneous anthrax; reference 24), in which case inhibition of t cell function may provide some advantages to the pathogen . In any case, because b. anthracis is most likely an accidental pathogen of mammals, it is unlikely that evolution may have played any role in directly shaping the effects of anthrax toxin on t cells . Therefore, this activity might be more a byproduct, relevant or not, of other activities of this toxin in the normal ecology of b. anthracis . Although every pathogen must contend with the onslaught of the innate immune responses, it is not necessarily the case that every pathogen must counteract the acquired immune response to fulfill its replication program . For most pathogens that cause acute infections, their life cycle within the host is most often over by the time the naive host mounts a meaningful acquired immune response capable of controlling the infection . In fact, most infections with pathogens that have coevolved with their hosts are indeed asymptomatic, do not lead to overt harm, and most often result in protective convalescent immunity . On the other hand, pathogens that cause persistent infections might be under strong evolutionary pressure to evolve specific mechanisms to avoid acquired immune responses . Indeed, mechanisms of antigenic variation or specific inhibition of antigen presentation evolved by microbial pathogens are well documented (25). For example, many viral pathogens specifically interfere, by a variety of mechanisms, with both major histocompatibility class i and ii antigen - presenting pathways (26, 27). In addition, many viral, bacterial, and protozoan pathogens undergo rapid antigenic variation to evade the onslaught of the acquired immune response (2830). Whether inhibition of t cell signaling by anthrax or yoph toxins should be added to the list of pathogenic mechanisms specifically evolved to counter the acquired immune response
Membrane proteins (mps) constitute about 30% of all the proteins encoded in the currently known genomes, and play critical roles in cell signaling, ion transport, and cell - cell communications, as well as assist the folding of other mps (1). Because of these biological significance, mps represent the most important class of drug targets about 50% of current molecular targets are membrane - bound (2). However, only about 2% (518 of 25,176) of the 3d structures deposited in the protein data bank (pdb; ref . 3) are for mps . And the number of high - resolution structures (from x - ray diffraction and more recently from nmr) remains even smaller, largely because of the difficulties in crystallizing mps . Recently, some new ideas and experimental approaches have been introduced in the area of mp crystallization (4), all of which exploit the spontaneous self - assembling properties of lipids and detergent as vesicles (vesicle - fusion method), discoidal micelles (bicelle method), and liquid crystals or mesophases (in meso or cubic - phase method). Despite these promising new methods, the current gap between need and supply of mp 3d structures makes prediction algorithms important and essential . Mps come in a variety of sizes and shapes, though the available 3d structure principles are far less diverse than those of the globular proteins . From a structural point of view one is the -helix bundle protein, in which one or several -helices span the membrane; and the other is -barrel protein, in which eight or more antiparallel tm -strands form a closed barrel 5 ., 6 .. two recent examples 7 . Since jhnig and edholm in 1992 presented one of the first methods using secondary structure prediction to build suitable model structures as initial conformations for molecular dynamic studies (9), several groups have tried computational approaches to elucidate mp structures . In 1993, milik and skolnick presented a method based on the combination of a hydropathy scale for the prediction of trans - bilayer fragments with dynamic monte carlo simulation techniques (10). In 1994, taylor et al . Adapted some programs originally developed for the prediction of globular protein structures to derive a method for the prediction of integral mp structures (11). Each step in the method is fully automated, from the initial sequence data bank searches to the final construction of 3d models . Consequently, estimates for prediction accuracy are perhaps overly optimistic . Here, we summarize recent attempts within the field of computational biology and bioinformatics to predict an mp s structure . Most current methods of theoretical mp structure prediction do not actually deal with predicting the 3d structure, but rather try to predict the most likely topology of the protein, that is to say, the in / out location of the n and c termini relative to the membrane, and the number and position of transmembrane (tm) segments . A high - quality model of secondary structure and topology is a prerequisite for experimental structure - function studies, and can be a starting point for attempts to model the 3d structure before molecular dynamics or simulated annealing simulations . In recent years, various accurate methods because the number of high - resolution structures of -barrel proteins is less than that of the -helix proteins, the neural network has been more frequently adopted in the -strand topology prediction . The details of some methods based on hidden markov models (hmms) are listed in table 2 . Many secondary structure prediction methods are based on statistical methods, physicochemical methods, sequence pattern maching, and evolutionary conservation (12). The main methods for identifing tm helices are on the basis of their hydrophobicity and known minimum length (at least 15 residues; ref . Membrane propensities were defined by a statistical analysis carried out on a set of 640 tm helices, belonging to 133 mps extracted from swiss - prot (14) that have experimentally defined topologies . The five widely used prediction methods for predicting the topology of -helix bundle mps are tmhmm (15), hmmtop (16), memsat (17), phdhtm (18), and toppred (19). Tmhmm, hmmtop, and memsat are all based on hmms with 5~7 types of structural states . Toppred was the first topology prediction method that combined hydrophobicity analysis and the positive - inside rule . Generally, these sequence - based methods for predicting the number and approximate location of tm helices within mps have about 85% accuracy . In 2003, karin meln et al . Tried to construct useful reliability scores for these methods (20). 21) algorithm can provide a solution to the problem that non - transmembrane query sequences may give false positive hits (20%-30%) in the prediction process . The upgraded and modified version of the das - prediction method, das - tmfilter algorithm, has been distributed (22). The new algorithm is designed to make distinction between protein sequences with and without tm helices at a reasonably low rate of false positive prediction (~1 among 100 unrelated queries) while the high efficiency of the original algorithm locating tm segments in queries is preserved (sensitivity of ~95% among documented proteins with helical tm regions). In 2003, xia and colleagues presented a new approach, conpred_elite (23), that can predict the whole topology with accuracies of 98% for prokaryotic and 95% for eukaryotic proteins as they reported . Besides the tm helix, another tm segments type is -barrel, which consists of several tm strands . Unlike -helical mps, there are no simple low - resolution experiments that yield large amounts of data for -barrel mps . In fact, the overall hydrophobicity for -barrel mps is similar to that of soluble proteins (13). Gromiha and colleagues combined amino acid preferences for -strands with the surrounding hydrophobicity of the respective residues to predict -strands (24). Diederichs and colleagues proposed to use a neural network to predict the topology of the bacterial outer membrane -strand proteins and to locate residues along the axes of the pores (25). Jacoboni and colleagues applied a method combining neural networks and dynamic programming to predict the location of membrane strands (26). The authors estimated that their system correctly predicted about 93% of all known membrane strands . More recently, martelli et al . Developed a sequence - profile - based hmm model that can predict the topology of -barrel mps cyclicing with 6 types of states (27). They reported that the accuracy of per residue of the model was about 83% . Lately the following protocol starting from secondary structure prediction and tm segments topology prediction are often used . Secondary structure prediction followed by tm segments identification along with prediction of loops connecting the segments, and molecular dynamics or simulated annealing simulations, may be finally used to refine these primal models . During the last refinement step, the protein is often inserted into a water / lipid bilayer / water or a water / n - octane / water environment to take into account the presence of the cell membrane . Charmm, gromos, amber, and cvff - insight are some widely used force fields in molecular dynamics calculation . The slow dynamics of lipid molecules in the bilayer might bring the difficulties in equilibrating the system (28). For globular proteins, the major successful methods for structure prediction include homology modeling, threading, and ab initio folding . Along with lucubrating the mechanism of mp folding and increasing the number of high - resolution mp structures, these methods will been applied to the direct prediction of whole mp 3d structures . The question of how the controlled integration of an mp into the lipid bilayer takes place is still not fully worked out, and there are certainly aspects of mp structures that will probably not be fully appreciated until this step has been accomplished . Some pursuers educed the viewpoint that the prediction of mp structures from amino acid sequences was, in large measure, a problem of physicochemistry (29). Physical influences that shape mp structures include interactions of the polypeptide chains with water, bilayer hydrocarbon core, bilayer interfaces, and cofactors . Studies on the mechanism of insertion and folding of mps into membranes are relatively rare and have been mostly performed with two model proteins: bacteriorhodopsin (br; ref . While br is a representative -helical bundle protein, ompa belongs to the class of -barrel protein . Homology modeling constructs structures (targets) that are homologous to other protein(s) whose 3d structure is known (templates). Because few high - resolution mp 3d structures are available to be used as templates, and the modeling can be unreliable when the sequence identity between the template and target proteins falls below 20%-30%, the applicability of homology modeling is limited . An ab initio method was presented (32), whose knowledge - based technique added a membrane potential to the energy terms (pairwise, solvation, steric, and hydrogen bonding). The method is based on the assembly of supersecondary structural fragments taken from a library of highly resolved protein structures using a standard simulated annealing algorithm . Results obtained by applying the method to small mps of known 3d structures showed that the method is able to predict, at a reasonable accurate level, both the helix topology and the conformations of these proteins . The structure prediction of membrane proteins still remains an interesting scientific problem . Because of the physical difference between mps and gps (globular proteins) current segment accuracy of reported algorithms are pretty high (above 90%), while the overall accuracy are still around 50%-60%, which gives birth to hand - raising methods to combine the reports from several other algorithms . The lack of both high - resolution and low - resolution experimental data of mp structures makes the algorithm development and their evaluation difficult, but the fact that most mp sequences are used as space blocks to get through the membrane bilayer that has predefined thickness makes the structure prediction of mps simple on functional aspects . New algorithms will emerge and reported algorithms will be refined to give a better answer to this problem.
Liver diseases are common all over the world as well as in india; and the prevalence of liver diseases is likely to increase in future . Liver plays an important role in metabolism of thyroid and gonadal hormones like conjugation, excretion, peripheral deiodination, and synthesis of thyroid - binding globulin (tbg) and sex hormone - binding globulin (shbg). Hence, it is not surprising that thyroid and gonadal dysfunction have been reported in various spectra of liver diseases and associated with the severity of liver disease . These changes represent changes in biochemical abnormalities due to liver dysfunction and pathological changes associated with end organ damage . Acute liver failure is associated with increased circulating endotoxins and proinflammatory mediators, which is quite similar to clinical state of sepsis and results in dysfunction of endocrinal glands like sick euthyroid syndrome also termed nonthyroidal illness syndrome (ntis) and hypogonadism . Also in stable cirrhosis, a state of hypothyroidism has been shown which correlates with slow progression of stage of cirrhosis . We conducted this study at a tertiary care centre to assess thyroid and gonadal function in subjects with acute hepatitis (ah), chronic liver disease (cld), and post - lt . This study was conducted as a cross - sectional study at the army hospital (research and referral), new delhi from may 2010 to dec 2011 . Subjects with ah, cld, and lt were recruited from outpatient department and inpatient ward / intensive care unit of gastroenterology department . Ah was diagnosed on the basis of presence of acute onset of jaundice with raised transaminases (3), no past history of liver disease, and absence of evidence of portal hypertension on ultrasonography of abdomen . Cld was diagnosed on the basis of evidence of liver disease of more than 6 months duration and/or evidence of portal hypertension on ultrasonography or upper gastrointestinal endoscopy . These subjects were classified as per child - pugh criteria child - pugh stage a (cld-1) and child - pugh stage b or c (cld-2). Patients on steroids or those who had received thyroid hormone or testosterone or had alcoholic liver disease were excluded from this study . This study was conducted according to the guidelines laid down in the declaration of helsinki and all procedures involving human subjects / patients were approved by the institutional human ethics committee at army hospital (research and referral). Written informed consent was obtained from all subjects / patients . Patient's fasting blood samples were collected and analyzed for hematological parameters (complete blood count with platelet counts), liver function tests [serum bilirubin, aspartate transaminases (ast), alanine transaminases, serum protein, serum albumin, and international normalized ratio (inr)]; blood glucose, serum creatinine, serum lipid profile, and electrolytes (serum sodium and potassium). Patient's blood was collected in fasting state and serum was separated and stored at 80c . Hormone levels were measured using commercial kit provided by bhabha atomic research centre, mumbai; and immunotech, beckman coulter company, france . Primary and subclinical hypothyroidism were defined as low free thyroxin (ft4) (normal 0.8 - 2.1 ng / ml) with raised thyroid - stimulating hormone (tsh) (normal 0.5 - 6.5 iu / ml) and normal ft4 with raised tsh, respectively . Gonadal failure was defined as primary when luteinizing hormone (lh), and follicular - stimulating hormone (fsh) were twice the upper limits of normal (> 30 iu / l and> 40 iu / l, respectively) or secondary when lh and fsh were low associated with low testosterone (te) levels in males (te <3.0 ng / ml) or amenorrhea in females . Data were presented as mean standard deviation or number (%) unless specified . All parametric data were analyzed by independent student's t - test in categorical groups (two groups) and analysis of variance test (> two groups). Pearson's correlation coefficient was calculated to assess the strength of relationship between thyroid and gonadal functions and other parameters . Patient's fasting blood samples were collected and analyzed for hematological parameters (complete blood count with platelet counts), liver function tests [serum bilirubin, aspartate transaminases (ast), alanine transaminases, serum protein, serum albumin, and international normalized ratio (inr)]; blood glucose, serum creatinine, serum lipid profile, and electrolytes (serum sodium and potassium). Patient's blood was collected in fasting state and serum was separated and stored at 80c . Hormone levels were measured using commercial kit provided by bhabha atomic research centre, mumbai; and immunotech, beckman coulter company, france . Primary and subclinical hypothyroidism were defined as low free thyroxin (ft4) (normal 0.8 - 2.1 ng / ml) with raised thyroid - stimulating hormone (tsh) (normal 0.5 - 6.5 iu / ml) and normal ft4 with raised tsh, respectively . Gonadal failure was defined as primary when luteinizing hormone (lh), and follicular - stimulating hormone (fsh) were twice the upper limits of normal (> 30 iu / l and> 40 iu / l, respectively) or secondary when lh and fsh were low associated with low testosterone (te) levels in males (te <3.0 ng / ml) or amenorrhea in females . Data were presented as mean standard deviation or number (%) unless specified . All parametric data were analyzed by independent student's t - test in categorical groups (two groups) and analysis of variance test (> two groups). Pearson's correlation coefficient was calculated to assess the strength of relationship between thyroid and gonadal functions and other parameters . In this study, we studied 25 patients of ah; 20 patients of cld with relatively preserved liver functions (cld1) which included child - pugh stage a and 30 patients with more advanced cld2 which included child - pugh stage b or c, and 10 patients who were post - lt . Most of the subjects with ah were acute viral hepatitis (n = 18); however, etiology could not be assessed in all cases . Four of these patients with ah had acute liver failure . Among 50 subjects with cld, 29 were hepatitis b virus (hbv)-related cirrhosis, 8 were hepatitis c virus (hcv)-related cirrhosis, 3 had hbv and hcv coinfection, and 10 had cryptogenic cirrhosis with probable autoimmune etiology in six . The indications for lts were hbv related cirrhosis in 5 patients, cryptogenic cirrhosis in 3 patients, and hcv - related cirrhosis in 2 patients . Basic parameters of various groups thyroid dysfunction was present in 16% (14/75) patients with liver diseases . Among thyroid dysfunction, the commonest was ntis 8% (6/75), which was present in four patients of ah (all with acute hepatic failure) and two patients of cld-2 . Subclinical hypothyroidism and primary hypothyroidism were present in three patients and one patient in cld group, respectively . Thyroid function test were evaluated after excluding three patients with overt hypothyroidism and thyrotoxicosis . Among patients with lt and ah group, cld-2 group had significantly lower levels of all thyroid hormones compared with controls and cld-1 group . Thyroid and gonadal hormones in subjects with liver diseases compared with controls hypogonadism was present in 40% (30/74) patients with liver diseases excluding a postmenopausal woman . Hypogonadism was commonest in patients with cld-2 (16/30; 53%), followed by lt (4/10; 40%), ah (6/25; 24%), and cld-1 (4/20; 20%). All the patients had secondary hypogonadism . Among patients with ah, four patients with acute liver failure had low testosterone and low lh (<5 the consistent abnormality detected was significantly lower testosterone levels in all groups with liver disease compared with controls . Serum estradiol levels were higher in all groups compared with controls, but were statistically significant only in ah and cld1 group [table 2]. Hypogonadism was predicted by older age, lower levels of serum albumin, total cholesterol and triglycerides and higher levels of plasma glucose, serum bilirubin, ast and inr [table 3]. Thyroid dysfunction was present in 16% (14/75) patients with liver diseases . Among thyroid dysfunction, the commonest was ntis 8% (6/75), which was present in four patients of ah (all with acute hepatic failure) and two patients of cld-2 . Subclinical hypothyroidism and primary hypothyroidism were present in three patients and one patient in cld group, respectively . Thyroid function test were evaluated after excluding three patients with overt hypothyroidism and thyrotoxicosis . Among patients with lt and ah group, cld-2 group had significantly lower levels of all thyroid hormones compared with controls and cld-1 group . Hypogonadism was present in 40% (30/74) patients with liver diseases excluding a postmenopausal woman . Hypogonadism was commonest in patients with cld-2 (16/30; 53%), followed by lt (4/10; 40%), ah (6/25; 24%), and cld-1 (4/20; 20%). All the patients had secondary hypogonadism . Among patients with ah, four patients with acute liver failure had low testosterone and low lh (<5 the consistent abnormality detected was significantly lower testosterone levels in all groups with liver disease compared with controls . Serum estradiol levels were higher in all groups compared with controls, but were statistically significant only in ah and cld1 group [table 2]. Hypogonadism was predicted by older age, lower levels of serum albumin, total cholesterol and triglycerides and higher levels of plasma glucose, serum bilirubin, ast and inr [table 3]. Clinical and biochemical predictors of hypogonadism in patients with chronic liver disease endocrine dysfunction are common with liver diseases, which is correlated with severity of liver dysfunction and improve after lt . In this study, we have assessed thyroid and gonadal functions in complete spectrum of severity of liver diseases and after lt . In present study, ntis was the commonest, was present in patients with acute liver failure and cld . Ntis is characterized by a normal total t4, normal / high free t4, low total t3, low free t3, and an elevated rt3 . Among patients with cld, cases with primary hypothyroidism and thyrotoxicosis were observed . Cld due autoimmune etiology is known to have associated autoimmune thyroid disease . In autoimmune hepatitis, both grave's disease (6%) and autoimmune hypothyroidism (12%) are common . Subclinical hypothyroidism has been reported in patients with hepatitis - c related cld, which has been attributed to direct cytopathic effect of hepatitis - c virus on thyroid cells, underlying latent autoimmune disease, and treatment related . Serum total t3 was lower in all forms of liver disease when compared with controls . This was due to decreased hepatic uptake and type-1 deiodinase activity (d1), which converts t4 to t3 . Liver disease is also associated with increase in inflammatory cytokines that negatively affect hypothalamothyroid axis, which may explain lower tsh levels (statistically not significant) observed in patients with liver disease as compared to controls in this study . Other studies have reported higher levels of tsh in clds, but these studies have not excluded patients with overt thyroid dysfunction, which we have excluded in this study before analysis . Serum total t3 was lower in ah and cld-2 compared with cld-1 in this study, reflecting associated severity of liver disease . Most consistent abnormality of thyroid functions reported is lower total and free ft3 and increase in rt3 in patients with cirrhosis of liver . The plasma t3:rt3 ratio has a negative correlation with the severity of cirrhosis . In present study thyroid hormone is associated with basal metabolic rate, and low total and free t3 levels may reflect adaptive hypothyroid state . This will reduce the basal metabolic rate and may help to preserve hepatocytes and liver function . Occurrence of hypothyroidism in cirrhotic patients has been shown to be associated with a biochemical improvement in liver function, and decreased rate of decompensation in cirrhosis . Total and free t4 was increased in cld-1 but decreased significantly in patients with cld-2 . Increase in total t4 has been observed in patients with acute and cld due to increase in tbg levels, which is synthesized as acute phase reactant . Patients with liver diseases have increased low - density lipoprotein (ldl) and lower high - density lipoproteins hdl, which improves after lt . Thyroid hormones increase the expression of ldl receptors on the hepatocytes and increase the activity of lipid - lowering liver enzymes, resulting in a reduction in low - density lipoprotein levels . Thyroid hormones also increase the expression of apolipoprotein a1, a major component of high - density lipoprotein . Hence, decrease in thyroid hormones associated with liver disease will adversely affect ldl disposal and decrease hdl synthesis . In this study, it was commonest with cld with child pugh stage b and c, and after lt . Serum total testosterone decreased progressively from acute liver disease to cld and correlated with the increasing severity of liver disease . Serum lh levels were significantly lower in patients with cld-1 but were comparable in other groups as was the fsh . Another study reported higher level of lh in patients with cirrhosis of liver but lower lh response to gonadotropin - releasing hormone indicating involvement of hypothalamo - pituitary - gonadal axis at all levels . There was no difference in fsh level among patients and controls . A study assessed pulsatile pattern of lh in patients with cld and found attenuated pattern of lh secretion, indicating hypothalamic dysfunction . On the contrary, few studies have suggested primary gonadal failure with compensatory in lh in patients with cld . Alcohol is known to affect gonadal functions, which were not included in this study . A study similar to ours, also noticed reduced levels of total and free testosterone and increased levels of shbg compared with controls with normal liver function in men with chronic nonalcoholic liver disease, which was related with severity of liver disease . In the present study, serum testosterone levels were higher in patients after lt, but remained lower than controls . Handelsman et al ., also reported gradual improvement in serum testosterone levels over 12 months post - lt, but levels remained subnormal . There was no change in lh and fsh after transplantation in this study . However, one study reported increase in gonadotropins and testosterone, and decrease in estradiol levels after transplantation . This was probably due to more severe gonadal dysfunction in their study, as 90% of patients had hypogonadism . In the present study, serum estradiol levels were higher in all patients with liver disease compared with controls, which was similar to observed by others . Mao et al ., observed that serum estradiol levels were correlated with severity of liver disease and deteriorating liver functions in males with hbv - related liver disease . The strengths of this study are the assessment of thyroid and gonadal functions at various stages of liver disease and exclusion of patients with alcoholic liver disease because alcohol is known to affect hypothalamo - gonadal axis . In the present study, most of the patients were stable and not critically ill except four patients with hepatic failure, leading to lesser confounding factors in thyroid and gonadal dysfunction . First, we have not measured free testosterone, tbg, and shbg due to financial constraints . Second, pituitary response to trh and gnrh was not assessed, which could have highlighted underlying mechanism . Third, as this was a cross - sectional study, we could not assess factors which could have predicted mortality or morbidity . In conclusion,
Adherence to antiretroviral therapy (art) is essential for maximal suppression of viral replication and avoidance of drug resistance . As such, good adherence is believed to be a critical determinant of long - term survival among hiv - infected individuals . Where availability of second - line or salvage antiretroviral drug regimens is limited early studies in africa reported adherence levels as high or higher than those observed in developed countries . However, measures used to assess adherence vary and many have not been validated in the context of rapid art scale - up . In this analysis, we describe the impact of art adherence on immunologic response and survival in a large population of adults initiating art in lusaka, zambia . We use the medication possession ratio a simple metric that has been correlated with virologic outcomes in developed countries to describe the adherence behaviour of our population . In lusaka, zambia, a large - scale public sector hiv care and treatment program was established by the zambian ministry of health in april 2004 . Clinical care, patient tracking and outcomes monitoring for the lusaka program have been described elsewhere . Briefly, care has been standardized across all sites according to the zambian national guidelines for adult hiv treatment . In the first months of the program, art was initiated in individuals with either a cd4 cell count <200 cells/l or who stage iii / iv . Since 2005, the zambian guidelines have been revised, so that art eligibility for patients in who stage 3 requires a cd4 cell count 350 cells/l . First - line drug regimens in zambia had comprised two nucleoside reverse transcriptase inhibitors (lamivudine with either zidovudine or stavudine) and one non - nucleoside reverse transcriptase inhibitor (nnrti; nevirapine or efavirenz). Since july 2007, however, the zambian ministry of health has incorporated combination tenofovir / emtricitabine as a first - line nucleoside reverse transcriptase inhibitor backbone for individuals newly initiating therapy; those continuing therapy remain on their previous regimen except in cases of toxicity or treatment failure . Antiretroviral drugs provided are free of charge and dispensed by pharmacy technicians or nurses within the art clinic itself . These individuals are authorized to collect a limited number of drug refills when the patient is unable to attend a pharmacy appointment . Medical information, including the number of pills dispensed and the date of next visit, is entered into an electronic patient tracking system . Patients on art who are> 10 days late for a scheduled appointment are followed up by community health workers at their homes . We calculated adherence based on a variation of the commonly - reported medication possession ratio (mpr). We divided the number of days late for pharmacy refills by total days on therapy, and then subtracted that percentage from 100% . The resulting figure represents the percentage of days a patient is known to have medication on hand and has been correlated with hiv virologic outcomes in africa . In the lusaka public sector, patients are provided a buffer stock of 3 days with each drug dispensation; therefore, we began calculating lateness on the fourth day after a missed pharmacy appointment . Since art is provided free charge by the zambian government, the patients were categorized as optimally adherent (95%), suboptimally adherent (8094%) and poorly adherent (<80%). Owing to the high rates of early mortality in our setting and others like it, we excluded patients from this analysis who had been on therapy for <12 months . We reasoned that poor patient outcomes associated with adherence would likely manifest later in the course of treatment, after viral drug resistance and treatment failure had developed . Early death also introduces bias into the mpr measurement, since these patients do not have the same opportunity to fully demonstrate long - term adherence behaviours . For these reasons, we considered only adherence behaviour during the first 12 months and evaluated outcomes after 12 months . Active if they attended their last scheduled clinical and/or pharmacy visit within 30 days of their appointment date . Dead patients are so classified in the medical record after confirmation from family members, friends or community health workers . Late patients are> 30 days overdue for their last scheduled clinical and/or pharmacy visit and cannot be located via community health worker contact tracing . In survival analyses and rate calculations, inactive patients are censored as of the date they became inactive, and late patients are censored 30 days after their last scheduled visit . We analysed programmatic data from treatment - nave adults (> 15 years) initiating art across 18 primary care sites in lusaka, zambia from april 1, 2004 to september 30, 2007 . To be included in the analysis, individuals had to be active for 12 months of follow - up, so that the mpr measure for adherence could be calculated . Those who had died, withdrew from the program or were lost to follow - up prior to 12 months were excluded . To compare demographic and clinical characteristics among members of the study population and those who were excluded, we analysed categorical variables using pearson's chi - square test . Non - parametric wilcoxon rank - sum tests were used to compare continuous variables . We used logistic regression to describe the association between these characteristics and adherence to therapy at 12 months . In this multivariable analysis, we included all covariates associated with adherence at a p - value <0.10 level . Log - rank tests were used to evaluate differences in survival among adherence categories using kaplan meier analysis; cox proportional hazards models were used to assess the risk of death . The proportional hazards assumption was confirmed via the kolmogorov - type supremum test (p = 0.83). All factors associated in crude analysis with p - value <0.10, or that have been previously shown to be associated with mortality in our setting, were included in the multivariable analysis . However, we excluded any such covariate if it was believed to be an intermediary step between the exposure and outcome . (for example, we include enrollment cd4 lymphocyte count in the multivariable models but not later measures of cd4). In all adjusted models, continuous covariates were categorized based on the conventions in the medical literature . Finally, we compared clinical and immunologic outcomes (i.e. Cd4 count, weight, haemoglobin) longitudinally according to mpr categories . Means and corresponding 95% cis at each time point were calculated; trends were evaluated at each 6-month interval using separate student's t - tests . Available patient data through november 1, 2008, the dataset freeze date, were considered in this report . All analyses were performed using sas version 9.1 (sas institute, cary, nc, usa), and were approved by the institutional review boards of the university of zambia (lusaka, zambia) and the university of alabama at birmingham (birmingham, al, usa), and by the us centers for disease control and prevention (atlanta, ga, usa). From april 1, 2004 to september 30, 2007, 37 039 art - nave adults (> 15 years of age) initiated treatment across 18 sites in lusaka, zambia . Of these, 27 115 (73.2%) remained active in the program at or after 12 months and were thus included in the analysis . A total of 9924 (26.8%) were excluded from the analysis because they had left the program before reaching the 12 month threshold: 1133 withdrew from the program, 5114 were late for patient visits and 3677 had died (crude mortality rate = 105.5/100 person - years, 95% ci: 102.1108.9). A comparison of those included and excluded from the analysis is shown in table 1 . Table 1characteristics of treatment - nave adults initiating art in lusaka, zambia, from april 1, 2004 to september 30, 2007 and continuing art for> 12 monthsincluded in analysisexcluded from analysisnvaluenvaluepage, median years (iqr)27 11535 (3041)992434 (2940)<0.001 1525271810.0%128512.9%<0.001 263512 15944.8%452645.6% 3645863631.8%288429.1% 4655287510.6%9019.1% 567272.7%3283.3%sex female16 88962.3%556656.1%<0.001 male10 22637.7%435843.9%adherence supporter no546920.2%328933.1%<0.001 yes21 64679.8%663566.9%cd4 lymphocyte count, median cells/l (iqr)26 068132 (69198)9409110 (48191)<0.001 200 cells/l635824.4%212122.5%<0.001 50199 cells/l15 27458.6%485551.6% <50 cells/l443617.0%243325.9%who stage i or ii874132.5%233023.8%<0.001 iii15 78958.6%605761.8% iv23988.9%142014.5%haemoglobin, median g / dl (iqr)23 79610.9 (9.612.3)837010.2 (8.711.8)<0.001 8.0 g / dl21 99592.4%712285.1%<0.001 <8.0 g / dl18017.6%124814.9%bmi, median kg / m (iqr)24 25020.0 (18.122.3)835818.9 (16.921.2)<0.001 20 kg / m12 14750.1%307836.8%<0.001 1819 kg / m614825.4%201524.1% 1617 kg / m425817.6%193023.1% <16 kg / m16977.0%133516.0%tuberculosis co - infection at enrollment no23 48886.6%856986.3%0.49 yes362713.4%135513.7%initial art regimen d4 t + 3tc + nvp10 69939.7%469148.0%<0.001 zdv + 3tc + nvp12 99648.3%350135.8% d4 t + 3tc + efv15235.7%8758.9% zdv + 3tc + efv10263.8%4234.3% tdf + ftc + nvp2290.9%981.0% tdf + ftc + efv4481.7%1942.0%adherence by mpr, median (iqr)27 11597.9 (91.2100.0)9924100.0 (92.6100.0)<0.00l 95100%17 06062.9%705171.0%<0.00l 8094%768228.3%141214.2% <80%23738.8%146114.7%for those included in the analysis, adherence by mpr was based on behaviour within the first 12 months from art initation . For those excluded because they were not active at 12 months, mpr was based on time from art initiation to censor date . Art = antiretrovinal therapy; d4 t = stavudine; zdv = zidovudine; 3tc = lamivudine; nvp = nevirapine; efv = efavirenz . Characteristics of treatment - nave adults initiating art in lusaka, zambia, from april 1, 2004 to september 30, 2007 and continuing art for> 12 months for those included in the analysis, adherence by mpr was based on behaviour within the first 12 months from art initation . For those excluded because they were not active at 12 months, mpr was based on time from art initiation to censor date . Art = antiretrovinal therapy; d4 t = stavudine; zdv = zidovudine; 3tc = lamivudine; nvp = nevirapine; efv = efavirenz . Of the 27 115 included in this analysis, 1419 (5.2%) withdrew from the program, 3067 (11.3%) were late for their most recent scheduled visit, and 823 (3.0%) were known to have died (crude post-12 month mortality rate = 2.1/100 person - years, 95% ci: 2.0, 2.3) after the initial 12-month period . The median observation period was 15.7 [interquartile range (iqr) = 7.526.4] months, from 12 months onward . Of 27 115 patients, 17 060 (62.9%) demonstrated optimal adherence, 7682 (28.3%) had suboptimal adherence and 2373 (8.8%) had poor adherence over the first 12 months on therapy (figure 1). Individuals with an mpr <80% were more likely to have a higher median cd4 count at enrollment than those with optimal adherence . When compared with those with optimal adherence, individuals in the 8094 and <80% mpr were generally younger and were less likely to report adherence support (i.e. Adherence the proportion of individuals with tuberculosis at time of art initiation was lower among individuals with suboptimal adherence (12.2 vs 14.0%; p <0.001) when compared with those with optimal adherence . The proportion of patients who withdrew from the program or were lost to follow - up after 12 months increased as adherence declined categorically (table 2). Figure 1distribution of adherence to art over the first 12 months on treatment among adults surviving to at least 12 months in lusaka, zambia, between april 1, 2004 and september 30, 2007 . Adherence was measured by the number of days a patient had antiretroviral drugs available according to pharmacy refill data table 2characteristics by adherence category for treatment - nave adults initiating art in lusaka, zambia, from april 1, 2004 to september 30, 2007 and continuing art for> 12 monthsoptimal (95100%)sub - optimal (8094%)poor (<80%)nvaluenvaluepnvaluepage, median years (iqr)17 06035 (3041)768234 (2941)0.03237333 (2840)<0.001 152516019.4%78210.2%0.1233514.1%<0.001 2635762044.7%345845.0%108145.6% 3645552532.4%244131.8%67028.2% 4655186310.9%78510.2%2279.6% 564512.6%2162.8%602.5%sex female10 73062.9%468561.0%<0.01147462.1%0.46 male633037.1%299739.0%89937.9%adherence supporter reported at enrollment14 05782.4%594377.4%<0.001164669.4%<0.001cd4 lymphocyte count, median cells/l (iqr)16 469132 (70196)7350129 (68198)0.592249139 (73207)<0.001 200 cells/l394323.9%180424.5%0.6061127.2%<0.01 50199 cells/l969958.9%429358.4%128257.0% <50 cells/l282717.2%125317.0%35615.8%who stage i or ii553232.6%243632.0%0.1977332.9%0.81 iii995658.7%446758.7%136658.1% iv14738.7%7139.4%2129.0%haemoglobin, median g / dl (iqr)15 18610.9 (9.612.3)665710.9 (9.612.3)0.42195311.0 (9.512.4)0.49 8.0 g / dl14 06092.6%615492.4%0.71178191.2%0.03 <8.0 g / dl11267.4%5037.6%1728.8%bmi, median kg / m (iqr)15 35620.0 (18.122.3)684620.0 (18.022.2)0.34204819.9 (18.022.3)0.53 20 kg / m772550.3%341849.9%0.42100449.0%0.75 1819 kg / m390925.5%170324.9%53626.2% 1617 kg / m265317.3%124118.1%36417.8% <16 kg / m10697.0%4847.1%1447.0%tuberculosis co - infection at enrollment238214.0%93712.2%<0.00130813.0%0.19initial art regimen d4 t + 3tc + nvp673139.7%304239.9%0.0792639.7%0.24 zdv + 3tc + nvp822248.5%365547.9%111947.9% d4 t + 3tc + efv9435.6%4405.8%1406.0% zdv + 3tc + efv6283.7%2983.9%1004.3% tdf + ftc + nvp1671.0%490.6%130.6% tdf + ftc + efv2711.6%1411.8%361.5%lost to follow - up or withdrawn after 12 months229413.4%140418.3%<0.00178833.2%<0.001defined at 12 months of therapy as the percentage of time on therapy with art via pharmacy claims.based on comparisons to the optimal adherence category.art = antiretroviral therapy; d4 t = stavudine; zdv = zidovudine; 3tc = lamivudine; nvp = nevirapine; efv = efavirenz . Distribution of adherence to art over the first 12 months on treatment among adults surviving to at least 12 months in lusaka, zambia, between april 1, 2004 and september 30, 2007 . Adherence was measured by the number of days a patient had antiretroviral drugs available according to pharmacy refill data characteristics by adherence category for treatment - nave adults initiating art in lusaka, zambia, from april 1, 2004 to september 30, 2007 and continuing art for> 12 months defined at 12 months of therapy as the percentage of time on therapy with art via pharmacy claims . Based on comparisons to the optimal adherence category . Art = antiretroviral therapy; d4 t = stavudine; zdv = zidovudine; 3tc = lamivudine; nvp = nevirapine; efv = efavirenz . Individuals who were> 35 years [odds ratio (or): 0.8; 95% ci: 0.70.9] or reported presence of an adherence buddy (or: 0.5; 95%ci: 0.50.6) were less likely to have mpr <80% . When compared with those with cd4 count> 200 cells/l, those with lower baseline cd4 counts were less likely to be poorly adherent over the first 12 months: 50199 cells/l (or: 0.9, 95% ci = 0.81.0) and <50 cells/l (or: 0.8, 95% ci: 0.70.9). Baseline world health organization (who) clinical stage, haemoglobin, body mass index (bmi) and tuberculosis status were not predictive of poor adherence (data not shown). In kaplan meier analysis, individuals with <80% adherence had a significantly higher risk of death when compared with those in other adherence categories (log rank p = 0.0001). In unadjusted proportional hazards regression, the relative hazard for death was 1.7 (95% ci: 1.42.1) among those with poor adherence, when compared with those with optimal adherence . These findings remained virtually unchanged in a multivariable model controlling for sex, age, adherence support, baseline cd4 lymphocyte count, baseline who stage and initial regimen dispensed [n = 25 836; adjusted hazard ratio (ahr): 1.7; 95% ci: 1.42.2]. Baseline haemoglobin and bmi were not included in this initial multivariable model, since early versions of our patient tracking software did not routinely collect these data . In a separate model that included these two parameters (n = 20 951), the hazard for mortality associated with poor adherence did not change appreciably (table 3). Figure 2post-12-month mortality by adherence category for art - nave adults initiating art in lusaka, zambia, between april 1, 2004 and september 30, 2007 . Adherence was measured by the number of days a patient had antiretroviral drugs available according to pharmacy refill data . The numbers shown at the bottom of the graph represent the number of patients active in each adherence category at 6-monthly intervals . Perfect survival in the first 12 months is a result of our study design . To be eligible for inclusion in this analysis, patients had to remain alive, active, and on art until at least 12 months table 3factors associated with post-12-month mortality among treatment - nave adults initiating art in lusaka, zambia, from april 1, 2004 to september 30, 2007 and continuing art for> 12 monthscrude hazard ratio (95% ci)adjusted hazard ratio (95% ci)adherence optimal (95100%)1.01.0 suboptimal (8094%)1.0 (0.91.2)0.9 (0.81.1) poor (<80%)1.7 (1.42.1)1.7 (1.42.3)age 35 years1.01.0> 35 years1.4 (1.21.6)1.4 (1.21.6)sex female1.01.0 male1.4 (1.21.6)1.3 (1.11.5)adherence supporter no1.01.0 yes0.7 (0.60.8)0.7 (0.60.9)cd4 count 200 cells/l1.01.0 50199 cells/l1.3 (1.01.5)1.2 (1.01.6) <50 cells/l1.9 (1.52.3)1.5 (1.22.0)who stage i or ii1.01.0 iii1.6 (1.41.9)1.4 (1.11.7) iv2.5 (2.03.2)2.0 (1.52.7)haemoglobin 8.0 g / dl1.01.0 <8.0 g / dl2.0 (1.62.5)1.6 (1.22.0)bmi 16 kg / m1.01.0 <16 kg / m2.2 (1.82.8)1.8 (1.42.2)initial art regimen d4 t + 3tc + nvp1.01.0 zdv + 3tc + nvp0.7 (0.60.8)0.7 (0.60.9) d4 t + 3tc + efv1.1 (0.81.4)0.9 (0.71.3) zdv + 3tc + efv0.8 (0.51.1)0.8 (0.51.3)adherence defined by mpr.in adjusted models, tenofovir - based regimens were not included due to relatively small number of patients who received these drugs (n = 636) and deaths documented among this group (n = 2).art = antiretroviral therapy; d4 t = stavudine; zdv = zidovudine; 3tc = lamivudine; nvp = nevirapine; efv = efavirenz . Post-12-month mortality by adherence category for art - nave adults initiating art in lusaka, zambia, between april 1, 2004 and adherence was measured by the number of days a patient had antiretroviral drugs available according to pharmacy refill data . The numbers shown at the bottom of the graph represent the number of patients active in each adherence category at 6-monthly intervals . Perfect survival in the first 12 months is a result of our study design . To be eligible for inclusion in this analysis, patients had to remain alive, active, and on art until at least 12 months factors associated with post-12-month mortality among treatment - nave adults initiating art in lusaka, zambia, from april 1, 2004 to september 30, 2007 and continuing art for> 12 months adherence defined by mpr . In adjusted models, tenofovir - based regimens were not included due to relatively small number of patients who received these drugs (n = 636) and deaths documented among this group (n = 2). Art = antiretroviral therapy; d4 t = stavudine; zdv = zidovudine; 3tc = lamivudine; nvp = nevirapine; efv = efavirenz . While trends in cd4 response, haemoglobin response and weight gain appeared similar for individuals in the optimal and suboptimal adherence categories, outcomes generally appeared worse among those with poor adherence (figure 3). When compared with individuals with 80% adherence, for example, those with poor adherence appeared to have an attenuated cd4 response at 18 months (185 vs 217 cells/l; p <0.001), 24 months (213 vs 246 cells/l; p <0.001), 30 months (226 vs 261 cells/l; p <0.001) and 36 months (245 vs 275 cells/l; p <0.01). Those with <80% mpr in their first 12 months also had attenuated increases in weight and haemoglobin at 18 months (5.0 vs 5.8 kg; p <0.001 and 1.7 vs 2.0 g / dl; p <0.001), 24 months (5.5 vs 6.0 kg; p <0.05 and 1.7 vs 2.0 g / dl; p <0.001), 30 months (5.8 vs 6.3 kg; p <0.17 and 1.8 vs 2.1 g / dl; p <0.01) and 36 months (5.6 vs 6.4 kg; p <0.06 and 2.0 vs 2.1 g / dl; p = 0.35). Figure 3immunologic and clinical responses by adherence category for art - nave adults initiating art in lusaka, zambia, between april 1, 2004 and september 30, 2007, and remaining on art for> 12 months . These include (a) cd4 lymphocyte count, (b) haemoglobin and (c) weight immunologic and clinical responses by adherence category for art - nave adults initiating art in lusaka, zambia, between april 1, 2004 and september 30, 2007, and remaining on art for> 12 months . These include (a) cd4 lymphocyte count, (b) haemoglobin and (c) weight from april 1, 2004 to september 30, 2007, 37 039 art - nave adults (> 15 years of age) initiated treatment across 18 sites in lusaka, zambia . Of these, 27 115 (73.2%) remained active in the program at or after 12 months and were thus included in the analysis . A total of 9924 (26.8%) were excluded from the analysis because they had left the program before reaching the 12 month threshold: 1133 withdrew from the program, 5114 were late for patient visits and 3677 had died (crude mortality rate = 105.5/100 person - years, 95% ci: 102.1108.9). A comparison of those included and excluded from the analysis is shown in table 1 . Table 1characteristics of treatment - nave adults initiating art in lusaka, zambia, from april 1, 2004 to september 30, 2007 and continuing art for> 12 monthsincluded in analysisexcluded from analysisnvaluenvaluepage, median years (iqr)27 11535 (3041)992434 (2940)<0.001 1525271810.0%128512.9%<0.001 263512 15944.8%452645.6% 3645863631.8%288429.1% 4655287510.6%9019.1% 567272.7%3283.3%sex female16 88962.3%556656.1%<0.001 male10 22637.7%435843.9%adherence supporter no546920.2%328933.1%<0.001 yes21 64679.8%663566.9%cd4 lymphocyte count, median cells/l (iqr)26 068132 (69198)9409110 (48191)<0.001 200 cells/l635824.4%212122.5%<0.001 50199 cells/l15 27458.6%485551.6% <50 cells/l443617.0%243325.9%who stage i or ii874132.5%233023.8%<0.001 iii15 78958.6%605761.8% iv23988.9%142014.5%haemoglobin, median g / dl (iqr)23 79610.9 (9.612.3)837010.2 (8.711.8)<0.001 8.0 g / dl21 99592.4%712285.1%<0.001 <8.0 g / dl18017.6%124814.9%bmi, median kg / m (iqr)24 25020.0 (18.122.3)835818.9 (16.921.2)<0.001 20 kg / m12 14750.1%307836.8%<0.001 1819 kg / m614825.4%201524.1% 1617 kg / m425817.6%193023.1% <16 kg / m16977.0%133516.0%tuberculosis co - infection at enrollment no23 48886.6%856986.3%0.49 yes362713.4%135513.7%initial art regimen d4 t + 3tc + nvp10 69939.7%469148.0%<0.001 zdv + 3tc + nvp12 99648.3%350135.8% d4 t + 3tc + efv15235.7%8758.9% zdv + 3tc + efv10263.8%4234.3% tdf + ftc + nvp2290.9%981.0% tdf + ftc + efv4481.7%1942.0%adherence by mpr, median (iqr)27 11597.9 (91.2100.0)9924100.0 (92.6100.0)<0.00l 95100%17 06062.9%705171.0%<0.00l 8094%768228.3%141214.2% <80%23738.8%146114.7%for those included in the analysis, adherence by mpr was based on behaviour within the first 12 months from art initation . For those excluded because they were not active at 12 months, mpr was based on time from art initiation to censor date . Art = antiretrovinal therapy; d4 t = stavudine; zdv = zidovudine; 3tc = lamivudine; nvp = nevirapine; efv = efavirenz . Characteristics of treatment - nave adults initiating art in lusaka, zambia, from april 1, 2004 to september 30, 2007 and continuing art for> 12 months for those included in the analysis, adherence by mpr was based on behaviour within the first 12 months from art initation . For those excluded because they were not active at 12 months, mpr was based on time from art initiation to censor date . Art = antiretrovinal therapy; d4 t = stavudine; zdv = zidovudine; 3tc = lamivudine; nvp = nevirapine; efv = efavirenz . Of the 27 115 included in this analysis, 1419 (5.2%) withdrew from the program, 3067 (11.3%) were late for their most recent scheduled visit, and 823 (3.0%) were known to have died (crude post-12 month mortality rate = 2.1/100 person - years, 95% ci: 2.0, 2.3) after the initial 12-month period . The median observation period was 15.7 [interquartile range (iqr) = 7.526.4] months, from 12 months onward . Of 27 115 patients, 17 060 (62.9%) demonstrated optimal adherence, 7682 (28.3%) had suboptimal adherence and 2373 (8.8%) had poor adherence over the first 12 months on therapy (figure 1). Individuals with an mpr <80% were more likely to have a higher median cd4 count at enrollment than those with optimal adherence . When compared with those with optimal adherence, individuals in the 8094 and <80% mpr were generally younger and were less likely to report adherence support (i.e. Adherence the proportion of individuals with tuberculosis at time of art initiation was lower among individuals with suboptimal adherence (12.2 vs 14.0%; p <0.001) when compared with those with optimal adherence . The proportion of patients who withdrew from the program or were lost to follow - up after 12 months increased as adherence declined categorically (table 2). Figure 1distribution of adherence to art over the first 12 months on treatment among adults surviving to at least 12 months in lusaka, zambia, between april 1, 2004 and september 30, 2007 . Adherence was measured by the number of days a patient had antiretroviral drugs available according to pharmacy refill data table 2characteristics by adherence category for treatment - nave adults initiating art in lusaka, zambia, from april 1, 2004 to september 30, 2007 and continuing art for> 12 monthsoptimal (95100%)sub - optimal (8094%)poor (<80%)nvaluenvaluepnvaluepage, median years (iqr)17 06035 (3041)768234 (2941)0.03237333 (2840)<0.001 152516019.4%78210.2%0.1233514.1%<0.001 2635762044.7%345845.0%108145.6% 3645552532.4%244131.8%67028.2% 4655186310.9%78510.2%2279.6% 564512.6%2162.8%602.5%sex female10 73062.9%468561.0%<0.01147462.1%0.46 male633037.1%299739.0%89937.9%adherence supporter reported at enrollment14 05782.4%594377.4%<0.001164669.4%<0.001cd4 lymphocyte count, median cells/l (iqr)16 469132 (70196)7350129 (68198)0.592249139 (73207)<0.001 200 cells/l394323.9%180424.5%0.6061127.2%<0.01 50199 cells/l969958.9%429358.4%128257.0% <50 cells/l282717.2%125317.0%35615.8%who stage i or ii553232.6%243632.0%0.1977332.9%0.81 iii995658.7%446758.7%136658.1% iv14738.7%7139.4%2129.0%haemoglobin, median g / dl (iqr)15 18610.9 (9.612.3)665710.9 (9.612.3)0.42195311.0 (9.512.4)0.49 8.0 g / dl14 06092.6%615492.4%0.71178191.2%0.03 <8.0 g / dl11267.4%5037.6%1728.8%bmi, median kg / m (iqr)15 35620.0 (18.122.3)684620.0 (18.022.2)0.34204819.9 (18.022.3)0.53 20 kg / m772550.3%341849.9%0.42100449.0%0.75 1819 kg / m390925.5%170324.9%53626.2% 1617 kg / m265317.3%124118.1%36417.8% <16 kg / m10697.0%4847.1%1447.0%tuberculosis co - infection at enrollment238214.0%93712.2%<0.00130813.0%0.19initial art regimen d4 t + 3tc + nvp673139.7%304239.9%0.0792639.7%0.24 zdv + 3tc + nvp822248.5%365547.9%111947.9% d4 t + 3tc + efv9435.6%4405.8%1406.0% zdv + 3tc + efv6283.7%2983.9%1004.3% tdf + ftc + nvp1671.0%490.6%130.6% tdf + ftc + efv2711.6%1411.8%361.5%lost to follow - up or withdrawn after 12 months229413.4%140418.3%<0.00178833.2%<0.001defined at 12 months of therapy as the percentage of time on therapy with art via pharmacy claims.based on comparisons to the optimal adherence category.art = antiretroviral therapy; d4 t = stavudine; zdv = zidovudine; 3tc = lamivudine; nvp = nevirapine; efv = efavirenz . Distribution of adherence to art over the first 12 months on treatment among adults surviving to at least 12 months in lusaka, zambia, between april 1, 2004 and september 30, 2007 . Adherence was measured by the number of days a patient had antiretroviral drugs available according to pharmacy refill data characteristics by adherence category for treatment - nave adults initiating art in lusaka, zambia, from april 1, 2004 to september 30, 2007 and continuing art for> 12 months defined at 12 months of therapy as the percentage of time on therapy with art via pharmacy claims . Based on comparisons to the optimal adherence category . Art = antiretroviral therapy; d4 t = stavudine; zdv = zidovudine; 3tc = lamivudine; nvp = nevirapine; efv = efavirenz . Individuals who were> 35 years [odds ratio (or): 0.8; 95% ci: 0.70.9] or reported presence of an adherence buddy (or: 0.5; 95%ci: 0.50.6) were less likely to have mpr <80% . When compared with those with cd4 count> 200 cells/l, those with lower baseline cd4 counts were less likely to be poorly adherent over the first 12 months: 50199 cells/l (or: 0.9, 95% ci = 0.81.0) and <50 cells/l (or: 0.8, 95% ci: 0.70.9). Baseline world health organization (who) clinical stage, haemoglobin, body mass index (bmi) and tuberculosis status were not predictive of poor adherence (data not shown). In kaplan meier analysis, individuals with <80% adherence had a significantly higher risk of death when compared with those in other adherence categories (log rank p = 0.0001). In unadjusted proportional hazards regression, the relative hazard for death was 1.7 (95% ci: 1.42.1) among those with poor adherence, when compared with those with optimal adherence . These findings remained virtually unchanged in a multivariable model controlling for sex, age, adherence support, baseline cd4 lymphocyte count, baseline who stage and initial regimen dispensed [n = 25 836; adjusted hazard ratio (ahr): 1.7; 95% ci: 1.42.2]. Baseline haemoglobin and bmi were not included in this initial multivariable model, since early versions of our patient tracking software did not routinely collect these data . In a separate model that included these two parameters (n = 20 951), the hazard for mortality associated with poor adherence did not change appreciably (table 3). Figure 2post-12-month mortality by adherence category for art - nave adults initiating art in lusaka, zambia, between april 1, 2004 and september 30, 2007 . Adherence was measured by the number of days a patient had antiretroviral drugs available according to pharmacy refill data . The numbers shown at the bottom of the graph represent the number of patients active in each adherence category at 6-monthly intervals . Perfect survival in the first 12 months is a result of our study design . To be eligible for inclusion in this analysis, patients had to remain alive, active, and on art until at least 12 months table 3factors associated with post-12-month mortality among treatment - nave adults initiating art in lusaka, zambia, from april 1, 2004 to september 30, 2007 and continuing art for> 12 monthscrude hazard ratio (95% ci)adjusted hazard ratio (95% ci)adherence optimal (95100%)1.01.0 suboptimal (8094%)1.0 (0.91.2)0.9 (0.81.1) poor (<80%)1.7 (1.42.1)1.7 (1.42.3)age 35 years1.01.0> 35 years1.4 (1.21.6)1.4 (1.21.6)sex female1.01.0 male1.4 (1.21.6)1.3 (1.11.5)adherence supporter no1.01.0 yes0.7 (0.60.8)0.7 (0.60.9)cd4 count 200 cells/l1.01.0 50199 cells/l1.3 (1.01.5)1.2 (1.01.6) <50 cells/l1.9 (1.52.3)1.5 (1.22.0)who stage i or ii1.01.0 iii1.6 (1.41.9)1.4 (1.11.7) iv2.5 (2.03.2)2.0 (1.52.7)haemoglobin 8.0 g / dl1.01.0 <8.0 g / dl2.0 (1.62.5)1.6 (1.22.0)bmi 16 kg / m1.01.0 <16 kg / m2.2 (1.82.8)1.8 (1.42.2)initial art regimen d4 t + 3tc + nvp1.01.0 zdv + 3tc + nvp0.7 (0.60.8)0.7 (0.60.9) d4 t + 3tc + efv1.1 (0.81.4)0.9 (0.71.3) zdv + 3tc + efv0.8 (0.51.1)0.8 (0.51.3)adherence defined by mpr.in adjusted models, tenofovir - based regimens were not included due to relatively small number of patients who received these drugs (n = 636) and deaths documented among this group (n = 2).art = antiretroviral therapy; d4 t = stavudine; zdv = zidovudine; 3tc = lamivudine; nvp = nevirapine; efv = efavirenz . Post-12-month mortality by adherence category for art - nave adults initiating art in lusaka, zambia, between april 1, 2004 and september 30, 2007 . Adherence was measured by the number of days a patient had antiretroviral drugs available according to pharmacy refill data . The numbers shown at the bottom of the graph represent the number of patients active in each adherence category at 6-monthly intervals . Perfect survival in the first 12 months is a result of our study design . To be eligible for inclusion in this analysis, patients had to remain alive, active, and on art until at least 12 months factors associated with post-12-month mortality among treatment - nave adults initiating art in lusaka, zambia, from april 1, 2004 to september 30, 2007 and continuing art for> 12 months adherence defined by mpr . In adjusted models, tenofovir - based regimens were not included due to relatively small number of patients who received these drugs (n = 636) and deaths documented among this group (n = 2). Art = antiretroviral therapy; d4 t = stavudine; zdv = zidovudine; 3tc = lamivudine; nvp = nevirapine; efv = efavirenz . While trends in cd4 response, haemoglobin response and weight gain appeared similar for individuals in the optimal and suboptimal adherence categories, outcomes generally appeared worse among those with poor adherence (figure 3). When compared with individuals with 80% adherence, for example, those with poor adherence appeared to have an attenuated cd4 response at 18 months (185 vs 217 cells/l; p <0.001), 24 months (213 vs 246 cells/l; p <0.001), 30 months (226 vs 261 cells/l; p <0.001) and 36 months (245 vs 275 cells/l; p <0.01). Those with <80% mpr in their first 12 months also had attenuated increases in weight and haemoglobin at 18 months (5.0 vs 5.8 kg; p <0.001 and 1.7 vs 2.0 g / dl; p <0.001), 24 months (5.5 vs 6.0 kg; p <0.05 and 1.7 vs 2.0 g / dl; p <0.001), 30 months (5.8 vs 6.3 kg; p <0.17 and 1.8 vs 2.1 g / dl; p <0.01) and 36 months (5.6 vs 6.4 kg; p <0.06 and 2.0 vs 2.1 g / dl; p = 0.35). Figure 3immunologic and clinical responses by adherence category for art - nave adults initiating art in lusaka, zambia, between april 1, 2004 and september 30, 2007, and remaining on art for> 12 months . These include (a) cd4 lymphocyte count, (b) haemoglobin and (c) weight immunologic and clinical responses by adherence category for art - nave adults initiating art in lusaka, zambia, between april 1, 2004 and september 30, 2007, and remaining on art for> 12 months . These include (a) cd4 lymphocyte count, (b) haemoglobin and (c) weight we investigated art adherence in an extremely busy, public sector art program in sub - saharan africa . When optimal adherence was defined as mpr 95%, 60% met this criterion . Less than 10% met criteria for poor adherence (mpr <80%); however, we observed poor clinical and immunologic response among individuals in this group . Although routine viral load data are not available in our setting, the 80% mpr threshold appears to hold some significance for crude measures of treatment response such as mortality and cd4 lymphocyte count improvement . Mpr and mpr - like measurements for adherence (e.g. Pharmacy refills, timeliness to clinical appointments) have been closely associated with virologic and clinical outcomes among patients on art . We observed increased mortality after 12 months among those with adherence <80%: a finding similar to other studies in the published literature . For example, hogg and colleagues found that intermittent use of art (defined as drug refills of <75%) was associated with a nearly 3-fold risk in mortality in british columbia . Nachega et al . Found that adherence to antiretroviral drug refills of <80% was associated with a 3-fold increase for death in the south african private sector . The risk for adverse outcomes observed in this cohort was of a slightly lesser magnitude when compared with other published series . To better understand the impact of adherence on later outcomes, we included only individuals enrolled in the program for> 12 months . Thus, there is selection bias towards longer term survivors, regardless of drug adherence behaviour . This is most evident in the baseline characteristics of the population, where those in the lower adherence stratifications had somewhat higher median cd4 lymphocyte counts . The attenuated effect may be a reflection of the higher baseline mortality risk in zambia regardless of immune status compared with canada or south africa . As in other studies, we observed an association between adherence and cd4 lymphocyte response . Individuals with adherence of <80% had an attenuated response at 18, 24, 30 and 36 months when compared with those at or above this mpr threshold . Haemoglobin and weight (usually described through bmi) are other clinical measures shown to be independently predictive of mortality among patients initiating art . The post-12-month mortality rate observed in this cohort (2.1 deaths/100 patient - years) was slightly higher than what has been reported among hiv - uninfected individuals in africa (0.60.8 deaths/100 patient - years), and well below that of hiv - infected individuals without access to treatment (1113 deaths/100 patient - years). Our analysis cohort comprised individuals who were likely healthier than the general population initiating art, having survived beyond periods of highest mortality risk (i.e. First 90 days on art) to at least 12 months . Although systems are in place to follow up those with missed visits, nearly 10% were late for appointments or withdrew altogether in the post-12-month period, a scenario common to other sub - saharan programs . If a significant proportion of follow - up losses were related to death a distinct possibility given the local tradition of returning to the rural family home at times of severe illness then this would lead to underestimated mortality . For individual patient care, we recognize that the utility of our findings may be limited . We focused on a survival outcome due to constraints of our programmatic database, but the ability to predict virologic suppression may be more relevant for clinical decision making . Bisson and colleagues, for example, demonstrated that pharmacy refill - based adherence measures may perform better than cd4 monitoring to predict virologic failure; overall performance of adherence, however, still led to significant misclassification of patient outcomes (i.e. Missed virologic failure, unnecessary regimen change) across several different thresholds . We observed similar findings among patients with discordant clinical and immunologic treatment responses in lusaka, suggesting that mpr measurements alone may be insufficient to predict virologic suppression . Instead, it should either be considered as a triaging tool or as part of more extensive algorithms in predicting virologic failure . In addition, while 95% adherence threshold is commonly referenced, recent studies have shown that even moderate levels (8090%) of adherence can result in viral suppression when combination regimens are used . Establishment of adherence thresholds that reliably predict virologic treatment failure and viral drug resistance could greatly assist individual patient care in resource - constrained settings . In contrast, our demonstrated association between mpr and mortality could provide an important framework for program monitoring in african settings . In lusaka, for example, we currently utilize a programmatic database to generate facility - level reports for clinical care quality and performance . An aggregate mpr metric could be generated for a given facility and serve as a clinic - level performance indicator . Sites with a high proportion of patients with poor adherence (e.g. <80%) could be identified and evaluated for potential interventions incorporating patient reminders, community education or adherence support . This feedback loop between patient outcomes and service improvement is a critical link to maintaining a high standard of clinical care, particularly in settings where hiv services are expanding rapidly and task shifting is employed to relieve critical health personnel shortages . Aggregate data could also assist policy makers and program managers at the district, provincial and national level, as they tailor specific interventions to optimize treatment . To our knowledge, this is the largest african cohort in the published literature evaluated for adherence, owing to the unprecedented resources made available through the zambian government, the global fund for aids, tuberculosis and malaria; and the us president's emergency plan for aids relief . We believe the pharmacy data to be complete because of the free antiretroviral drugs provided by the zambian ministry of health (thus reducing the probability of drug collection at non - study sites), and standardized data capture across all sites . One limitation of this analysis is our reliance on mpr; in many ways, it is a crude measure of adherence . Although simple to calculate, it does not describe the actual ingestion of medication or the pattern of non - adherence (e.g. Frequency, duration). Patient outcomes in this urban population may also not be representative of those in rural areas, where a growing number of programs are now expanding in africa . Further work is needed to understand the role of adherence measurements such as mpr in such settings . Lastly, we were unable to fully account for the impact of food supplementation programs that were slowly rolled out in lusaka over the observation period . Recent work demonstrated a clear impact of food supplementation on individual - level adherence; however, immunologic and clinical outcomes were unchanged at 6 and 12 months following art initiation . Whether food supplementation might have an impact on survival is unknown . In summary, we demonstrate that mpr correlates with patient outcomes in the settings of rapid service scale - up, task shifting and high disease burden . Our findings are thus applicable to the many urban public sector programs in africa seeking to expand services in the face of significant resource constraint . Although 80% mpr appears to be an important threshold for clinical outcomes and survival, its utility for individual patient care may be limited . Mpr thresholds that correlate with virologic failure and viral drug resistance should be delineated, given their direct relevance to clinical care . Centers for disease control and prevention (cooperative agreement u62/ccu12354) through the president's emergency plan for aids relief, and a grant for operations research for aids care and treatment in africa from the doris duke charitable foundation (2005047). Additional investigator salary or trainee support is provided by the national institutes of health (k01-tw06670; k23-ai01411; p30-ai027767; d43-tw001035); doris duke clinical scientist development award (2007061). The findings and conclusions included herein are solely the responsibility of the authors and do not necessarily represent the official position of the centers for disease control and prevention.
Nowadays, anterior ankle arthroscopy is routinely used for the treatment of the anterior ankle impingement syndrome and talar osteochondral defects [9, 27], although the technique can be used for a wide variety of ankle pathology . The anterolateral portal in anterior ankle arthroscopy is created lateral to the peroneus tertius tendon or, if not present, lateral to the extensor digitorum longus tendon . The superficial peroneal nerve or its terminal branches, mainly the intermediate dorsal cutaneous nerve, is at risk with regard to this routine anterolateral portal [4, 9, 10, 18, 19, 23] (fig . A. anatomical relations of the anterolateral portal . B. anatomical relations of the anteromedial portal 1 lateral malleolus, 2 medial malleolus, 3 talus, 4 tibialis anterior tendon, 5 extensor hallucis longus tendon, 6 extensor digitorum longus and peroneus tertius tendons, 7 peroneus brevis tendon, 8 peroneus longus tendon, 9 achilles tendon, 10 tibialis posterior tendon, 11 flexor digitorum longus, 12 flexor hallucis tendon (musculotendinous), 13 deep peroneal nerve and anterior tibial artery and veins, 14 medial dorsal cutaneous nerve (medial terminal branch of superficial peroneal nerve), 15 intermediate dorsal cutaneous nerve (lateral terminal branch of superficial peroneal nerve), 16 posterior tibial nerve and posterior tibial artery and veins, 17 sural nerve and small saphenous vein, 18 saphenous nerve and great saphenous vein, 19 posterior peroneal artery and 20 anterior peroneal artery transverse section at the level of the ankle joint . A. anatomical relations of the anterolateral portal . B. anatomical relations of the anteromedial portal 1 lateral malleolus, 2 medial malleolus, 3 talus, 4 tibialis anterior tendon, 5 extensor hallucis longus tendon, 6 extensor digitorum longus and peroneus tertius tendons, 7 peroneus brevis tendon, 8 peroneus longus tendon, 9 achilles tendon, 10 tibialis posterior tendon, 11 flexor digitorum longus, 12 flexor hallucis tendon (musculotendinous), 13 deep peroneal nerve and anterior tibial artery and veins, 14 medial dorsal cutaneous nerve (medial terminal branch of superficial peroneal nerve), 15 intermediate dorsal cutaneous nerve (lateral terminal branch of superficial peroneal nerve), 16 posterior tibial nerve and posterior tibial artery and veins, 17 sural nerve and small saphenous vein, 18 saphenous nerve and great saphenous vein, 19 posterior peroneal artery and 20 anterior peroneal artery the superficial peroneal nerve provides motor innervation to the peroneus longus and brevis muscles and passes along the lateral intermuscular septum, subsequently perforating the crural fascia to function as a sensory nerve to the vast majority of the dorsal foot (fig . 2) after fascia perforation, this nerve is usually divided into its terminal branches; the medial dorsal cutaneous and intermediate dorsal cutaneous nerve [12, 14]. The level at which the superficial peroneal nerve divides into its terminal branches relatively to the level of the ankle joint was subject to numerous anatomical studies and different classification types [1, 5, 14, 19, 24]. The superficial peroneal nerve, and if divided, its terminal branches are the only nerves in the human body that can be made visible (fig . 3). Preoperative identification of this nerve should assist in preventing iatrogenic damage during foot and ankle surgery, rapid localization prior to local anaesthetic blocks, external fixator applications as well as surgical decompressions .fig . 2anatomical dissection of the cutaneous nerves of the foot and ankle . 1 superficial peroneal nerve, 2 fascial piercing of the superficial peroneal nerve, 3 superficial peroneal nerve before piercing the crural fascia, 4 anterior compartment of the leg, 5 lateral compartment of the leg, 6 medial dorsal cutaneous nerve (medial terminal branch of superficial peroneal nerve), 7 intermediate dorsal cutaneous nerve (lateral terminal branch of superficial peroneal nerve), 8 lateral dorsal cutaneous nerve (terminal branch of sural nerve), 9 sural nerve (the saphenous vein was removed), 10 medial calcaneal nerve (branco or sural nerve), 11 common nerves digital of medial dorsal cutaneous nerve (medial terminal branch of superficial peroneal nerve), 12 cutaneous branch (medial terminal branch) of deep peroneal nerve, 13 superior extensor retinaculum, 14 inferior extensor retinaculum, 15 tip of lateral malleolus, 16 inferior peroneal retinaculum, 17 achilles tendon and 18 tuberosity of the v metatarsal bonefig . 3using the combined ankle plantar flexion and inversion position, the superficial peroneal nerve (the intermediate dorsal cutaneous nerve or lateral terminal branch of superficial peroneal nerve) could be visualized in 3 out of the 10 examined ankles (30%) anatomical dissection of the cutaneous nerves of the foot and ankle . 1 superficial peroneal nerve, 2 fascial piercing of the superficial peroneal nerve, 3 superficial peroneal nerve before piercing the crural fascia, 4 anterior compartment of the leg, 5 lateral compartment of the leg, 6 medial dorsal cutaneous nerve (medial terminal branch of superficial peroneal nerve), 7 intermediate dorsal cutaneous nerve (lateral terminal branch of superficial peroneal nerve), 8 lateral dorsal cutaneous nerve (terminal branch of sural nerve), 9 sural nerve (the saphenous vein was removed), 10 medial calcaneal nerve (branco or sural nerve), 11 common nerves digital of medial dorsal cutaneous nerve (medial terminal branch of superficial peroneal nerve), 12 cutaneous branch (medial terminal branch) of deep peroneal nerve, 13 superior extensor retinaculum, 14 inferior extensor retinaculum, 15 tip of lateral malleolus, 16 inferior peroneal retinaculum, 17 achilles tendon and 18 tuberosity of the v metatarsal bone using the combined ankle plantar flexion and inversion position, the superficial peroneal nerve (the intermediate dorsal cutaneous nerve or lateral terminal branch of superficial peroneal nerve) could be visualized in 3 out of the 10 examined ankles (30%) arthroscopic surgery, as compared to open surgical approaches, offers the advantage of direct and magnified visualization of the anatomical structures, less postoperative morbidity, faster and functional rehabilitation, earlier sport resumption, and the possibility of outpatient treatment [8, 10, 20]. Despite these obvious advantages, complications do occur . The overall complication rate varies in between 8 and 17% [2, 4, 6, 811, 13, 15, 17, 18, 20], of which iatrogenic superficial peroneal nerve damage, related to anterolateral portal placement, is reported most frequently [4, 10, 18]. Reported an overall complication rate of 9%, of which iatrogenic superficial peroneal nerve damage accounts for 27% . Good anatomical knowledge, the use of vertical incisions only affecting the skin parallel to the tendons and neurovascular structures with subsequent blunt dissection up to the level of the joint have been postulated [9, 12]. Furthermore, minimal endurance of distraction and tourniquet inflation within a protocol ensuring routine portal placement and usage of proper arthroscopic instrumentation are other considerations [9, 10, 16, 18, 22, 24]. Preoperatively, the superficial peroneal nerve, or its terminal branches, can be made visible with ankle plantar flexion and inversion, while preoperatively it can be visualized with cutaneous transillumination [9, 21, 24, 25]. If visible, it is recommended to mark the course of the nerve on the skin preoperatively . It is noteworthy that despite improved arthroscopic techniques and the fact that the superficial peroneal nerve can be visualized transcutaneously, damage to this nerve remains the most frequently reported complication in anterior ankle arthroscopy [4, 10, 18]. Therefore, the purpose of this study was to perform an anatomical study to determine the course of the superficial peroneal nerve in different positions of the foot and ankle to possibly explain and clarify this rather contradiction, and eventually to diminish future complication rates . Ten lower legs (five left and five right), amputated approximately 15 cm below the knee, from seven men and three women fresh frozen specimens were carefully examined in the department of pathology and experimental therapeutic unit of human anatomy and embryology in barcelona, spain . The specimens were from the caucasian origin with a median age of 77(range 6590 years). Specimens were excluded in case of deformities or scars following ankle surgery and/or limited range of motion . The superficial peroneal nerve with its surrounding soft tissue was fixed with a mosquito clamp at the amputation level . An experienced anatomist (pg) determined the course of the superficial peroneal nerve, or if divided its terminal branches, by creating a window (20 mm wide and 15 mm long), only affecting the skin without manipulating the nerve . The localization of the window was at the level of the anterior joint line, lateral to the peroneus tertius tendons or if not present, lateral to the extensor digitorum longus tendon . The ankle was subsequently moved from 10 plantar flexion and inversion to the neutral position (90) and then to 5 dorsiflexion to determine whether the course of the nerve changed . The reference point was regarded being the combined plantar flexion and inversion position . In this position, the medial edge of the nerve was marked with a pin (0.5 mm). Possible nerve movements, relatively to the reference point, were recorded with a digital caliper (australian measuring instruments 0200 mm). Manipulating the ankle in the different ankle positions was standardized by means of the telos stress device (telos equipment, weiterstadt, germany) (fig . The course of the superficial peroneal nerve, or if divided its terminal branches, was determined by creating a window (20 mm wide and 15 mm long), only affecting the skin without manipulating the nerve . Manipulating the ankle in the different ankle positions was standardized by means of the telos stress device (telos equipment, weiterstadt, germany). The reference point was regarded being the combined plantar flexion and inversion position . In this position, the medial edge of the nerve was marked with a pin (yellow in the photography) photograph showing the used method in the study . The course of the superficial peroneal nerve, or if divided its terminal branches, was determined by creating a window (20 mm wide and 15 mm long), only affecting the skin without manipulating the nerve . Manipulating the ankle in the different ankle positions was standardized by means of the telos stress device (telos equipment, weiterstadt, germany). The reference point was regarded being the combined plantar flexion and inversion position . In this position, the medial edge of the nerve was marked with a pin (yellow in the photography) subsequently, the ankle was positioned in the neutral position (90) to assess whether the course of the superficial peroneal nerve, or its terminal branches, changed with manual tension on the skin covering the lateral aspect of the ankle, plantar flexion of the 4th toe and plantar flexion of all the toes . Each of the measurements was performed by three independent observers, and inter - observer reliability was assessed through an intraclass correlation coefficient (icc). An one sample t test was performed to determine whether the nerve movement was significantly different from 0 (p <0.05). Each of the measurements was performed by three independent observers, and inter - observer reliability was assessed through an intraclass correlation coefficient (icc). An one sample t test was performed to determine whether the nerve movement was significantly different from 0 (p <0.05). The superficial nerve or its terminal branches consistently moved to lateral while the ankle was moved from 10 plantar flexion and inversion to the neutral and then to the 5 dorsiflexion position . From 10 plantar flexion and inversion to the neutral position, the average nerve movement was 2.4 mm to the lateral side (sd 0.9, range 0.954.19). The standardized ankle movement from 10 plantar flexion and inversion to the 5 dorsiflexion resulted in an mean nerve movement of 3.6 mm to the lateral side (sd 1.8, range 1.387.68). Both displacements to the lateral side were significant (p <0.01) (table 1).table 1general specimen characteristics with superficial peroneal nerve movement (mm) from combined 10 plantar flexion and inversion to neutral and to 5 ankle dorsiflexionspecimenagegendersuperficial peroneal nerve movement (mm) from 10 plantar flexion and inversion toneutral5 dorsiflexionobserver 1observer 2observer 3averageobserver 1observer 2observer 3average1791.791.921.961.893.743.623.383.582831.641.571.681.631.892.091.811.933761.381.461.631.491.501.591.381.494710.951.000.990.981.951.911.751.875653.223.583.193.333.253.453.863.526812.492.462.372.443.623.553.333.507903.132.963.093.066.096.186.126.138722.042.322.332.233.273.253.023.189672.602.542.402.513.333.293.493.3710783.983.924.194.037.497.397.687.52median77average2.363.61sd0.921.97icc (intraclass correlation coefficient)0.980.99one sample t test (p <0.05)<0.01<0.01 general specimen characteristics with superficial peroneal nerve movement (mm) from combined 10 plantar flexion and inversion to neutral and to 5 ankle dorsiflexion the inter - observer reliability coefficients (icc) for the measurement of nerve displacement for both ankle movements were 0.98 (95% ci; 0.9530.998) and 0.99 (95% ci; 0.9800.998), respectively . With the ankle in the neutral position the superficial peroneal nerve or its terminal branches did not move while applying tension on the skin, flexion of the 4th toe or flexion of all toes . The most important finding of the present study is that the anatomical orientation of the superficial peroneal nerve consistently changes by altering the ankle position . The superficial peroneal nerve, or if divided the intermediate dorsal cutaneous nerve, and the medial dorsal cutaneous nerve, are the only nerves in the human body that can be made visible in the majority of humans . Numerous manoeuvres have been described to visualize the superficial peroneal nerve or its terminal branches [3, 7, 9, 13, 17, 21, 24]. These were introduced to attempt avoidance of iatrogenic damage during anterolateral portal placement, external fixator applications, surgical decompressions and rapid localization prior to local anaesthetic blocks . One of these manoeuvres includes combined plantar flexion and inversion of the ankle [9, 17, 24]. The possibility to visualize the superficial peroneal nerve has clinical implications in ankle surgery (open and arthroscopically) and in local regional anaesthetic techniques . As described, different manoeuvres were introduced to visualize this nerve, being able to mark its course preoperatively, with the aim of preventing complications . Despite the possibility to visualize the nerve, complication rates in ankle arthroscopy are still relatively high, ranging from 817%, mostly regarding the superficial peroneal nerve and especially the intermediate dorsal cutaneous nerve [2, 4, 6, 811, 13, 15, 17, 18, 20]. One of the possible limitations of our research was that the specimens were amputated below the knee . The present investigation concerns the movement of the superficial peroneal nerve in different foot and ankle positions, in below the knee amputated specimens . We assumed that with the amputation the normal nerve anatomy might was disturbed, and for this reason the nerve was fixed to its surrounding soft tissue with a mosquito clamp . However, we hypothesize that the amputation did not have a major influence on our results, since from proximal to distal the nerve divides several times to innervate the peroneal muscles . Marking the superficial peroneal nerve in ankle plantar flexion and inversion provides a false sense of safety when making the anterolateral portal in the normal or slight dorsiflexion position, since the marking does not correspond to the subcutaneous position of the nerve in this position . When the ankle is moved from plantar flexion and inversion to the neutral position (90), the nerve consistently moves lateral relatively to the marking . If the anterolateral portal is made medial to the marking, this will prevent iatrogenic damage to the superficial peroneal nerve . Placing the anterolateral portal lateral to the marking places the nerve at risk to iatrogenic damage . The current study shows that the superficial peroneal nerve consistently moves lateral when the ankle is manoeuvred from combined plantar flexion and inversion to the neutral or dorsiflexed position . If visible, it is therefore advised to create the anterolateral portal medial from the preoperative marking, in order to prevent iatrogenic damage to the superficial peroneal nerve.
Marine sponges (porifera) have been prolific sources of many interesting bioactive substances and are still an important resource for marine natural product chemistry . In the course of our studies on biologically active natural products of marine organisms, we have recently isolated four new melophlins p (1), q (2), r (3), and s (4) together with seven known melophlins a (5), d (6), e (7), g (8), h (9), i (10), and o (11) from two marine sponges of the genus melophlus collected in palau . Melophlins are tetramic acid derivatives possessing a long alkyl chain and have been isolated from melophlus sarasinorum [13]. Compounds 111, except 8, showed weak to modest growth inhibitory activity against a murine leukemia cell line l1210 . Melophlins a (5) and b (possessing the c11 alkyl chain and two methyl groups at c-5 and c-9) were reported to have moderate cytotoxicity against hl-60 and arrest nih3t3 fibroblasts in the g1 phase of the cell cycle . Furthermore, melophlins a and b were found to reverse the morphology of h - ras - transformed hih3t3 cells to normal and might act on the components of the ras - mediated signal transduction pathway . Therefore, we intended to investigate the influence of 11 compounds (111) on the colony formation of chinese hamster v79 cells . We also observed the effect of 11 compounds (111) on the production of an inflammatory cytokine, il-8, in pma - stimulated hl-60 cells since the inhibition of cell proliferation sometimes correlates with il-8 production . In this paper, we would like to describe some structure - activity relationships of these compounds against v79 and l1210 cells . Melophlins were isolated from two marine sponges of the genus melophlus collected in palau as described previously . Dimethylsulfoxide (dmso) was purchased from pierce chemical co. (rockfield, il), and fetal bovine serum (fbs) was obtained from gibco after checking the lot . Chinese hamster v79 cells were grown as a monolayer culture in eagle s mem (nissui seiyaku co., ltd ., the hl-60 cell line was obtained from the japanese cancer research resources bank (jcrb, kamiyoga, tokyo, japan) and maintained in tissue culture dishes in rpmi 1640 medium (nissui seiyaku, tokyo, japan) supplemented with 10% heat - inactivated fbs, 2 mm glutamine, 100 u / ml of penicillin g, and 100 g / ml of streptomycin . The relative plating efficiencies against v79 cells were determined as the ratio of the number of colonies in various concentrations of samples to that in the sample - free control, as described in previous papers . Two hundred cells were seeded onto a 60/15-mm plastic plate with 4 ml culture medium and incubated overnight at 37c . After each sample in dmso (4 l) was added to the culture medium, cells were further cultured for four days . The numbers of colonies in the sample plates were counted and compared with those in the control cultures . The il-8 concentrations of the culture supernatants under control and various test conditions were measured by elisa using a combination of monoclonal and polyclonal antibodies . Melophlins were isolated from two marine sponges of the genus melophlus collected in palau as described previously . Dimethylsulfoxide (dmso) was purchased from pierce chemical co. (rockfield, il), and fetal bovine serum (fbs) was obtained from gibco after checking the lot . Chinese hamster v79 cells were grown as a monolayer culture in eagle s mem (nissui seiyaku co., ltd ., the hl-60 cell line was obtained from the japanese cancer research resources bank (jcrb, kamiyoga, tokyo, japan) and maintained in tissue culture dishes in rpmi 1640 medium (nissui seiyaku, tokyo, japan) supplemented with 10% heat - inactivated fbs, 2 mm glutamine, 100 u / ml of penicillin g, and 100 g / ml of streptomycin . The relative plating efficiencies against v79 cells were determined as the ratio of the number of colonies in various concentrations of samples to that in the sample - free control, as described in previous papers . Two hundred cells were seeded onto a 60/15-mm plastic plate with 4 ml culture medium and incubated overnight at 37c . After each sample in dmso (4 l) was added to the culture medium, cells were further cultured for four days . The numbers of colonies in the sample plates were counted and compared with those in the control cultures . The il-8 concentrations of the culture supernatants under control and various test conditions were measured by elisa using a combination of monoclonal and polyclonal antibodies . Melophlin a (5) has been reported to inhibit the cell proliferation of hl-60 at the ic50 value of 0.2 g / ml (0.57 m). This compound arrested nih3t3 fibroblast cells in the g1 phase of the cell cycle at 1 g / ml (2.85 m) and reversed the morphology of h - ras transformed nih3t3 cells to normal at 5g / ml (14.2 m). On the other hand, melophlins c, e (7), g (8), h (9), i (10), m, n, and o (11) were not cytotoxic to hl-60, hela, or tf-1 cells . In our experiment, compounds 17 and 911 showed weak to modest inhibitory activity against l1210 cells (table 1); therefore, we examined the effect of compounds 111 on the rate of colony formation using v79 cells . Since the production of an inflammatory cytokine, il-8, is, in some cases, correlated with the inhibition of cell proliferation, we also observed the influence of 11 compounds on the il-8 production system mediated by pma - stimulated hl-60 cells . Although the inhibitory activities of the 11 compounds on the colony formation of v79 were not remarkable, melophlins h (9) and o (11) showed activity at ec50 values of 8.5 and 9.6 m, respectively (table 1); however, compounds 111 did not affect the production of il-8 . Linear - chain - type compounds (1, 5, 6, and 8) were least active among the four chain types (table 1). The methyl group at c-5 reduced the activity of linear - chain - type and iso - type compounds (2, 7, and 9), while the activity of anteiso - type compounds (3, 10, and 11) were increased by c-5 methylation . Activity was decreased as the chain length increased for linear - chain - type compounds . On the other hand, activity was increased as the chain length decreased for iso - type and anteiso - type compounds . These structure - activity relationships were detected against both v79 and l1210 cells except for the iso - type compound with the c-5 methyl group (compound 2), which showed stronger activity against l1210 cells than compound 9 . The activity of the 10-methyl compound (4) against v79 and l1210 cells was stronger than that of the iso - type compound (2) and weaker than that of the anteiso - type compound (3). Therefore, it will be interesting to examine the in vitro growth inhibitory activity of compounds possessing iso - type or anteiso - type chains with a chain - length of less than c12 against human solid tumor cell lines.
Recently, esthetic requirements in dental restorative treatment are on the rise . For this reason, recent attention - drawing material is zirconia which is a biocompatible material with stable structure . Flexural strength and fracture toughness of zirconia reaches up to 900 - 1200 mpa and 9 - 10 mpam1,2 respectively, and zirconia has been proven to be much more excellent in its strength than that of glass infiltrated full porcelain which is widely used in esthetic restoration.1,2 material properties of zirconia like these show that zirconia can be substituted for metal and can be surely used even in long span fixed prosthesis.3,4 cad / cam system which was introduced to dentistry in 1980's has brought cost and time reduction in fabricating restoration by using computer for inputting, designing and cutting the form of restoration . In order to overcome disadvantage of previous method of framework fabrication in which post - sintered zirconia block was cut, pre - sintered zirconia block is being used recently . By doing so, fabrication time and consumption of bur this method, however, has disadvantage in that approximately 15 - 30% shrinkage occur in the procedure of sintering and hardening the cut block using cam unit for the increased density of zirconia block.5 when using partially sintered block, therefore, it is more important to calculate shrinkage rate accurately than any other things in order to compensate shrinkage which inevitably occurs in sintering process, and also to gain precise product when using partially sintered block . It is considered that internal and marginal fit plays a key role in longevity of prosthesis . In previous studies, many authors said that 120 m or less marginal gap of prosthesis was clinically acceptable.6,7 according to a study on fit of zirconia framework which was used in all ceramic crown, marginal gap was reported to be 64 - 83 m,8,9 and in another study on clinical fit of 3-unit fixed prosthesis, zirconia prosthesis (lava, 3 m espe, seefeld, germany) for which cad / cam was used reportedly showed 80 m of mean marginal gap.10 the reason why marginal fit is important is because increased marginal leakage may cause secondary caries, periodontitis, pulpitis etc . And there was a report from one previous study saying the thicker the cement became due to a large internal gap, the weaker the porcelain became.11 this effect is shown also in zirconia . Excessive thickness of cement increases radial crack12,13 and causes fracture of veneering porcelain.14 fracture of veneering porcelain is considered to be one of the greatest reasons for removing zirconia restoration.15 - 18 as opposed to this, too small internal gap may cause unstable seating of prosthesis.19 abduo et al.20 said that factors which influenced fit of prosthesis were fabrication system of zirconia, veneering, configuration, span length of zirconia etc . Although the use of zirconia has increased and are being used more in multiple areas where the tooth are missing, studies on fit of zirconia fixed partial denture until the now have been limited to 3-unit fixed partial denture with nearly linear form; there has been few study on internal or marginal fit of 4 or more unit fixed partial denture with curved form.20 particularly, there was no study in which both internal and marginal fit depending on the span length were simultaneously evaluated . The purpose of this study, therefore, was to measure marginal and internal fit of single, 4-unit, 6-unit zirconia fixed partial denture core which had been fabricated using cad / cam system by using replica technique and to evaluate the effect that span length on fit while evaluating whether measured marginal gap was in clinically acceptable range or not . Experimental groups were divided into single, 4-unit and 6-unit groups, and preparation of each abutment teeth were done in order to make single crown of upper right central incisor, 4-unit fixed partial denture with abutment teeth of upper right and left lateral incisor where upper right and left central incisors were missing, and 6-unit fixed partial denture with abutment teeth of upper right and left canines where upper right, left central and lateral incisors were missing (table 1). For tooth preparation, surveyor and diamond bur were used, and total taper of each abutment teeth was intended to be 6. preparation amount was 2 mm on incisal part and 1 mm on axial wall, and 1 mm width deep chamfer margin was formed . In order to prevent possible wear or fracture of master models from repetitive impression taking procedure, dentiformmodels (nissin dental prod . Japan) where abutment preparation were done were duplicated and titanium master model weremade (addtech co., seoul, korea) (fig . 1). Impression of titanium model were taken with conventional method by using addition silicone impression material (imprint ii, 3 m espe, st . Paul, mn, usa) and readymade tray, and total 30 improved stone (fuji rock ep, gc corp, tokyo, japan) models including 10 models per each group were made (fig . 2). After scanning of the manufactured plaster model using cad / cam system, partially sintered zirconia blocks were cut and sintered, and finally zirconia cores were fabricated (fig . 3). When fabricating cores, cad / cam system of orapix (seoul, korea) and zirconia blocks were used . Thickness of core and cement space were set as 0.6 mm and 40 m respectively . Internal adjustment was not done so that fit of core that was fabricated by only cad / cam could be merely evaluated . After core fabrication was done, whether there were remnants inside, defects or distortions were checked and then cleaned with high pressure steaming . After positioning fabricated zirconia cores on each models, jigs for positioning the zirconia coreon the model were made using pattern resin (gc dental, japan) so that zirconia could always be positioned at a same spot on the models . After filling fit checking material (fit checker, gc dental, japan) in inner surface of core after that, pre - made resin jig was positioned above the core . While maintaining adapted position by hand pressure, it was place in universal testing machine (shimadzu corporation, kyoto, japan) immediately . Universal testing machine was set to measurement mode, and its compressive force was limited to 40 n, and regular force (40 n) was maintained for 5 minutes until fit checker was completely hardened . When doing so, the silicon film of fit checker has to be fully attached to silicone core . Filling inner surface of silicone film of core with regular bodied addition silicone impression material (aquasillv, densply caulk, usa) and hardening the filled material increase strength andalso enables gaining of stable film layer . Each 8 measuring points were established for labial and mesio - distal side respectively (fig . 4), and cutting was done right in the center of the model labialy and mesio - distally (fig . 5). By using measuring microscope (axio, carl zeiss, rochester, ny, usa) and i - solution (imt i - solution inc ., vancouver, bc, canada), thickness of fit checker was measured at 16 measuring points of each abutment teeth (fig . Mean value was documented after 3 times measured by 2 experimenter for each measuring points . Mean value and standard deviation value were calculated by adding measurement values of right and left abutment teeth of each group which was divided into bucco - lingual and mesio - distal . Whether there was statistically significant difference between groups which were divided into bucco - lingual and mesio - distal or not was analyzed by multivariate analysis . After that, if there had been statistically significant difference, post - hoc test was done using dunnett t3 test per each measuring points . In all analyses, twenty seven specimens including nine single, nine 4-unit and nine 6-unit were used in fit measurement . There existed 1 specimen per each group of which measurement could not be done because fracture had occurred in the process of applying force after putting fit checker inside the core and placing it on abutment . Statistical analysis was performed by adding each measurement values of right and left abutment teeth of each group . By doing so, number of specimen of each group became nine single, and eighteen 4-unit and eighteen 6-unit . Mean value and standard deviation value of gap at bucco - lingual measuring point were calculated (table 2). As a result of multivariate analysis, statistically significant difference was shown at point 2, 4, 7 and 8 . In order to investigate between which groups of point 2, 4, 7 and 8 showed the difference, post - hoc test was performed using dunnett t3 test (table 3). Mean value and standard deviation value of gap at mesio - distal measuring point were calculated (table 4). The result of anova showed statistically significant differences at point 2, 4, 7 and 8 by span length factor . In order to investigate between which groups of point 2, 4, 7 and 8 showed the difference, post - hoc test was performed using dunnett t3 test (table 5). As a result of measuring point analysis which showed statistically significant difference, measurement value increased from single, 4-unit and 6-unit in order in point 2, 4, 7 when bucco - lingual analysis was done . But in point 8, the gap of 4-unit group was measured to be the smallest . In the result of mesio - distal analysis, measurement value increased from single, 6-unit and 4-unit in order in point b, zirconia is an esthetical and biocompatible material, and its physical property is so strong that it can be used for fixed prosthesis of posterior region . Because of its property like this, it has become one of the most noteworthy materials in esthetic prosthetics . In order for zirconia to be used as prosthetic restoration material that can last long and function stably, not only its physical property but also the fit of prosthesis made by it should meet clinical requirements . In fabrication of zirconia prosthesis using cad / cam, completely sintered, this method has disadvantages in time and cost although fit could be more excellent than when partially sintered block which is used in most fabrication system is used . On the other hand, if partially sintered block is used, it will be better off when it comes to time and cost factor . However, if cutting is not done by accurate calculation due to shrinkage in sintering process, level of fit will decrease significantly . According to previous studies, 120 m or smaller marginal gap is clinically accepted.6,7 in the studies on fit of single or 3-unit fixed partial denture zirconia prosthesis fabricated by cad / cam system, marginal gap showed approximately 64 - 83 m as a result.8,9 these show that zirconia can be used for fixed partial denture restoration adequately . However, these were the studies which had been mostly proceeded on linear type fixed prosthesis which was shorter than 3-unit . Study on prosthesis of which span is longer than this has not been done.10 in this study, therefore, whether fit of zirconia prosthesis is influenced by the span length was investigated using anterior teeth model . As methods for measuring marginal or inner fit, a method of direct inspection, a method of inspection after cutting, a method of evaluation by impression taking, a method of evaluation using explorer and so on although a method of inspection after cutting may be the most accurate way, there is a shortcoming in that more specimens have to be made for the increase of the number of measurement . Therefore, replica technique where measurement of fit of various parts could be done easily with only small number of specimens was used in this study . This method had been considered to have low accuracy in the past,21 but it became known to be reliable compared to other methods for measuring fit by rahme et al.22 and laurent et al.23 study . Specimens for measurement were made using replica technique, and thickness of fit checker was measured at 16 measuring points which were designated per each abutment teeth, and 2 experimenters recorded mean value after 3 times of measurements so that error could be minimized . As a result of measurement, overall fit was acceptable (small gap) in marginal and axial wall area, but connection part between margin and axial wall and incisal part showed relatively large gap . Because it was considered that whether it was either right abutment tooth or left abutment tooth could not be a variable for analysis, mean value and standard deviation were calculated by adding measurement values of right and left abutment teeth together for statistical analysis in 4-unit and 6-unit group . In the case of this study, multivariate analysis was used because it was considered that gap of inner surface and margin could not be independent to each other although independent variable was a span length . As a result of statistical analysis of bucco - lingual (1 - 8) and mesio - distal (a - h) measuring points, there were statistically significant differences at point 2, 4, 7, 8 and b, d, e, f, g among the groups . From this result, the fact that span length influenced fit of zirconia core was confirmed . As a result of analysis of each measuring points which showed statistically significant difference, measurement value increased from single, 4-unit to 6-unit in order at 2, 4, 7 point, but measurement value of 4-unit group came out smallest at 8 point . As opposed to this, 4-unit group showed the largest value at b, f, g point as a result of mesio - distal analysis . This is considered to be because of small number of specimens that were used in the experiment and measurement error as well . Also, there were more points which showed significant difference inmesio - distal part than inbucco - lingual part . It is guessed that the reason for this is because more error occurred in the process of shrinkage at mesio - distal part since the form of zirconia core was not bucco - lingually but mesio - distally long . Although there was not enough number of specimen for more accurate multivariate analysis in statistical analysis process, kruskal - wallis test was performed to compensate it, and same result as that of multivariate analysis was gained . It can be said that the meaning of this study lays in the fact that evaluation of effect of span length on fit in fabrication of prosthesis . However, the number of specimen was not enough for securing statistical significance which is the weak point of this study . Also, if fit of prosthesis had been measured after veneering, the study would have been closer to clinical setting . In some specimens, measurement was difficult because boundary of silicone film and impression material was blurred . For more accurate measurement, this method needs improvement . Also, in case of fixed partial denture, fit is not something that is independent to one another . Therefore, it is considered that comparative evaluation on volume will be more necessary than to just observe some part after cutting it . Also, if analysis such as micro ct24 and so on is additionally used, appearance of transformation or distortion in shrinkage process will be able to be evaluated . The further studies to investigate the difference of fit among the different manufacturers with increased number of specimens will be necessary . And change of the span length influenced marginal fit and internal fit to some degree . In single or 4-unit fixed partial denture group, mean value of marginal fit was within clinically acceptable range . In 6-unit group, however, some margins showed values that were out of clinically acceptable range . Therefore, it was analyzed that the increase of the span length could possibly decrease fit between zirconia core and abutment tooth when fixed partial denture was 6-unit or longer.
Only a few cases have been reported, being described for the first time by kersting and helming . Though the majority of eccrine spiradenomas involve the head, trunk, and extremities, they can also appear all throughout the skin, mostly as painless solitary nodules . Our case report is a malignant eccrine spiradenoma (mes / spiradenocarcinoma) diagnosed in an asymptomatic woman on the basis of breast cancer prevention programme . Because of its rare incidence and lack of imaging workups our aim is therefore to discuss the main characteristics of it, and to review the literature available on this issue . A 48-year - old woman, asymptomatic, with no medical history of interest, presented in a mammography made as part of a breast cancer screening programme a lesion in the left upper - breast . 1) demonstrated a well - defined, isodense nodule located in the upper - outer quadrant of the left breast . Longitudinal and transverse scans with colour doppler images were obtained . The images (fig . 2) showed a well - defined lobulating mass measuring 18 13 15 mm with homogeneous hypo echogenicity . In the colour doppler sonography a histologic examination of these showed a characteristic biphasic population of outer small cells with darkly staining nuclei surrounding larger cells with pale cytoplasm without epidermal connection . Intraoperative radiologic control of the excised lesion showed integrity of the nodule with broad resection margins . Macroscopically the sample measured 4 2.5 cm in which on serial sectioning a 2 1.5 cm white nodule was noted . Microscopic examination showed cells arranged in ragged sheets, nests, cords, and solid masses along with occasional irregular glandular structures . Benign areas, was seen as sharply demarcated lobules composed of 2 cell populations (peripheral basaloid cells and central pale cells) arranged in nests with focal cystic changes . Adjoining to this 2-cell population focus (gradually or abruptly) proliferation cells with histopathological malignant characteristics transited to solid masses of a homomorphous large cell population with hiperchromatic and pleomorphic nuclei, eosinophilic cytoplasm (fig . Inmunohistochemically, both cellular areas exhibited p63 and were positive for smooth muscle actin (sma). Focal reactivity for ema and cea was found in the central, large, pale cells . Luminal cells expressed muc-1, ck 7, ema, cea, ck cam5.2 and gcdfp-15 . Around 4050% of the cells composing the carcinomatous area showed positivity to ki-67 (fig . S-100, ck 20, vimentin, cd3, cromogranin and ck34betae12, estrogen / progesterone receptors were not expressed . These features were consistent with a low - grade mes noted to be arised from a pre - existing benign long - standing eccrine spiradenoma . The sentinel lymph node retrieved from axillary dissection was submitted for intraoperative histological examination obtaining a negative result for metastatic carcinoma . Metastatic workup detection (bone scan, computed tomographic scans of the chest, abdomen, and pelvis) was also negative . The patient has been seen in follow - up every 36 months and has had yearly sonographic examinations . After 32 months of follow - up, there is no evidence of recurrent or metastatic disease . Eccrine spiradenomas are usually benign, well - defined sweat - gland neoplasms that affect both men and women, showing no sex predilection (male to female ratio 1:1). Age at presentation is variable, but most commonly arising at an age of 50 . Spiradenoma lesions tend to arise on the head; neck; trunk; and, less commonly, the arms followed by the legs . However, the development of neoplasms with eccrine sweat gland phenotype in the mammary ducts is not surprising in as much as the breast is considered a modified sweat gland . Since the original report, 102 cases of mes have been documented in the literature . Of these, only six are described affecting the breast . There are only three cases, including ours, diagnosed as malignant transformation in the breast (table 1). Although, four of the five cases of breast spiradenomas were benign, 2 of these were reported to suffer malignant transformation, and one carcinosarcomatous conversion . Reviewing the reports, the lesions usually show a typical history of a long - standing (often decades); unchanged cutaneous solitary nodule that suddenly becomes enlarged . The average size of malignant spiradenomas at presentation is 3.9 cm (range 0.515 cm) in diameter, but usually 12 cm as in our patient . It generally begets medical attention when a pre - existing undiagnosed lesion rapidly enlarges, changes colour, ulcerates, or becomes painful and tender . The imaging findings of breast eccrine spiradenomas have not been clearly demonstrated because of the rare incidence of eccrine spiradenomas and the lack of imaging workup . The findings reported were: round or oval shape, well - demarcated masses hypo, or hyper - dense in mammography . The us findings of the current case were characterized as well - circumscribed, oval - shaped, hypoechoic masses, surrounded by echogenic lines that represent the dermal layer . Reported the first description of the sonographic findings of a mes in 1993 as a hypoechoic, heterogeneous lesion . Mri, ct, and radiography can be used to assess the presence of metastatic foci in the case of malignant spiradenoma . Although, an eccrine spiradenoma may resemble an epidermal inclusion cyst on mammography and us, these may be identified on the basis of mri findings . The mri findings of epidermal inclusion cysts and eccrine spiradenomas are different [810] and so they might be used in making the differential diagnosis . Diagnosis of mes is based on histopathologic examination and requires finding a focus of benign spiradenoma within or adjacent to the malignant tumour . Nowadays, high - grade tumours show multiple foci of florid squamous differentiation, including proliferation of solid masses consisting of large cells with hyperchromatic nuclei, loss of pas - positive basement membrane, sarcomatous cell metaplasia and surrounding cell reaction . The mesenchymal elements are non - specific and are often described as rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, and chondrosarcoma . On the other hand, in the absence of a benign focus, tumours can be confused with other skin malignancies . The diagnosis of these is much more difficult and requires finding benign focus on histopathology and an inmunohistochemical myoepithelial benign profile in consonance with the finding of two cell populations . In our case, histologically proliferation of cells with hyperchromatic pleomorphic vesicular nuclei, variably prominent nucleoli, increased mitoses, invasion of surrounding membrane and adipose mammary tissue characterized malignant transformation . Inmmunohistochemically, the cells express immunophenotypes similar to mioepithelial cells with a variable expression of p63 and sma, and ductal differentiation cells expressing cea, ema, ck7, muc-1, ckcam5.2 y gcdfp-15 . S-100 protein positivity in basal epithelial cells has been demonstrated, but was not evidenced in our case . We also identified ki-67 and d1-cyclin expression, as a result of a low - grade differentiation tumour . While the inmmunohistochemical profile of benign or malignant eccrine spiradenoma is not diagnostic of an eccrine neoplasm, it is helpful in elucidating the cellular differentiation and narrowing the differential diagnosis . Radiation and hyperthermic chemotherapy can also be administered as medical treatment to prevent focal recurrence . In our case, malignant eccrine spiradenoma metastasizes (40%) to regional lymph nodes, bone, lungs, brain, and liver (in descending order of frequency). Distant metastases portend an ominous prognosis, but if possible, resection should be done . We did not implement an axillary dissection, as sentinel node biopsy turned out negative . Due to the high risk of developing metastases, close follow up of these patients for early detection of recurrence should be carried out . Malignant eccrine spiradenoma metastasizes (40%), so a close follow up is recommended . The authors declare that there is not any source of funding for this surgery case report . Written informed consent was obtained from the patient for publication of this case report and accompanying images . Francisco villalba ferrer, andrs garca vilanova, carlos fuster diana, jose medrano gonzlez: surgery, follow up and surgical comment on the clinical case.
Upper and lower limb innervation is greatly affected by brachial and lumbosacral plexus lesion, leading to loss of motor and sensory function [17]. In the neonate, such injuries are even more harmful, since surgical treatment is limited and neuronal loss is particularly enhanced by neurotrophic factors deprivation . Nonexistent or limited handling of neonatal proximal lesions results in limb dysfunction and neuropathic pain [812]. Experimentally, a well - accepted model to mimic axotomy injury retrograde repercussion to spinal neurons is the neonatal peripheral nerve axotomy [1318]. Sciatic nerve transection, early after birth, results in significant degeneration of spinal motoneurons as well as sensory neurons present in the dorsal root ganglia . Also, a number of interneurons, present in deep spinal cord laminae, degenerate due to the loss of input and/or output . Importantly, exogenous treatment with neurotrophins transiently rescues a significant number of neurons, leading to the possibility of axonal regrowth and regeneration [19, 20]. To date, neonatal peripheral nerve repair following neurotmesis is largely limited due to technical drawbacks . In fact, to perform end - to - end coaptation by epineural / perineural suturing in the newborn represents a major challenge . On the contrary, reconnection of transected stumps by using biocompatible sealants may significantly facilitate the process, as well as taking the advantage of employing the adhesive as scaffold to engraft stem cells, as well as neurotrophic substances, to the injury site . Fibrin sealant has been applied in neurosurgery for decades, being effective and biocompatible, with no side effects to the nervous system microenvironment . Together, when activated, their interaction mimics the coagulation cascade, forming a stable and adhesive clot . Regard, much effort by the center for the study of venoms and venomous animals (cevap) has been made to produce a new fibrin sealant that uses a serine protease from a brazilian snake (crotalus durissus terrificus) with thrombin - like activity . Also, in order to avoid human blood, bubaline - derived blood serum cryoprecipitate has been used as the fibrinogen source . Among several advantages, the nonhuman fibrin glue hampers transmission of hepatitis and hiv / aids [2327]. Also, it has a customizable formulation that can be adjusted to increase or decrease fibrin clot formation and stability . Although anatomical repair of spinal roots and other lesioned plexus components constitute the primary approach, additional strategies are necessary to enhance neuroprotection and to improve the regenerative response of severed neurons . This may be achieved by stem cell therapy that has shown particularly important positive effects following nervous system injury [2837]. In this regard, we have already shown evidence that mononuclear cell therapy produced neuroprotection, preservation of synapses in the spinal cord, and reduction of glial reaction following dorsal and ventral horn adult lesion [38, 39]. The present work investigated the viability and efficiency of neonatal sciatic nerve end - to - end coaptation, performed with the application of fibrin sealant . We show that both commercial and customized nonhuman fibrin adhesives perform equally well and allow motor and sensory recovery up to 12 weeks after injury . Nerve stumps coaptation was performed by using either a commercially available fibrin sealant - tissucol (baxter, vienna, austria) or a patented fibrin sealant derived from snake venom, kindly supplied by the center for the study of venoms and venomous animals (cevap) of unesp (patent registration numbers br1020140114327 and br1020140114360). At the time of use, the components were previously thawed, reconstituted, mixed, and applied [2427, 31]. Two - day - old (p2) neonatal lewis rats were anesthetized by hypothermia and subjected to unilateral sciatic nerve transection at mid - thigh level . Animals were divided into three groups: axotomy alone (ax, n = 30), in which a 2 mm segment of the proximal stump was resected in order to assure absence of contact between stumps; axotomy followed by end - to - end cevap's fibrin sealant coaptation (ax+fs, n = 30); axotomy followed by end - to - end commercial fibrin sealant coaptation (ax+cfs, n = 30). An overview of the surgical procedure is illustrated in supplementary figure s1 (in supplementary material available online at http://dx.doi.org/10.1155/2016/9028126). An additional nonoperated control group was specifically utilized for the motor test evaluation (n = 5). Also, in order to evaluate the retrograde degeneration of motoneurons following nerve transection without proximal stump resection, an additional axotomy control group was evaluated following stump reapposition (ax - sr; n = 5). Both fibrin sealants used herein are composed of three separate solutions: (1) fibrinogen, (2) calcium chloride, and (3) thrombin or thrombin - like (in the case of the serine protease derived from snake venom). During surgical repair, the first two components were applied and the proximal and distal stumps were approximated in an end - to - end fashion . The sciatic nerve was gently shifted to assure the stability of the coaptation and to evaluate the success of the repair . The institutional committee for ethics in animal (ceua / unicamp) and the center of experimental and ethical animal (ceea / unesp) approved all experiments (proc . Number 2593 - 1 and 904 - 2011, resp . ), which were performed in accordance with the guidelines of the nih and the brazilian college for animal experimentation . The animals were housed using a 12 h light / dark cycle and controlled temperature (23c), with free access to food and water . Animals were anaesthetized with an overdose of kensol (xylasine, kning, argentina, 10 mg / kg) and vetaset (ketamine, fort dodge, usa, 50 mg / kg), and the vascular system was rinsed by transcardial perfusion with phosphate buffer 0.1 m (ph 7.4). For neuronal survival counting and the immunohistochemical evaluation, subjects (n = 5 for each group) were fixed by vascular perfusion of 10% formaldehyde in phosphate buffer (ph 7.4). The lumbar intumescence was dissected out, postfixed overnight, washed in phosphate buffer, and stored in sucrose 10, 20, and 30% for 24 h each, before freezing . Transverse cryostat sections (12 m thick) of the lesioned spinal cord segments (l4, l5, and l6) were obtained, transferred to gelatin coated slides, and stored at 22c until use . For sciatic nerve regeneration analysis, the rats were transcardially perfused with 200 ml of a fixative containing 2.5% glutaraldehyde and 1.0% paraformaldehyde in 0.1 m phosphate buffer (ph 7.4). The sciatic nerve ipsi and contralateral to the lesion were removed and stored overnight in the same fixative at 4c . The specimens were then osmicated, dehydrated, and embedded in durcupan (fluka acs, steinheim, switzerland). The resin blocks were trimmed and semithin sections (0.5 m) were obtained and stained with 0.7% sudan black in 70% of ethanol solution for morphometric analysis . Counting of motoneurons survival was performed on spinal cord sections (n = 5 for each group, ipsi and contralateral ventral horn, 4, 8, and 12 weeks after lesion). Cryostat transverse sections were stained for 35 min in aqueous 1% cresyl fast violet solution at room temperature . The sections were then washed in distilled water, dehydrated, and mounted with entellan (merck, darmstadt, germany). The motoneurons were identified based on their morphology and location in the ventral horn (dorsolateral lamina ix). Only cells with visible nucleus were counted for every twentieth section (totalizing an interval of 240 m between sections) along the lumbar enlargement, both on the ipsilateral and contralateral sides of each spinal cord . The percentage of surviving cells was analyzed by the ratio of absolute numbers of motoneurons, counted per section, on the lesioned and nonlesioned sides, respectively, and multiplying the result by 100 . Abercrombie's formula was used to correct the duplicate counting of neurons: n = nt/(t + d), where n is the corrected number of neurons counted, n is the number of cells counted, t is the thickness of the sections (12 m), and d is the average diameter of neuronal nuclei . Since the difference in size significantly affects cell counts, the value of d was calculated specifically for each experimental group (ipsilateral and contralateral). In this sense, the nuclear diameter of 15 randomly picked neurons from each group was measured with imagej software (version 1.33, national institutes of health, usa) and the mean value was calculated . Slides containing transverse spinal cord sections were incubated for 45 min in a 3% bsa solution followed by incubation with the following primary antibodies: mouse anti - synaptophysin (dako, glostrup, denmark 1: 200) rabbit anti - gfap (abcam, 1: 1500), and rabbit anti - iba1 (waco, 1: 700). The primary antibodies were diluted in a solution containing 1% bsa (bovine serum albumin) and all sections were incubated for three hours in a moist chamber at room temperature . After rinsing in pb, the sections were incubated with a cy3-conjugated secondary antiserum (1: 250, jackson immunoresearch, west grove, pa, usa) for 45 min in a moist chamber at room temperature . The sections were then rinsed in pb, mounted in a mixture of glycerol / pb (3: 1), and observed with a leica dm5500b microscope coupled with a leica dfc345 fx camera . For quantitative measurements, three representative images of the ipsilateral and contralateral ventral horn were captured from each animal for all experimental groups (n = 5 for each group, ipsi and contralateral ventral horn, 4, 8, and 12 weeks after lesion). For analysis of synaptophysin immunolabeling, the integrated density of pixels was measured in eight areas surrounding each lateral motor nucleus motoneuron, in the anterior horn of the spinal cord . For the analysis of anti - glial fibrillary acidic protein (gfap) and anti - ionized calcium binding adaptor molecule 1 (iba1) antibodies, the integrated density of pixels was measured in the lateral region of the spinal cord ventral horn, as described by oliveira et al . And freria et al . The integrated density of pixels was calculated for each section and then as a mean value for each spinal cord . The data are represented as the mean standard error (se) for each group . Morphometry, regenerated axon area, and counting analyses were performed by sampling at least 30% of each nerve cross - section (magnification of 1,000x) using a bright field microscope (n = 5 for each group, ipsi and contralateral ventral horn, 4, 8, and 12 weeks after lesion). Sampling bias was avoided by spreading the micrographs systematically over the entire cross - section, according to the procedure proposed by mayhew and sharma . These images were used for counting the total number of myelinated fibers . For each specimen, two fields were used to measure the perimeter and diameter of myelinated fibers and the myelin area, using adobe photoshop cs6 extended . These values were used to calculate the myelinated axon diameter, fiber diameter, myelin thickness (fiber diameter, axon diameter/2), and g ratio (axon diameter / fiber diameter) that indicate the relative success of the regeneration process as well as myelination process . Lumbar spinal cords (n = 3 for each group, ipsi and contralateral ventral horn, 4 weeks after lesion) were dissected out and stored overnight in fixative at 4c . The specimens were then trimmed, osmicated, dehydrated, and embedded in durcupan (fluka). Ultrathin sections from the l4l6 segments were collected on formvar coated copper grids, counterstained with 2% uranyl acetate and lead citrate, and examined under a transmission electron microscope operating at 120 kv (tecnai biotwin g2 spirit, fei company, the netherlands). Neurons with large cell bodies (~35 m in diameter), found in the sciatic motoneuron pool and cut in the nuclear plane, were identified as -motoneurons by the presence of c - type nerve terminals and chromatolysis (axotomized side). The cell surfaces were then sequentially digitalized at a magnification of 11,000x with an eagle 2k video camera (fei company, the netherlands) connected to a computer system, and the images mounted together using vector graphics software . Synaptic terminals opposing the motoneuron somata were identified and their numbers per 100 m of cell membrane, as well as the membrane covering of all the terminals (calculated in percentage of membrane length), were determined using the measurement tool of image j software (version 1.33u, national institutes of health (nih), usa). The terminals were typed as f - type (flattened synaptic vesicles or flattened and spherical vesicles that contain glycine / gamma - aminobutyric acid (gaba) as neurotransmitter), s - type (spherical synaptic vesicles that contain glutamate as the neurotransmitter), or c - type (presence of a subsynaptic cistern and acetylcholine as the neurotransmitter), according to the procedure described by conradi in 1969 (supplementary figure s2). The distance between consecutive nerve terminals covering the motoneurons a total of 24 sciatic -motoneurons from three animals per group (two neurons per animal in four groups, ax, ax+fs, ax+cfs, and contralateral) were quantified . Motor function was analyzed using the peroneal functional index (pfi) by the walking track test (catwalk system, noldus inc . A high speed video camera gevicam (gp-3360, usa) equipped with a wide - angle lens (8.5 mm, fujicon corp ., china) is positioned underneath the walkway and the paw prints are automatically recorded and classified by the software . The paw prints from each animal were obtained twice a week from the third week of life . Pfi was calculated as the distance between the third toe and hind limb pads (print length) and the distance between the first and fifth toes (print width). Measurements of these parameters were obtained from the right (unlesioned) and left (lesioned) paw prints, and the values were calculated using the following formula described by bain and colleagues in 1989: (1)pfi=174.9eplnplnpl+80.3etsntsnts13.4,where n: normal or nonoperated side; e: experimental or operated; pl: print length; ts: total toe spread, or distance between first to fifth toes ., ribeiro preto, sp, brazil) was utilized to evaluate sensory function recovery (n = 5 for each group). For that, the rats were kept inside acrylic boxes for twenty minutes before the experiment for habituation . Nociception threshold was measured at the plantar region by touching the skin with a pipette tip adapted to a force transducer, which is connected to a digital counter that shows the force, in grams, used to trigger the paw flinch . Hyperalgesia was measured by stimulating the paw surface for six times, and the results were averaged and shown as a mean se . In order to avoid foot pad lesion and inflammation, the maximum stimulation weight (cut - off threshold) was set to 70 g. each data collection was carried out in duplicate and in a blinded manner . The data are presented as mean se, and the differences between groups were considered significant when the p value was <0.05 (), <0.01 (), and <0.001 (). Statistical analysis was performed with graphpad prism 4.0 software . In this sense, data were subjected to anova followed by bonferroni post hoc test for parametric data or mann - whitney u test for nonparametric data . Neuronal survival was investigated as the ipsi / contralateral ratio of motoneurons present at ventral horn lamina ix . After 4, 8, and 12 weeks, a severe degeneration motoneuron was observed in the ax group . Coaptation groups performed equally well and presented statistically significant rescue of axotomized motoneurons (figure 1, table s1). No significant differences between the numbers of motoneurons on the contralateral side in the different experimental conditions were observed . Additionally, axotomy alone with immediate reapposition of the stumps and followed by 2 mm proximal stump resection did not differ with regard to motoneuron survival (supplementary figure s3). Quantitative measurements of synaptophysin immunoreactivity in the sciatic motor nuclei after axotomy (ax) and after axotomy followed by coaptation (ax+fs; ax+cfs) were carried out 4, 8, and 12 weeks after injury . Axotomy group showed significant reduction of immunoreactivity ipsilateral to the lesion side . On the contrary, synaptic covering was preserved following coaptation in all survival times analyzed (figure 2, supplementary data table s2). A significant increase in astrocyte reactivity after axotomy was observed four weeks after lesion (figure 3, supplementary data table s3). Similar results were observed in microglial reactivity, where groups with coaptation showed decreased microglial reaction, particularly at four weeks after lesion (figure 4, supplementary data table s4). Although absence of spontaneous regeneration following axotomy alone was observed, acute coaptation (ax+fs, ax+cfs) resulted in axonal growth with sensorimotor recovery . Area measurements showed no significant difference between ax+fs and ax+cfs groups, although such nerves presented reduced dimensions as compared to the contralateral (nonlesioned) samples (table 1). Additionally, the estimation of total number of axons revealed no significant differences between coaptation groups (figure 5, supplementary data table s5), although significantly reduced in comparison to the normal nerve . Of note, commercial fibrin adhesive displayed the worst values for all parameters analyzed at four weeks after lesion, indicating slower axonal regeneration progress (figures 6, 7, and 8). Such initial difference was not present eight and twelve weeks after lesion, so that both coaptation groups performed in a similar fashion at later stages . Statistical differences between groups were only observed at four weeks after sciatic nerve coaptation . In this sense, ax+fs displayed better recovery as compared to ax+cfs . Such statistical evaluation is presented in detail as supplementary material: figure s4, fiber diameter; figure s5, axon diameter; and figure s6, myelin thickness . No statistical differences were depicted regarding the g ratio (figure 9), although cfs group presented mostly under myelinated fibers four weeks after injury . All analyzed motoneurons showed at least one cholinergic presynaptic terminal (type c) in apposition to the plasmatic membrane surface . Lesioned neurons, affected by axotomy, showed changes compatible with chromatolysis, such as displacement of the nucleus to the periphery and decrease in cytoplasm electron density . Glial projections were observed, in the ax group, intermingling the space between part of the presynaptic terminals and the postsynaptic membrane (figure 10(a)). Fully apposed terminals were depicted in the other experimental groups (figures 10(b)10(d)). The total synaptic covering, which represents the motoneuron body surface in contact with presynaptic terminals, revealed reduction of excitatory terminal covering in all groups analyzed, when compared to the contralateral side (figure 10(e)). Four weeks after lesion, nontreated neurotmesis (ax group), presented a 41.22% covering reduction of type s inputs when compared with contralateral side . Nerve coaptation resulted in excitatory input preservation, with no statistical differences between commercial and cevap sealant (figures 10(f) and 10(g)). Fibrin sealant nerve repair promoted even more prominent effects on inhibitory inputs (type f boutons), showing close to normal covering (figures 10(f) and 10(g)). Axotomy alone led to greater distance between terminals that contrasted with coaptation repair, where clusters of terminals were close to each other (figures 10(h)10(k)). The recovery of motor function was studied by the walking track test, using the catwalk system (noldus inc . ). Statistical analysis from peroneal functional index showed a significantly greater peroneal functional index (pfi) following coaptation (ax+fs, ax+cfs), regardless the nature of the fibrin sealant (figure 11). Pfi has been chosen over sciatic functional index (sfi), because measuring the intermediate toe spread (its) was not possible . We regard that to muscle atrophy since nerve lesion was performed in the neonatal period . The nociceptive recovery was analyzed by stimulation of the plantar surface of the paw with electronic von frey . Contralateral paw showed withdraw reflex around 30 g. axotomy alone (ax) rats did not show withdraw behavior at the cut - off weight, indicating lack of sensory perception (70 g, figure 12(a)). Both groups subjected to coaptation with fibrin sealant (ax+fs and ax+cfs) showed lower threshold withdrawal response (around 45 g, figures 12(b)-12(c)). It is well known that neonatal axotomy results in devastating neuronal loss in the spinal cord and dorsal root ganglia (drg). This is particularly because of the interruption of neurotrophic factors production and retrograde flow, mostly at the target organs . In fact, administration of neurotrophic factors, such as brain derived neurotrophic factor (bdnf) and ciliary neurotrophic factor (cntf) rescue motoneurons from degeneration . Also, the use of cannabidiol (cbd), a cannabinoid with neuroprotective properties, is also able to avoid both motoneuron and drg neurons death, following neonatal peripheral nerve lesion . In this way, early neonatal nerve axotomy has been broadly accepted as a consistent model for studying neuroprotection [46, 48]. Nevertheless, due to the reduced size of pups, combined with the thin and delicate structure of the peripheral nerves at perinatal stage, repair following transection is hardly accomplishable . The present work shows that end - to - end nerve coaptation can be performed in a reproducible fashion by using fibrin sealant as a connecting structure between proximal and distal stump . It is important to highlight that in humans nerve size is larger, allowing direct suturing . In this scenario, the use of fibrin glue may enhance repair stability and function as a scaffold for cell migration . In the present study, we compared two different fibrin adhesives, one commercial brand and another recently developed by cevap . The latest is based on nonhuman components, avoiding transmission of infectious blood - borne diseases . The results have shown that both sealants (commercial and cevap) are excellent for the coaptation process, although cevap's fibrin sealant has proven to be easier to handle at the moment of surgery . Surgeries involving neonatal subjects ought to be particularly fast, and clotting speed is a crucial parameter . Due to the fact that cevap derived adhesive uses bubaline fibrinogen, the concentration of such protein in the cryoprecipitate is specially higher than the human counterpart . In turn this in turn possibly allowed early regrowth of regenerating axons, originated from the proximal stump, resulting in improved morphometric parameters, four weeks after repair . In line with the positive results described in the present work, the fibrin sealant from cevap has been tested in humans, for autologous skin grafting of the nasogenian sulcus showing promising results . Moreover, it has been tested for the immobilization of free periodontal gingival grafts in lower premolars and for the treatment of chronic venous ulcers [5054]. Besides, recent studies also show the efficacy of this new sealant on peripheral nerve regeneration [55, 56], treatment of skull defects, and following ventral root avulsion . It has been shown that this scenario can be partially reversed by local or systemic administration of trophic substances, such as neurotrophins, antioxidants, cannabinoids, and gangliosides [47, 59, 60]. Nevertheless, the regrowth of axons from the spinal motoneurons and drg neurons towards the periphery is dependent on anatomical restauration of pns structures . We show that coaptation at the neonatal stage is neuroprotective by itself and generates a permissive scaffold through which regeneration can be successfully accomplished . The results shown herein indicate significant preservation of motoneurons in the spinal cord, assessed by direct counting at 4, 8, and 12 weeks after lesion, reinforcing the long lasting positive effects of the repair procedure . Nevertheless, since nerve repair was carried out at p2, motor and sensory evaluation could only be initiated from the third (catwalk) or fourth (von frey) week of age due to animal size and sensory - motor coordination . In turn, our data reflect already established recovery that happened before behavior tests could be started, what explains the rather constant peroneal and von frey results . Importantly, restauration of neonatal lesion, which mimics obstetric brachial plexus injury, led to preservation of synapses, as seen by synaptophysin immunostaining . Reduction of astrogliosis and microglial reaction also contribute to synapse stability and functionality, correlating with substantial motor and sensory recovery in ax+fs and ax+cfs groups up to twelve weeks after surgery . In this regard, improved peroneal index following coaptation was observed with either fibrin sealant treatment, when compared with axotomy alone . Such improvement indicates reestablishment of muscle innervation as well as suprasegmental control [61, 62]. After an injury, retraction of presynaptic boutons that appose spinal motoneurons takes place during the first week after lesion . This event reduces synaptic transmission [6469] and allows the axotomized neurons to focus metabolism on survival and regrowth of a new axon and dendrites . Herein, synaptic covering was significantly preserved in coaptation groups, in line with the immunohistochemical observations (synaptophysin). A quantitative analysis of the synaptic covering for each type of presynaptic terminal showed significant preservation of s and f type of synapses . However, the proportion of preserved inhibitory synapses was greater than excitatory . Such scenario, according to lind et al ., facilitates neuroprotection by reducing glutamatergic excitotoxicity [70, 71]. Besides the prevention of excitotoxicity, the number of preserved presynaptic terminals/100 m in apposition to the alpha - motoneuron membrane was also increased in both coaptation groups . Synapse dynamics also depend on astrocytes and microglia that act in favor of maintenance of homeostasis that is necessary to transmission and synaptic plasticity . One well - known retrograde effect of peripheral axotomy is the development of central astrogliosis and microglial reaction, directly interfering on synapse stability [7275]. Gfap and iba1 are highly expressed proteins in astrocytes and microglia, respectively, and are upregulated after lesion [73, 76]. The fact that coaptation downregulates glial reaction at lumbar spinal cord level is in line with the positive anatomical and behavioral findings previously discussed . Overall, the present data suggest that acute repair of neonatal peripheral nerve with both fibrin sealant analyzed, namely, a commercial brand and a nonhuman derived adhesive produced by cevap / brazil, promotes neuroprotection and regeneration of motor and sensory axons . Also, the present study demonstrates that neonatal end - to - end nerve coaptation is feasible and may in turn be of use for repairing obstetric brachial plexus injuries.
Pathogen bioinformatics have been developed and applied as a vehicle to discover novel genes and the search for virulence - associated genes combining approaches that assay gene expression, adaptive evolution and gene transfer [1 - 3]. In this study, layers of data about plasmodium falciparum, obtained with gene transcript and genome sequencing as well as gene and protein expression profiling technologies, were integrated to reveal insights into previously undiscovered regulation during intraerythrocytic development . This integrative approach uses an evidence - based assessment of disparate datasets similar to gene structure prediction approaches that rely on accumulation of evidence such as similarity to known genes, nucleotide compositional features, intron / exon boundaries and promoter sequences . The high malaria burden in africa necessitates increased efforts to understand the biology of the pathogen with a view to discovering new drugs, candidate vaccines and diagnostics, as well as improving existing ones . The publication of the genomes of the human malaria parasite p. falciparum and the rodent malaria parasite plasmodium yoelii as well as ongoing sequencing projects of other plasmodium species presents new opportunities to achieve the above - mentioned goals [7 - 9]. In addition, there have been efforts to obtain and analyse on a large - scale, gene expression profiles (transcriptome) of plasmodium species using expressed sequence tags (ests) [1,10 - 13], full length cdnas, serial analysis of gene expression (sage) and microarrays [17 - 19]. Protein expression profiles (proteome) on particular stages of the p. falciparum life cycle are also available . The random single - pass sequencing of a cdna library to generate short (200500 bp) nucleotide sequences that tag an expressed gene sequence is an established method of gene discovery . Est gene indices are generated by computer - based methods to organise these tags by assigning them into groups to remove redundancies and yield reconstructed transcripts that represent consensus sequences of each group . These indices are being used to understand the complexity of the human genome, especially in providing information on alternative transcripts, non - translated transcripts, truly unique genes and extremely short genes that will complement the genome data . The availability of the complete genome of p. falciparum 3d7 makes it possible to provide similar information for the parasite . In fact, additional est and full - length cdna sequences are required to improve the current annotation and verify predicted genes . Est sequencing projects on plasmodium have identified novel genes but only limited analyses have been performed on ests for coordinate and differential gene expression . Plasmodium ests from a variety of cdna libraries are available in the genbank est database (dbest). As of february 2003, 11 libraries comprising of nine asexual, one sporozoite and one gametocyte were available in dbest . Microarrays, mrna differential display and est - based analysis have been used to study transcriptional differences between asexual and gametocyte stages of p. falciparum, revealing stage - specific genes . These studies were done prior to the publication of the genome sequence of strain 3d7 . Furthermore, in the case of li and colleagues, the functional annotation was selective . An est - based analysis with an improved functional annotation that combines the automated annotation from p. falciparum gene indices and the curated annotation in the plasmodium genome database (plasmodb) is needed . In addition, integration of proteomic data with such analysis has been recognized as an important component in drug target identification and validation in the human genome . The number of ests used to generate a consensus sequence in a gene index can provide a rough estimate of the mrna abundance in the tissue or cell of origin . Furthermore, statistical tests have been developed to identify genes that are differentially expressed (significantly overexpressed) in a particular tissue compared to one or more other tissues . The differences in est counts have been applied to understand gene expression in different metabolic pathways, tissues or stages [32 - 34]. Microarray and sage methods are more narrow but sensitive for differential gene expression studies and can be used to validate broader est - based analysis . The life cycle of p. falciparum involves stages in the female anopheline mosquito vector and stages in the human host . The pre - erythrocytic stage involves invasion and growth within liver cells, whereas the intraerythrocytic cycle is a multi - stage process, which includes differentiation into asexual stages (rings, merozoites, trophozoites and schizonts) as well as sexual stages (male and female gametocytes). The clinical symptoms of malaria are produced primarily as a consequence of the asexual life cycle, while the sexual cycle, which can be divided into early (i - ii) and late (iii - v) gametocyte stages, is necessary for the development of the parasite in the mosquito . The intensive research on gene expression in the asexual stage compared to gametocyte stage can be inferred from the number of cdna libraries deposited in the dbest as mentioned above . The late (mature) stage gametocyte cdna library (id:10054) should contain transcripts important for gametocyte maturation and also formation of gametes and fertilization . The availability of a cdna library of 3d7 (id:9765) asexual mixed stage (rings, trophozoites and schizonts) and genome data from the same strain presents an opportunity to determine differentially expressed transcripts between the two libraries . Transcription and translation in malaria parasites is complex and characterized by features such as multiple transcripts, antisense transcripts, stage - specific transcripts, chromosomal clusters encoding co - expressed proteins, unspliced mrna, gene family member - specific expression and translational control . Understanding the role of these features in the regulation of important intraerythrocytic biological processes can deliver new tools for malaria control . For example, a proportion of genes involved in glycolysis, proteolysis and apicoplast targeting of nuclear encoded genes are thought to be regulated during the transition from asexual to sexual stages . The integration of data from est sequencing with those from genomic, microarray and proteomic technologies could provide insights into molecular mechanisms that contribute to the regulation of these processes . The significant increase in disparate datasets from genome sequencing and post - genomic analysis of p. falciparum necessitates delivery of integrated analysis from which biological processes important to the survival of the parasite can be determined . The integrated approach developed has identified stage - overexpressed genes with computational and experimental evidence to support their functional analysis . Furthermore, the approach is demonstrated as a means to appraise critically the data quality of the increasing number of post - genomic datasets from malaria parasites . The integrative analysis approach that was used to combine genomic, expressed sequence tag, microarray, proteomic and gene ontology data from p. falciparum 3d7 is presented in figure 1 . The starting integrative criterion was significant overexpression of a transcript in a stage relative to the other stage . Criteria used and their acceptable ranges are presented in table 1 . Simplified flowchart of integrative analysis of plasmodium falciparum data . Flowchart symbols: rounded rectangle, start or end; rectangle, process; diamond, decision . Threshold values for steps in integrative analysis of plasmodium falciparum data expressed sequence tags derived from p. falciparum 3d7 mixed asexual stage (dbest i d: 9765) and gametocyte (iii - v) stages (dbest i d: 10054) cdna libraries were retrieved using sequence retrieval system (srs) version 7.02 from embl database (release 74, march 2003). A total of 15,126 ests consisting of 11,872 asexual and 3,254 gametocyte ests were downloaded . Transcript reconstruction of these ests was performed using stackpack clustering system version 2.2 as described previously for reconstructing plasmodium transcripts . Briefly, the process starts with removal of artifactual sequences such as repeats and vector sequences . The " clean " sequences are grouped using a loose clustering approach into clusters and the clusters assembled into contigs . The alignments of sequences that make up these assembled clusters are analysed to produce consensus sequences of maximal length representing the reconstructed transcripts . Stackpack was chosen for its ability to provide extended consensus sequences (hide et al . In preparation). A gene index, manufactured by such a method, is therefore a non - redundant representation of a set of reconstructed gene fragments that approximates to the best available representation of genes for that organism . The clustering was unsupervised in that known sequences such as mrna, full - length cdna, previously reconstructed ests or exon constructs were not used to guide the process . This type of clustering was required to provide valid input data for the software used to calculate the differential expression statistics applied in this study . Audic - claverie (ac) and the chi - square () 2 2 statistical tests for differential gene expression were used to identify stage - overexpressed transcripts . These pairwise tag statistics are based on est counts of contigs (assembled clusters) with at least five ests since for a 95% confidence interval, the first value that is significantly different from 0 is 5 . The calculation of these statistics was implemented with the web version of ideg6 software; with a significance threshold of 0.05 . A suite of perl scripts was written to extract est counts from output of stackpack 2.2 and present the input dataset in the format required by ideg6 . Data extracted from the output file of ideg6 were (1) contig description; (2) observed and normalised est counts from the two libraries; and (3) probability that a transcript is differentially expressed as represented by p - values for the two tests . Transcripts for which the p - values for both statistics were less than 0.05 were taken as differentially expressed . Since these statistics determined transcripts differentially expressed, the terms asexual - overexpressed and gametocyte - overexpressed were used for transcripts (or genes) with significant overexpression in mixed asexual stage and late stage gametocytes respectively . Annotated protein predictions (release 4.0) of the whole genome sequence of p. falciparum 3d7 was obtained from the plasmodb website; . The overview page for each gene was retrieved using wget and saved as a hypertext markup language (html) file on a local computer to allow ease of manipulation without accessing the database over the internet . A perl script was used to query each page for the words sporozoite, merozoite, trophozoite or gametocyte preceded by an apostrophe (') followed by a specific text as for the gametocyte;' gametocyte stage peptide fragment(s) detected by mass spectrometry' . A match of this text was taken as evidence of expression and protein expression at the stage was assigned 1 or else 0 for no evidence . Thus, a 4-digit binary accession that indicates evidence for expression in sporozoite, merozoite, trophozoite and gametocyte is used to represent the 15 protein expression profiles presented by florens et al . And an additional accession for lack of evidence in all stages (0000). Reconstructed transcripts were annotated on the basis of similarity searches using ncbi blastx version 2.2.1 against predicted proteins of p. falciparum 3d7 . Statistical significance cut - off was set at an e - value of 10following that of carlton et al . . Since an unsupervised clustering was performed, to support the functional annotation, the annotations obtained were correlated with the tigr p. falciparum gene index; (version 6.0, release date the correlation was done by computational extraction of associated annotation of the tigr tentative consensus (tc) followed by manual checking to determine if the annotation obtained in our analysis was identical to that of the tigr tcs . If the annotations were not identical, the reconstructed sequence was excluded from further analysis . Genes classified as being involved in glycolysis (go:0006096), proteolysis (go:0006508) or targeted to the plastid (go:0009536) were retrieved by searching plasmodb gene overview page for the respective go identification (i d) number in a similar way as described for the protein expression profile except the search text was the respective go i d preceded by the greater than sign (>) for example> go:0006096 . This text limits the search to the gene ontology section of the gene overview page . The number of genes retrieved was: 20 for glycolysis, 98 for proteolysis and 553 for plastid component . The numbers of ests used to generate a reconstructed sequence were retrieved from the fasta sequence description line of all reconstructed sequences generated by stackpack 2.2 . The levels of expression or average signal intensities obtained from microarray experiments on the serine repeat antigen (sera) gene family of p. falciparum [19,42 - 44] were used to compare the levels of expression obtained using ests . This gene family is characterised by a cysteine proteinase framework and was selected because its members are annotated as being involved in proteolysis . Published microarray studies on this family have been obtained that facilitated comparative analysis with est data . The integrative analysis approach that was used to combine genomic, expressed sequence tag, microarray, proteomic and gene ontology data from p. falciparum 3d7 is presented in figure 1 . The starting integrative criterion was significant overexpression of a transcript in a stage relative to the other stage . Criteria used and their acceptable ranges are presented in table 1 . Simplified flowchart of integrative analysis of plasmodium falciparum data . Flowchart symbols: rounded rectangle, start or end; rectangle, process; diamond, decision . Threshold values for steps in integrative analysis of plasmodium falciparum data expressed sequence tags derived from p. falciparum 3d7 mixed asexual stage (dbest i d: 9765) and gametocyte (iii - v) stages (dbest i d: 10054) cdna libraries were retrieved using sequence retrieval system (srs) version 7.02 from embl database (release 74, march 2003). A total of 15,126 ests consisting of 11,872 asexual and 3,254 gametocyte ests were downloaded . Transcript reconstruction of these ests was performed using stackpack clustering system version 2.2 as described previously for reconstructing plasmodium transcripts . Briefly, the process starts with removal of artifactual sequences such as repeats and vector sequences . The " clean " sequences are grouped using a loose clustering approach into clusters and the clusters assembled into contigs . The alignments of sequences that make up these assembled clusters are analysed to produce consensus sequences of maximal length representing the reconstructed transcripts . Stackpack was chosen for its ability to provide extended consensus sequences (hide et al . In preparation). A gene index, manufactured by such a method, is therefore a non - redundant representation of a set of reconstructed gene fragments that approximates to the best available representation of genes for that organism . The clustering was unsupervised in that known sequences such as mrna, full - length cdna, previously reconstructed ests or exon constructs were not used to guide the process . This type of clustering was required to provide valid input data for the software used to calculate the differential expression statistics applied in this study . Audic - claverie (ac) and the chi - square () 2 2 statistical tests for differential gene expression were used to identify stage - overexpressed transcripts . These pairwise tag statistics are based on est counts of contigs (assembled clusters) with at least five ests since for a 95% confidence interval, the first value that is significantly different from 0 is 5 . The calculation of these statistics was implemented with the web version of ideg6 software; with a significance threshold of 0.05 . A suite of perl scripts was written to extract est counts from output of stackpack 2.2 and present the input dataset in the format required by ideg6 . Data extracted from the output file of ideg6 were (1) contig description; (2) observed and normalised est counts from the two libraries; and (3) probability that a transcript is differentially expressed as represented by p - values for the two tests . Transcripts for which the p - values for both statistics were less than 0.05 were taken as differentially expressed . Since these statistics determined transcripts differentially expressed, the terms asexual - overexpressed and gametocyte - overexpressed were used for transcripts (or genes) with significant overexpression in mixed asexual stage and late stage gametocytes respectively . Annotated protein predictions (release 4.0) of the whole genome sequence of p. falciparum 3d7 was obtained from the plasmodb website; . The overview page for each gene was retrieved using wget and saved as a hypertext markup language (html) file on a local computer to allow ease of manipulation without accessing the database over the internet . A perl script was used to query each page for the words sporozoite, merozoite, trophozoite or gametocyte preceded by an apostrophe (') followed by a specific text as for the gametocyte;' gametocyte stage peptide fragment(s) detected by mass spectrometry' . A match of this text was taken as evidence of expression and protein expression at the stage was assigned 1 or else 0 for no evidence . Thus, a 4-digit binary accession that indicates evidence for expression in sporozoite, merozoite, trophozoite and gametocyte is used to represent the 15 protein expression profiles presented by florens et al . And an additional accession for lack of evidence in all stages (0000). Reconstructed transcripts were annotated on the basis of similarity searches using ncbi blastx version 2.2.1 against predicted proteins of p. falciparum 3d7 . Statistical significance cut - off was set at an e - value of 10following that of carlton et al . . Since an unsupervised clustering was performed, to support the functional annotation, the annotations obtained were correlated with the tigr p. falciparum gene index; (version 6.0, release date the correlation was done by computational extraction of associated annotation of the tigr tentative consensus (tc) followed by manual checking to determine if the annotation obtained in our analysis was identical to that of the tigr tcs . If the annotations were not identical, the reconstructed sequence was excluded from further analysis . Genes classified as being involved in glycolysis (go:0006096), proteolysis (go:0006508) or targeted to the plastid (go:0009536) were retrieved by searching plasmodb gene overview page for the respective go identification (i d) number in a similar way as described for the protein expression profile except the search text was the respective go i d preceded by the greater than sign (>) for example> go:0006096 . This text limits the search to the gene ontology section of the gene overview page . The number of genes retrieved was: 20 for glycolysis, 98 for proteolysis and 553 for plastid component . The numbers of ests used to generate a reconstructed sequence were retrieved from the fasta sequence description line of all reconstructed sequences generated by stackpack 2.2 . The levels of expression or average signal intensities obtained from microarray experiments on the serine repeat antigen (sera) gene family of p. falciparum [19,42 - 44] were used to compare the levels of expression obtained using ests . This gene family is characterised by a cysteine proteinase framework and was selected because its members are annotated as being involved in proteolysis . Published microarray studies on this family have been obtained that facilitated comparative analysis with est data . A total of 210 transcripts that were differentially expressed were manually checked for correlation with tigr and/or apiestdb p. falciparum gene indices . However, the mixed asexual stage had the highest percentage (83%) of genes with evidence of protein expression in the same stage (stage - correlated protein expression) compared to 31% for the late gametocyte stage . The 194 transcripts differentially expressed between the two libraries consisted of 51 from the mixed asexual stage and 143 from the late gametocyte stage . The complete list with transcript identification used in this study, correlated transcripts in the tigr p. falciparum gene index, gene locus name, gene product description, representative est or ests (for genes with representation from both libraries), observed and normalized est counts for the two stages, as well as protein expression profile, are presented in the additional files 1 and 2 for mixed asexual stage and late gametocyte stage respectively . A list of stage - overexpressed transcripts that match those of li et al . Summary of functional annotation and protein expression of plasmodium falciparum transcripts asexual - overexpressed plasmodium falciparum transcripts transcript generated by stackpack 2.2 . Distribution of protein expression profiles for plasmodium falciparum stage - overexpressed genes 4-digit binary accession for protein expression evidence in sporozoite, merozoite, trophozoite and gametocyte . A total of 128 gametocyte - overexpressed and 48 asexual - overexpressed transcripts had a significant match with the predicted p. falciparum 3d7 proteins . Seventy - four genes (40 asexual - overexpressed, 34 gametocyte - overexpressed) showed evidence of stage - correlated protein expression (tables 3 and 4). The well - studied s - antigen (pf10_0343) is one of the 8 asexual - overexpressed genes without stage - correlated protein expression . Four gametocyte - overexpressed genes (pfb0730w, pfi1210w, pf10_0115 and pfl0105w) had more than one reconstructed transcript . Multiple transcripts were generated when the reconstructed transcripts associated with a gene are not contiguous, and thus were not assembled into the same contig . Fifty - three of the 74 genes were classified as novel in that either the description of the gene product is labelled hypothetical protein or have the word putative . In order to identify gametocyte - overexpressed genes that also have stage - correlated protein expression in the proteomics data of lasonder et al ., the spreadsheet file containing 1,289 unique malaria proteins from that study was processed to yield a 3-digit binary accession representing evidence for protein expression of genes in trophozoites / schizonts, gametocytes and gametes . Fifteen of the 34 gametocyte - overexpressed genes were detected by both proteomic analyses (table 6). Our analysis points to the need to clarify potential confusion in the annotation of the sexual stage specific protein precursor or pfs16 (pfd0310w), a known marker for the earliest events of sexual differentiation . The locus name (pf11_0318) of another gene, pf16, may be assigned to this gene . Pf16 has sequence similarity to a sperm flagella protein localized to the central pair of the axoneme . The gametocyte - overexpressed gene identified in this study was confirmed to be pfs16 and not pf16 by the identical functional annotation of the associated consensus sequence from this study and that in the tigr p. falciparum gene index . Gametocyte - overexpressed plasmodium falciparum genes with correlated protein expression in two proteomic studies evidence of expression: 0, no evidence; 1, with evidence . 4-digit binary accession for protein expression evidence in sporozoite, merozoite, trophozoite and gametocyte . The identified asexual - overexpressed genes that have been experimentally characterised have known roles in protein degradation, purine salvage, rhoptry biogenesis and protein trafficking, schizont rupture, merozoite invasion, phospholipid biosynthesis, nuclear metabolism, oxidative stress defense, cell proliferation and membrane biogenesis . Glyceraldehyde-3-phosphate dehydrogenase (pf14_0598) and atp - dependent phosphofructokinase (pf11_0294) are two of 20 genes known to be involved in glycolysis . Microarray average intensities available in plasmodb for pf11_0294 support its gametocyte - overexpression when compared to a closely related gene, pfi0755c that also codes for a phosphofructokinase and shows protein expression in intraerythrocytic stages . The microarray expression values for pfi0755c in trophozoite and schizont stages are 17,223.33 and 7,894 respectively in contrast to ~1,600 in both stages for pf11_0294 . Inspection of the predicted protein features of pf11_0294 revealed the presence of two protein domains: gonadotropin - releasing domain, gnrh (pfam i d: pf00446) and laminin n - terminal (domain vi) (pfam i d: pf00055). These domains are found in proteins that are extracellular and have a role in regulation of germ cell development . Pfb0340c, a cysteine protease and member of the sera gene family was significantly overexpressed in mixed asexual stage . Other genes in the sera family for which est data were available were checked for correlation of functional annotation and their est count retrieved . As shown in table 7, the est counts were variable across the gene family consistent with microarray - based studies [42 - 44]. There was est evidence for expression of pfb0345c (sera4), pfb0340c (sera5) and pfb0335c (sera6), the three central genes that were demonstrated to be essential for asexual stage growth . The genbank accession numbers of a representative est from these genes are bi936220, bi815392 and bq633262 respectively . Pfb0340c showed the highest est count and microarray intensity values during asexual development of the parasite . Correlation of est abundance and microarray intensity associated with sera gene family -, no ests observed . Comments on gene expression: -/+, low or absent expression; +, expression confirm by rt - pcr and microarray . R, rings; t, trophozoite; s, schizont; asyn, asynchronous culture; -, no expression value reported . Central genes in the sera locus that could not be disrupted in study . Gene with multiple transcripts, tc6886 (bi670678) tc6962 (bi814535). Out of the 17 transcripts (four asexual and 13 gametocyte) associated with genes targeted to the apicoplast, only two genes: mal13p1.281 and pfe0145w have similarities to known genes (glutamate - trna ligase and 50s ribosomal subunit protein l28). There was evidence of protein expression in at least one asexual stage for two (pf07_0087, pf14_0543) of the four asexual - overexpressed genes (table 3). Six gametocyte - overexpressed genes showed evidence for expression in the sporozoite stage while only pf11_0525 showed evidence in the sporozoite and gametocyte stages . The domains are iq (calmodulin - binding motif, pfam i d: pf00612) and lysm (lysin motif, pfam i d: pf01476), which is a general peptidoglycan - binding module . A list of apicoplast - targeted genes with stage - overexpressed transcripts is presented in additional file 4 . A total of 210 transcripts that were differentially expressed were manually checked for correlation with tigr and/or apiestdb p. falciparum gene indices . The majority of the stage - overexpressed transcripts were from the late gametocyte stage . However, the mixed asexual stage had the highest percentage (83%) of genes with evidence of protein expression in the same stage (stage - correlated protein expression) compared to 31% for the late gametocyte stage . The 194 transcripts differentially expressed between the two libraries consisted of 51 from the mixed asexual stage and 143 from the late gametocyte stage . The complete list with transcript identification used in this study, correlated transcripts in the tigr p. falciparum gene index, gene locus name, gene product description, representative est or ests (for genes with representation from both libraries), observed and normalized est counts for the two stages, as well as protein expression profile, are presented in the additional files 1 and 2 for mixed asexual stage and late gametocyte stage respectively . A list of stage - overexpressed transcripts that match those of li et al . Summary of functional annotation and protein expression of plasmodium falciparum transcripts asexual - overexpressed plasmodium falciparum transcripts transcript generated by stackpack 2.2 . Distribution of protein expression profiles for plasmodium falciparum stage - overexpressed genes 4-digit binary accession for protein expression evidence in sporozoite, merozoite, trophozoite and gametocyte . A total of 128 gametocyte - overexpressed and 48 asexual - overexpressed transcripts had a significant match with the predicted p. falciparum 3d7 proteins . Seventy - four genes (40 asexual - overexpressed, 34 gametocyte - overexpressed) showed evidence of stage - correlated protein expression (tables 3 and 4). The well - studied s - antigen (pf10_0343) is one of the 8 asexual - overexpressed genes without stage - correlated protein expression . Four gametocyte - overexpressed genes (pfb0730w, pfi1210w, pf10_0115 and pfl0105w) had more than one reconstructed transcript . Multiple transcripts were generated when the reconstructed transcripts associated with a gene are not contiguous, and thus were not assembled into the same contig . Fifty - three of the 74 genes were classified as novel in that either the description of the gene product is labelled hypothetical protein or have the word putative . In order to identify gametocyte - overexpressed genes that also have stage - correlated protein expression in the proteomics data of lasonder et al ., the spreadsheet file containing 1,289 unique malaria proteins from that study was processed to yield a 3-digit binary accession representing evidence for protein expression of genes in trophozoites / schizonts, gametocytes and gametes . Fifteen of the 34 gametocyte - overexpressed genes were detected by both proteomic analyses (table 6). Our analysis points to the need to clarify potential confusion in the annotation of the sexual stage specific protein precursor or pfs16 (pfd0310w), a known marker for the earliest events of sexual differentiation . The locus name (pf11_0318) of another gene, pf16, may be assigned to this gene . Pf16 has sequence similarity to a sperm flagella protein localized to the central pair of the axoneme . The gametocyte - overexpressed gene identified in this study was confirmed to be pfs16 and not pf16 by the identical functional annotation of the associated consensus sequence from this study and that in the tigr p. falciparum gene index . Gametocyte - overexpressed plasmodium falciparum genes with correlated protein expression in two proteomic studies evidence of expression: 0, no evidence; 1, with evidence . 4-digit binary accession for protein expression evidence in sporozoite, merozoite, trophozoite and gametocyte . The identified asexual - overexpressed genes that have been experimentally characterised have known roles in protein degradation, purine salvage, rhoptry biogenesis and protein trafficking, schizont rupture, merozoite invasion, phospholipid biosynthesis, nuclear metabolism, oxidative stress defense, cell proliferation and membrane biogenesis . Glyceraldehyde-3-phosphate dehydrogenase (pf14_0598) and atp - dependent phosphofructokinase (pf11_0294) are two of 20 genes known to be involved in glycolysis . Microarray average intensities available in plasmodb for pf11_0294 support its gametocyte - overexpression when compared to a closely related gene, pfi0755c that also codes for a phosphofructokinase and shows protein expression in intraerythrocytic stages . The microarray expression values for pfi0755c in trophozoite and schizont stages are 17,223.33 and 7,894 respectively in contrast to ~1,600 in both stages for pf11_0294 . Inspection of the predicted protein features of pf11_0294 revealed the presence of two protein domains: gonadotropin - releasing domain, gnrh (pfam i d: pf00446) and laminin n - terminal (domain vi) (pfam i d: pf00055). These domains are found in proteins that are extracellular and have a role in regulation of germ cell development . Pfb0340c, a cysteine protease and member of the sera gene family was significantly overexpressed in mixed asexual stage . Other genes in the sera family for which est data were available were checked for correlation of functional annotation and their est count retrieved . As shown in table 7, the est counts were variable across the gene family consistent with microarray - based studies [42 - 44]. There was est evidence for expression of pfb0345c (sera4), pfb0340c (sera5) and pfb0335c (sera6), the three central genes that were demonstrated to be essential for asexual stage growth . The genbank accession numbers of a representative est from these genes are bi936220, bi815392 and bq633262 respectively . Pfb0340c showed the highest est count and microarray intensity values during asexual development of the parasite . Correlation of est abundance and microarray intensity associated with sera gene family -, no ests observed . Comments on gene expression: -/+, low or absent expression; +, expression confirm by rt - pcr and microarray . R, rings; t, trophozoite; s, schizont; asyn, asynchronous culture; -, no expression value reported . Central genes in the sera locus that could not be disrupted in study . Gene with multiple transcripts, tc6886 (bi670678) tc6962 (bi814535). Out of the 17 transcripts (four asexual and 13 gametocyte) associated with genes targeted to the apicoplast, only two genes: mal13p1.281 and pfe0145w have similarities to known genes (glutamate - trna ligase and 50s ribosomal subunit protein l28). There was evidence of protein expression in at least one asexual stage for two (pf07_0087, pf14_0543) of the four asexual - overexpressed genes (table 3). Six gametocyte - overexpressed genes showed evidence for expression in the sporozoite stage while only pf11_0525 showed evidence in the sporozoite and gametocyte stages . The domains are iq (calmodulin - binding motif, pfam i d: pf00612) and lysm (lysin motif, pfam i d: pf01476), which is a general peptidoglycan - binding module . A list of apicoplast - targeted genes with stage - overexpressed transcripts is presented in additional file 4 . An integrative approach was used to determine genes associated with transcripts differentially expressed between mixed asexual stage and late stage gametocyte parasites . The publication of the genome sequence of two malaria parasites presents opportunities for post - genomic era malaria research including gene discovery and comprehensive understanding of gene expression . The study has revealed (1) possible regulatory mechanisms in malaria parasites' gametocyte maturation, (2) correlation between est and microarray data for a p. falciparum gene family to present unique est - derived information, (3) candidate genes on which computational and experimental studies can be performed, and (4) the need for more empirical studies on gene and protein expression in malaria parasites . These presents 366 more contigs than described by li et al . Reflecting inclusion of new mixed asexual stage ests deposited after march 2002 . Only 21 of the 24 significantly stage - specific transcripts identified by li et al . Were among our stage - overexpressed transcripts after correlation of functional annotation . Both studies demonstrate the asexual - overexpression of the gene for glyceraldehyde-3-phosphate dehydrogenase (gapdh), an important gene in the glycolytic pathway . Gene and protein expression were observed, as well as protein domain evidence for specialization or adaptation of atp - dependent phosphofructokinase (pf11_0294) for metabolic coupling of glucose utilization and maturation of gametocytes in malaria parasites . This enzyme is of major regulatory importance in plasmodium and has been characterised only in plasmodium berghei . In addition, it has been proposed as a potential drug target in protozoan parasites . Two genes (pf11_0294, pfi0755c) annotated as phosphofructokinase are present in the genome . This is consistent with the fact that many key enzymes in the glycolytic pathway occur as isoenzymes . Interestingly, pf11_0294 possesses a gonadotropin - releasing domain gnrh and laminin n - terminal (domain vi) that are thought to regulate germ cell development . Pf11_0525 is the only apicoplast - targeted gene associated with a gametocyte - overexpressed transcript that showed stage - correlated protein expression . The fact that germ cell biology is conserved in evolution enables us to speculate on the possible roles of this protein . The calmodulin (cam) binding site has been extensively studied in a sperm autoantigen (sp17), which is a zona binding protein and a member of the family of cam binding proteins that contain the iq motif in the cam binding domain . This domain has a regulatory role and undergoes proteolytic processing at the initiation of an acrosome reaction . Some bacterial proteins such as hydrolytic enzymes contain the general peptidoglycan - binding module (lysm) and have a role in cell - wall penetration . Pf11_0525 does not have evidence of a bipartite peptide for apicoplast targeting and thus may be targeted via a different mechanism to the organelle or it may no longer function in the plastid . The est counts of the sera gene family are comparable with the gene expression levels observed in microarray experiments . Both technologies agree that expression levels of members are variable as is expression of central genes during the asexual stage of the parasite . Pfb0340c (sera5) is the first described member of the family and is also a malaria vaccine candidate . The est counts for pfb0340c observed is consistent with high gene expression levels in trophozoites and schizonts in published microarray experiments . The increasing amount of published and unpublished data from microarray, sage, est and differential display on malaria parasites shows that pairwise correlation is required . Comparison of such datasets obtained from different gene expression technologies can complement less sensitive technologies, hence adding value to data generation from these methods . For example, this study provides identity of ests and also potential alternative transcripts that can be used to further characterize the sera central genes . Furthermore, pfb0325c (sera8) did not have est evidence consistent with low or absent expression observed in the microarray studies . However, there was evidence of its expression in the sporozoite stage, indicating the gene may be functional in other stages of the life cycle as speculated by miller et al . . Large - scale comparative expression analysis of gene families in multiple malaria parasites is needed to advance the knowledge of their evolution and their role during intraerythrocytic development . The two uncharacterized genes from which we speculate functional insights, pf11_0294 and pf11_0525, have putative orthologues in p. yoelii yoelli (py05918 and py06990 respectively) and were also detected in two independent proteomic analysis as expressed in the mature gametocyte stage . These observations strengthen the need for further studies on these genes and the possibility of studies with model malaria parasites . In general, various categories of candidate genes were provided that can be intensively studied as drug targets, antigenic targets, epidemiological or clinical markers . Eighty - seven of the 121 gametocyte - overexpressed genes did not show evidence of stage - correlated protein expression while 15 of those with such evidence were corroborated by the two proteomics studies . These corroborated genes represent a set of gametocyte - overexpressed genes with correlated transcription and translation data and thus candidates for studies on gametocyte maturation in malaria parasites . A shortlist of stage - overexpressed genes targeted to the plastid is presented to facilitate studies to understand the regulation of plastid metabolism in malaria parasites . This study has identified the lack of correlation between gene and protein expression of the asexual - overexpressed s - antigen, consistent with observations from published proteome analysis . This observation and those from the gametocyte - overexpressed transcripts as well as comparing outputs from est clustering efforts demonstrate that our integrative approach has the utility to compare outputs of different post - genomic analysis . The analysis indicates the need for additional empirical studies on gene and protein expression in malaria parasites . Such studies could improve current understanding on discrepancies between gene and protein expression profiling data as well as the detection of proteins with unique characteristics such as proteolytic processing, post - translational modification and sub - cellular location . The value of integrating a variety of datasets to unravel undiscovered regulation in biological processes during the gametocyte maturation stages of p. falciparum was demonstrated . Furthermore, comparative analysis of est and microarray data was performed on the sera gene family to advance the knowledge of their gene regulation and additional functional genomics reagents were presented to facilitate their study . Finally, the integrative approach was shown as a means to appraise critically the data quality of the increasing number of post - genomic datasets from malaria parasites . Plasmodium falciparum asexual - overexpressed transcripts plasmodium falciparum gametocyte - overexpressed transcripts correlated stage - overexpressed transcripts in this study and that of li et al . Plasmodium falciparum candidate genes for studies into plastid metabolism the authors thank colleagues at the south african national bioinformatics institute for useful suggestions and staff of electric genetics for stackpack support . Rdi is a claude harris leon foundation fellow and thanks the undp / world bank / who special programme for research and training in tropical diseases (tdr) and the malaria research and reference reagent resource center (mr4) for grants to attend workshops on malaria bioinformatics and microarrays.
Desktop computer and laptop use is becoming common, and computing - related musculoskeletal symptoms are considered important health problems in university students and office workers1,2,3,4 . Previous studies have indicated that improper sitting posture during visual display terminal (vdt) work (e.g., desktop computer or laptop use) can induce musculoskeletal disorders, especially low - back pain3, 4). Carter and banister3 suggested that increased tension in ligaments and discs during a slumped sitting posture may lead to low - back pain . Cross - legged sitting, a commonly adopted posture during daily living and vdt work, has been reported to induce a slumped sitting posture5, 6 . Lee et al.5 showed that the cross - legged sitting position leads to a greater slumped posture compared with an upright sitting posture during vdt work . To prevent the unwanted slumped posture, it is crucial to identify factors that contribute to slumped posture during cross - legged sitting . Limited hip flexion range of motion (rom) could be one possible risk factor for excessive lumbar flexion . Because the lumbar spine and hip joint are connected via the pelvis, limited hip flexion can cause greater lumbar flexion through pelvic posterior tilt during the trunk flexion - related posture7 . Although it seems reasonable that limited hip flexion rom leads to greater lumbar flexion during the cross - legged sitting posture, no study has investigated how hip flexion rom influences the kinematics of the lumbar spine in the cross - legged posture during vdt work . Furthermore, previous studies have not determined whether greater lumbar flexion during vdt work with a cross - legged sitting posture changes trunk muscle activities as measured by electromyography (emg). Only one emg study revealed decreased emg activity in the internal oblique (io) muscles during the static cross - legged sitting posture compared with the static normal sitting posture8 . Thus, the aim of the present study was to compare the lumbar flexion angle and trunk muscle activity in individuals with and without limited hip flexion rom during vdt work with a cross - legged sitting position . All subjects showed right - leg dominance and performed computer work more than 20 hours per week . Measurements of the right hip flexion rom were used to classify subjects into the experimental or control groups . According to previous findings9, sufficient hip flexion rom was defined as more than 120 of hip flexion, and limited hip flexion rom was defined as 110 in this study . Subjects with more than 120 of right hip flexion rom (one female, six males) were classified into the control group, and subjects with no more than 110 of right hip flexion rom (eight males) were classified into the experimental group . Subjects were excluded if they had acute low - back pain, orthopedic damage, or lower extremity injury during the last 6 months . In addition, individuals with hip flexion rom between 110 and 120 were also excluded in this study . The inje university faculty of health science human ethics committee approved this study, and each subject signed an informed consent form before participation . To measure hip flexion rom, subjects were placed in the supine position on a table, and an examiner passively flexed the right hip of subjects until further hip flexion was limited by firm end feel9 . The fulcrum of a goniometer was placed on the greater trochanter, and the proximal and distal arms were aligned with the lateral midlines of the pelvis and femur, respectively . The lumbar flexion angle was measured using eight vicon mx - t10 motion capture systems (vicon motion systems ltd ., three reflective markers were placed on the bilateral anterior superior iliac spines and on the midpoint between the bilateral posterior superior iliac spines for the pelvic segment . Additionally, four reflective markers were attached to the first and second lumbar spinous processes and 3 cm bilaterally from the second lumbar spinous process for the lumbar segment . The lumbar flexion angle was calculated by assessing the anterior rotation of the lumbar segment with respect to the pelvic segment using the cardan angle10 . The trunk muscle activity of the bilateral rectus abdominis (ra), external oblique (eo), and io muscles was recorded using a synchronized surface emg system (delsys inc ., prior to attachment of the electrodes, skin preparation was performed by shaving the hair and cleansing with an alcohol swab . Each electrode was attached along the direction of the muscle fiber based on placements suggested by criswell11 . Emg signals were acquired at a sampling rate of 1,000 hz with a bandwidth of 20450 hz and converted into root - mean - square (rms) data . To normalize emg data of the trunk muscles, reference voluntary contraction (rvc) data of trunk muscles were collected when subjects were seated on a chair in a comfortable sitting posture with 90 of hip and knee flexion for 40 s. the rvc maneuver was repeated three times, and the mean value of the average muscle activity for the middle 30 s of the three trials was used to normalize trunk muscle activity . Prior to vdt work, a laptop (xnote r400, lg, seoul, korea) was placed on a 73-cm - high desk, and subjects sat on a height - adjustable chair without a backrest with 90 of hip and knee flexion . The examiner confirmed the 90 hip and knee flexion position using a goniometer . For vdt work with a cross - legged sitting posture, subjects were instructed to cross the right leg over the left by putting the right knee on the left knee5, 8 . Following cross - legged sitting, subjected performed typing work in which they copied some text provided on the monitor by the korean version of hansoft . The subjects typed for 1 min for each trial, and three test trials were conducted with 30-s rest periods between trials . The average values of the lumbar flexion angle and emg activity of the trunk muscles for the middle 40 s of each trial were collected, and the mean values of three test trials were used for data analysis . The subjects characteristics (age, height, weight, hip flexion rom), lumbar flexion angle and trunk muscle activity during vdt work with cross - legged sitting in the experimental and control groups were compared using independent t - tests ., chicago, il, usa) was used, and the statistical significance level was set at p = 0.05 . The general characteristics of the subjects in the control group (mean age 25.1 1.5 years; mean weight 62.6 5.7 kg; mean height 171.9 6.9 cm) and the experimental group (mean age 23.6 1.5 years; mean weight 67.4 4.1 kg; mean height 175.1 4.5 cm) were not significantly different (p> 0.05), except for the hip flexion rom (123.6 2.4 versus 102.5 3.8; p <0.001). The lumbar flexion angle was significantly greater in the experimental group compared with the control group during vdt work with cross - legged sitting (p = 0.017) (table 1table 1 . Comparison of the lumbar flexion angle and trunk muscle activity between the two groups during visual display terminal work with cross - legged sittingvariablemean sdcontrol groupexperimental grouplumbar flexion ()15.2 7.023.3 4.3right ra activity (% rvc)101.2 5.9100.5 8.2right eo activity (% rvc)98.7 6.2113.3 30.4right io activity (% rvc)91.4 7.9109.4 22.3left ra activity (% rvc)101.3 7.1101.7 8.2left eo activity (% rvc)98.3 5.0115.3 35.0left io activity (% rvc)99.8 10.897.2 11.3rvc, reference voluntary contraction; ra, rectus abdominis; eo, external oblique; io, internal oblique . however, there were no significant differences in emg measures of trunk muscle activity between the control and experimental groups (p> 0.05). Rvc, reference voluntary contraction; ra, rectus abdominis; eo, external oblique; io, internal oblique . * in the present study, the subjects with limited hip flexion rom showed greater lumbar flexion compared with subjects with sufficient hip flexion rom during vdt work with cross - legged sitting; however, these differences in hip flexion rom did not influence emg activity in trunk muscles . The lumbo - pelvic - hip complex is connected via a kinematic link termed the lumbo - pelvic rhythm7, 12 . During forward bending, the lumbar spine is flexed anteriorly with respect to the pelvis, while the pelvis is flexed anteriorly on the femur12 . When lumbar flexion is limited, greater hip flexion is required with pelvic anterior tilt throughout the lumbo - pelvic rhythm7 . In other words, greater lumbar flexion is caused throughout the pelvic posterior tilt when hip flexion is limited . The results of our study imply that greater lumbar flexion compensated for insufficient hip flexion in subjects in the experimental group during vdt work with cross - legged sitting . Despite significant differences in lumbar flexion between the experimental and control groups, emg activity in the trunk muscles was not significantly different between the two groups . Snijders et al.8 reported that the cross - legged sitting position decreased emg activity in io muscles due to increased stability in the sacroiliac joint . The cross - legged sitting posture is performed by the combined motion of hip flexion and adduction . Increased tension in the biceps femoris, gluteus maximus and piriformis by hip flexion and abduction influences sacroiliac joint compression and tension in the sacrotuberous ligament, which can contribute to increased stability in the sacroiliac joint13,14,15 . Although the hip flexion angle may have been influenced by differences in hip flexion rom in subjects in this study, sufficient hip adduction may have been possible for all subjects during vdt work with cross - legged sitting . We suggest that the sacroiliac joint compression by hip adduction during cross - legged sitting may not differ between subjects with and without limited hip flexion rom, resulting in the absence of significant differences in emg activity in trunk muscles between the two groups . First, our study included a small sample size; therefore, it is difficult to generalize our results . However, a previous study by lee et al.5 showed that trunk kinematics were immediately changed after assuming a cross - legged sitting posture during vdt work, and the trunk flexion angle then did not significantly change for 30 min . Based on previous findings, we consider that the kinematic data of the lumbar spine and emg data of the trunk muscles during vdt work with cross - legged sitting for short periods may provide meaningful information for individuals who prefer cross - legged sitting.
Laparoscopic supracervical hysterectomy (lsh) has continued to represent a favorable alternative to total abdominal hysterectomy (tah) for the treatment of benign gynecologic conditions, particularly due to the reduced complication rates, shorter surgery / hospital stay, and prompt resumption of patient daily living activities . Nevertheless, studies continue to suggest that lsh should not be used as a treatment for benign gynecological conditions, particularly cervical dysplasia . The purpose of this commentary is to address the primary objections against lsh and further illustrate the benefits inherent in this procedure . Initially, the primary impetus for removal of the cervix at the time of hysterectomy in patients with benign conditions was to prevent cervical cancer . However, the incidence of cancer in the cervical stump is extremely low and primarily preventable due to latent disease progression, pap smear technology, and hpv screening . Therefore, removing this organ solely for the purpose of preventing cervical cancer appears counterintuitive, especially considering that both at - risk patients and the general nonhysterectomized population receive the same recommended screening guidelines . Since lsh involves the removal of the uterine section ostensibly related to the specific condition, the operation fixes many gynecologic problems while it conserves the patient's anatomy and sexual function by retaining the cervix and its mucous - secreting glands . Furthermore, the cervix is not typically associated with pelvic pain or bleeding, and thus patients can thereby avoid the common complaints of vaginal dryness and dyspareunia . Studies have further indicated that removal of the normal cervix can cause untoward bladder and bowel consequences, including prolapse and urinary incontinence . Additionally, prior research has reported that lsh outcomes coincide with favorable rates of prolapse and vaginal cuff dehiscence (vcd). In particular, hur et al examined the prevalence of vcd in a large hysterectomy study, indicating that the condition has a significantly following tah compared with lsh . Randomized controlled trials and metaanalyses have documented that lsh is associated with a higher incidence of cervical stump complications (eg, cyclical bleeding and urinary incontinence). However, the cyclical bleeding with lsh is often slight and can be tolerated if the patient receives adequate preoperative counseling . In terms of stress urinary incontinence, tah appears to be associated with more favorable outcomes compared with lsh, whereas there were no reported lower urinary tract symptom (luts) differences between the 2 procedures . We contend that because vaginal suspension alters the bladder neck angle and reduces postoperative incontinence, when performing lsh, consideration for suspending both the vagina and the cervical stump may significantly mitigate stress urinary incontinence . While there were no reported differences between tah and lsh regarding the incidence of luts, urinary tract infections, incomplete bladder emptying and voiding complications increased after tah at 1-year follow - up but decreased in the lsh patients . In an earlier surgical study, gimbel et al also reported a much higher incidence of serious adverse events and perioperative blood loss in patients treated with tah compared with those treated with lsh . Furthermore, the tah group exhibited more bladder / ureteral injuries, underwent longer operative times . Patients who present with recurrent cervical dysplasia should consider having their cervix removed if a total hysterectomy is warranted . However, when a patient initially presents with cervical dysplasia, lsh may be preferable to hysterectomy particularly given the reportedly lower complication rates, reduced surgical time, and earlier recovery . The combination of improved prevention programs, patient adherence to annual screening recommendations, and an informed community appreciation of the virus's vaccination distribution may further render this issue inconsequential . We suspect that the controversy surrounding the removal of the cervix is partially attributed to both insufficient lsh outcome studies and because many gynecologic surgeons are not formerly trained or experienced with this treatment option . While we recognize that both the american college of obstetrics and gynecology and a recent cochrane analysis clearly state that tah is more beneficial than lsh in treating benign gynecologic conditions, clinicians should strongly consider the several encouraging lsh findings and emerging studies that continue to substantiate the efficacy of lsh for treating many common benign gynecologic conditions.
A 76-year - old woman with hypertension was admitted to the hospital with complaints of chest pain and dyspnea . Arterial blood gas analysis revealed the following findings of hypoxemia and hypocapnia: po2, 36.7 mmhg; and pco2, 30.4 mmhg . The patient's cardiac enzyme values were: creatine kinase - mb, 105 ng / ml; and troponin i, 0.38 ng / ml . The b - type nitriuretic peptide test result was 64 . A diagnosis of recent myocardial infarction was made based on the following electrocardiographic findings: t - wave inversions and q waves in leads ii and iii and a vf, and t - wave inversions in leads v1 to v4 . Transthoracic echocardiography revealed akinesia in the apex free wall of the right ventricle, moderate pulmonary hypertension (right ventricular systolic pressure [rvsp], 55 mmhg), and a 2.72.8 cm mobile mass in the free wall of the right atrium . Pulmonary computed tomography (ct) angiography revealed pulmonary emboli occluding the pulmonary arteries in the upper and lower lobes on both sides, and a suspicious intra - atrial cardiac tumor with pulmonary thromboembolism was revealed according to the radiologic findings (fig . To detect other possible causes of the pulmonary embolism, abdominal ct and tumor marker tests were performed, the results of which were all normal . The patient was diagnosed with pulmonary embolism due to the mass in the right atrium based on the result that the patient's d - dimer was <0.35 . However, the patient refused surgical treatment . After 26 months, she was intubated due to severe dyspnea and was then referred to ajou university hospital . Transthoracic echocardiography revealed: 1) hypokinesia in the right ventricle, 2) a dilated right ventricle, 3) mild pulmonary hypertension, 4) a 3.52.8 cm mass in the free wall of the right atrium, which had grown compared to previous findings, and 5) tricuspid regurgitation . Pulmonary ct angiography revealed emboli occluding the right pulmonary artery and segmental arteries in the upper, middle, and lower lobes with a right atrial mass . The radiological characteristics were similar to that of a previous study (an intra - atrial cardiac tumor with pulmonary thromboembolism) (fig . Pulmonary perfusion imaging showed decreased perfusion in the majority of the upper and lower right lobes, in part of the anterior segment of the left upper lobe, and in part of the superior and posterior basal segments of the left lower lobe (fig . Transesophageal echocardiography revealed a 4.82.6 cm hypermobile mass with multilobular heterogeneous echogenicity in the upper lateral aspect of the free wall of the right atrium, which was not affecting the blood flow in the superior vena cava, inferior vena cava, or tricuspid valves (fig . 2c, coronary angiography showed findings of moderate coronary artery disease, which was treated with drug therapy alone . We decided to perform removal of the right pulmonary arterial emboli and the mass in the right atrium . The emboli in the left pulmonary artery decreased in size compared to the previous ct findings, and it was not considered a critical lesion because it was confined to the inferior lingular segmental artery . A standard sternotomy incision was made . The patient's body temperature was lowered to 23 (moderate hypothermia) through cardiopulmonary bypass, and then the myxoma with a 2 to 3 mm pedicle on the free wall of the right atrium was excised along with adjacent normal tissue . At 23, we opened the right pulmonary artery, which revealed that the myxoma segment was not adhered to the intima of the artery (fig . An embolus within the right pulmonary artery was removed en bloc in a bloodless surgical field (fig . 3b). After we confirmed that there were no further masses in the arteries supplying each segment, the right pulmonary artery and atrium were closed . Histopathological examination revealed that both the mass resected from the right atrium (4.33.53 cm) (fig . 3c, white arrow) and the embolus removed from the right pulmonary artery (721.5 cm) (fig . In addition, the size of the right atrium had decreased and the pulmonary artery pressure had slightly decreased to 35 mmhg . However, tricuspid regurgitation remained stationary (grade 2/4). Imaging studies, the right lung had improved, but the left lung showed no changes . The patient is currently on out - patient follow - up without any significant symptoms . According to the echocardiography performed in december 2010, there was no evidence of pulmonary embolism, and the rvsp was 35 mmhg . Pulmonary emboli are mainly caused by deep vein thromboses and embolus in the right heart or at the tip of a catheter . Chitwood were the first to perform a successful resection of a myxoma through cardiopulmonary bypass . The recurrence rate of pulmonary embolism after resection has been reported to be 0.4% to 5% . Pulmonary emboli frequently recur when 1) surgical resection is incomplete, 2) emboli detached during surgery adhere to the intima of the heart, 3) emboli are of multicentric origin, 4) there is a family history of myxoma, or 5) a new myxoma develops from myxoma precursor cells . Because myxoma tissue is extremely friable, it is frequently detached and it adheres to a new site [2 - 5]. Approximately 75% of all the myxoma cases occur in the left atrium, 23% in the right atrium, and 2% in the ventricle . Myxoma can manifest obstruction symptoms due to blood flow blockade, symptoms due to embolism, arrhythmia, and other generalized symptoms . Syncope or sudden death can develop when pulmonary or systemic circulation is disturbed or when blood flow to the atrioventricular valve is blocked . Thrombosis can be induced by myxoma segments, thrombi that has formed within the tumor, or infected lesions within the tumor . Embolism occurs in 30% to 45% of the patients with myxoma of the left atrium . Emboli originating from myxoma in left atrium are mainly found in the brain, kidney, and branches of the aorta and lower extremities . Embolism occurs in approximately 10% of the patients with myxoma in the right atrium, and even pulmonary embolism can occur as in our case . In our case, the pulmonary embolism that had been diagnosed in may 2008 was different from the one detected by pulmonary ct angiography in march 2006 . The former is thought to have been induced by thrombi formed by the myxoma in the right atrium, but not by myxoma segments, because the ct density (region of interest [roi] property) was significantly higher in the myxoma detected in may 2008 (roi, -20 for the myxoma in the right atrium as well as for the myxoma in the right pulmonary artery) than in the myxoma detected in march 2006 (roi, 80). In march 2006, heparin administration at admission and maintenance anticoagulant therapy with coumadin resolved thrombi in the pulmonary artery on both sides were performed; however, in may 2008, the myxoma mass in the right atrium was separated and occluded the right pulmonary artery, causing severe dyspnea that required endotracheal intubation . Removal of myxoma in the right side is important for preventing pulmonary embolism, maintaining contractibility, and restoring dilation functions of bilateral atria . Special precautions should be taken during tumor resection in order to prevent thrombus formation and pulmonary embolism . Jones et al . Proposed that complete resection of myxoma should be performed under adequate exposure and minimal manipulation . Furthermore, pulmonary embolectomy should be considered in cases of myxoma associated with pulmonary embolism in order to improve patient symptoms . We reported a case of a 76-year - old woman with myxoma in the free wall of the right atrium . The patient's initial bilateral pulmonary embolism was developed due to thrombi formed by the myxoma; however, the embolism that occurred again two years later was due to myxoma segments.
Cancer remains one of the major causes of death in children between the ages of 1 15 years.1 pediatric cancers differ markedly from adult cancers in their nature, distribution and prognosis . Pediatric oncologists face unique challenges because treatment with irradiation, surgery and chemotherapy can adversely affect the children's growth and development . Though lower compared with the incidence of some adult cancers, it comes next to accidents as the leading cause of death among children in the developed world.1 the patterns of childhood cancer in america and europe are almost the same, with leukemia and tumors of the central nervous system accounting for over one - half of the new cases . However, there is a dearth of data on the incidence and patterns of childhood cancer in africa . Although many papers have been published on this in some african countries,29 reports on the patterns and incidence of childhood cancer in sudan are very few . The objective of this study is to determine the patterns of childhood cancer in gezira state, central sudan . The patterns of cancer were studied focusing on the prevalence of tumors according to age, sex, geographic and ethnic distribution and relating the cancer to environmental and genetic causative factors . All children with cancer, aged one to 15 years diagnosed by means of histological or cytological examination and admitted to the institute of nuclear medicine and molecular biology and oncology from may 1999 december 2004, were included in the study . Gezira is the second largest state in sudan, with an estimated population of 3,962,000 with a 50:50 male to female ratio (49.3:50.7) according to the last population census (1993). Gezira state was considered the richest state in the country before the discovery and extraction of oil . The institute of nuclear medicine, molecular biology and oncology (inmo) which was founded in 1993, has a new department established in 1997 to manage and care for cancer patients in a modern multidisciplinary approach . This is the second oncology centre in sudan and it caters for patients with cancer from gezira state and the surrounding states in the central region of sudan . The results of this study showed a pattern of childhood cancer in patients admitted to inmo during the period (may 1999 dec . 2004). Lymphoma was the most prevalent (42.8%) followed by acute lymphoblastic leukemia (19.8%) and kidney tumors (12.8%). Prevalence of childhood cancer over the period may 1999 december 2004 at inmo distribution of childhood cancer according to place of residence and gender distribution of children according to their tribes types of cancer by age of children admitted to inmo over the period may 1999-december 2004 association between common types of cancers and child tribes the prevalence of tumor in children in our study was higher among boys (64.7%) than girls (35.3%). Most of the children admitted with cancer came from the rural areas (66.1%) compared to 33.9% from urban areas . The distribution of children according to their tribes showed that the majority of them belonged to central sudan tribes (41.8%), followed by kordofan and northern sudan tribes, (both of them recorded 20.3%). Children from the darfur tribes constituted 14.8%, while those from eastern sudan tribes were only 2.7% . The results showed that lymphoma, acute lymphoblastic leukemia and bone tumor commonly occurred in children above five years of age in contradistinction to kidney tumor and retinoblastoma which commonly occurred in children younger than 5 years . The prevalence of non - hodgkin lymphoma in males were found to be 65% while it was 35% in females . There was also a significant association between the children's tribes and the types of cancers at a level of 5% . Although the causes of childhood cancers are largely unknown, a few conditions can be explained with specific chromosomal and genetic abnormalities, and ionizing radiation exposure . Environmental causes have long been suspected by many scientists but have been difficult to determine because it is difficult to identify past exposure levels in children particularly during potentially important periods such as pregnancy or even the time prior to conception . In addition, each of the distinctive types of childhood cancers develops unique clinical course in terms of age, race, gender and many other factors.1 it has been shown that in many developing countries, the reported prevalence of childhood cancer in boys is substantially higher than in girls . The ratio of boys to girls registered with childhood cancer, increased with decreasing gross domestic product and with increasing infant mortality, suggesting that boys are increasingly more likely to be affected than girls with increasing economic disadvantages.10 the ratio of boys to girls in our study is 1.8:1 which agrees with the african trend but differs from the trend in western countries where the female to female ratio is 1:1.1012 the pattern of the cancer in gezira - sudan is like other african countries, with lymphoma as the commonest followed by acute lymphoblastic leukemia and kidney tumor . This was also the pattern found in a study conducted in khartoum in the early nineties.1316 the distribution of the most common three cancers according to age and gender in our study, is in consonance with international trends for lymphoma as a common cancer in children aged more than five years, with non - hodgkin lymphoma being twice more common in males than in females, while hodgkin is five times more common in males than in females . In this study, acute lymphoblastic leukemia differed from the international trend in that it was more common among the children aged more than five years . However, the gender distribution was similar to the international pattern which has females and males equally affected . The prevalence of kidney tumors as evident in our study is in accord with the international pattern which shows that the tumor is prevalent in children less than 5 years of age with slight difference in male to female ratio 1.7: 1 . There was a significant relationship of the prevalence of different cancers in relation to the different sudan tribes . In this study it was evident that the: patterns of cancer in gezira state is like other african patterns but different from those of western countries.lymphoma is the most common cancer in gezira state, with males three times more affected than females . Children above five years of age were shown to be the most commonly affected.in contrast to western countries leukemia is more prevalent in children above five years of age . Patterns of cancer in gezira state is like other african patterns but different from those of western countries . Lymphoma is the most common cancer in gezira state, with males three times more affected than females . In contrast to western countries leukemia is more prevalent in children above five years of age . There is a need for research to determine the annual incidence of different cancers in children in gezira and determine mortality and five year survival rates.a detailed study of environmental risk factors is necessary since gezira state has the largest agricultural scheme in africa and middle east . There is a need for research to determine the annual incidence of different cancers in children in gezira and determine mortality and five year survival rates . A detailed study of environmental risk factors is necessary since gezira state has the largest agricultural scheme in africa and middle east.
It was observed in 1910 that a sufficiently high concentration of guanosine could form a gel, unlike the other nucleobases, and in 1962 it was discovered that four guanosine can self - assemble to form a hydrogen - bonded square, with bonds between the n1o6 and n2n7 positions . This structure is known as a g - tetrad or g - quartet . Like any nucleobase, there is also a strong propensity for these structures to stack on each other via - interactions, forming four - stranded helices called g - quadruplexes, with the phosphate backbone perpendicular to the plane of the g - quartets . The four strands may be from separate molecules, or they may be from only 2 or 1, with loops joining them together [37]. They form with great thermal stability, and have been found experimentally to form from genomic sequences in critical regions such as telomeres, gene promoters and utrs, [9, 10] and to have physiological effects in each of these regions . In telomeres, their formation reduces the activity of telomerase, the upregulation of which has been associated with 85% of cancers, and has led to much pharmaceutical interest . G - quadruplexes in gene promoters, such as the oncogenes c - myc and c - kit, [12, 13] have been shown to control transcriptional activity in vitro, although interestingly their formation can lead to the increase or decrease of activity in different systems . It has been shown that g - quadruplex formation in the 5 utr can decrease translational activity, and there have been suggestions of other physiological effects . A wide variety of proteins have been found to interact specifically with them, and they have been shown experimentally to form in vivo [1618]. G - quadruplexes have also been employed as biosensors (e.g., for thrombin) and in other nanotechnological applications (e.g., [20, 21]). Some of these uses are reviewed in . In parallel with the experimental work being developed, computational techniques have also been developed to predict which sequences will form g - quadruplexes [2224]. There are a variety of different algorithmic rules that can be used to predict which sequences can form g - quadruplexes, [25, 26] although some are more widely used and accepted . There is not sufficient evidence for any of them to be held as absolutely true, and it is only recently that any work has been done to try to predict relative stabilities of possible g - quadruplex structures, rather than just whether they could form or not . Despite this limitation, computational methods have led to a number of discoveries, including the observations that g - quadruplexes are relatively rare in the human genome, but more prevalent than expected in gene promoters . Some of the computational discoveries have been recently reviewed [25, 26]. The field as a whole has grown very significantly in recent times, with a roughly exponential rise in publications (see figure 1), including over 350 in 2009 . A dedicated book has been produced, together with special issues of some journals focused on this topic, and some databases on particular aspects of g - quadruplexes . A series of international conferences has been initiated, the first two hosted in louisville, ky [29, 30]. At the first of these, it was suggested that a central and coherent website to store and provide data related to g - quadruplexes should be produced, and we volunteered to provide such a repository, hosted at the url http://www.quadruplex.org/. Here, we describe the features available at that website, and in particular the core databases to describe predicted g - quadruplexes, and a new tool to estimate the thermal stability of these structures computationally . We also describe the other online sources of predictive data for g - quadruplexes, so that researchers may chose the most appropriate tool for their work . The core quadruplex database (quaddb, http://www.quadruplex.org/?view=quadbase) provides both static and searchable data for researchers on computationally predicted g - quadruplexes (putative quadruplex sequences, pqs). These have been generated as previously described, using our favoured predictive algorithm, which identifies sequences on either strand of the form (g3+n17g3+n17g3+n17g3 +). This has been shown experimentally to be a good predictor of in vitro g - quadruplex formation . It aims to identify specific g - quadruplexes that may form, providing a testable in vitro hypothesis that can be tested using simple biophysical methods . For any researcher interested in identifying pqs in specific sequences, we provide the quadparser program pre - compiled for ms windows and mac os x with detailed instructions . The program is customisable, so that different patterns can be searched for . Different loop length constraints, g - tract lengths and so forth may all be set, so that the algorithm can be adjusted to fit with the particular context desired . Quadparser has a variety of output styles for different uses, and reads sequence data in fasta format . The data search section allows a researcher to identify any pqs in gene promoters (defined as the 1 kb upstream of the tss) or utrs for their gene of interest . The output provides full details of the gene, including genomic parameters, and the location and sequence of pqs in the appropriate regions of every transcript of the gene . Figure 2 displays the output when searching the human genome for pqs in the promoter or utrs of c - kit (hgnc nomenclature kit). Currently, searches may be performed against the human, chimpanzee and mouse genomes . As a convenient alternative to gene - by - gene searches or using the quadparser program we currently offer this data for human (builds 34, 35 and 36 for back compatibility), chimpanzee (2.1), mouse (37), rat (3.4), dog (2), chicken (2), zebrafish (7), fruitfly (5.4), roundworm (180) and yeast (1.01) genomes . In each case the data provides a genomic coordinates for each pqs, together with the strand, sequence and a unique identifier . The thermal stability of g - quadruplexes varies with the concentration of monovalent cations, specifically na and k. however, even for fixed concentrations, the exact details of the sequence, and hence the structure formed, make a very large difference . G - quadruplexes can vary from those which are too unstable to form at 5c to those which will resist temperatures above 95c . It is therefore necessary not just to predict which sequences can form g - quadruplexes at all, but also the stability with which such sequences can form . Such experiments are relatively easy to perform, and have led to a series of studies of different aspects of the relationship between sequence and stability [3134]. However, this does not enable prediction of unmeasured sequences, forcing researchers to make informed guesses as to the stability of novel sequences . We recently developed a bayesian learning algorithm that is capable of making accurate predictions of thermal stability for new sequences, having been trained on a collection of measured sequences . We provide an interface to this system at http://www.quadruplex.org/?view=quadpredict, enabling researchers to make easy predictions of melting temperatures under various conditions for any desired sequence . One feature of the bayesian inference we use is that in addition to predictions of the melting temperature, we also provide uncertainties in the values for each sequence . In general, the uncertainty increases for sequences that are highly unlike those in our training set . This therefore enables researchers to decide rationally how much faith to place in a particular prediction . We intend to develop the training data further, and have already employed a rational active learning protocol to collect more data and reduce the uncertainties below that originally presented . We will continue to do this, and also provide an opportunity for researchers to contribute their own data, so that the bayesian inference can be increasingly accurate . We hope that depositing data publicly may become a standard requirement for publication of g - quadruplex thermal data . We allow researchers to discover whether particular sequences they are interested in are already in our database of measurements, with information about exactly how such an experiment was performed . We hope that these facilities will prove useful to all those working in this field . As well as those interested in biological aspects of g - quadruplexes, we feel this facility may be particularly helpful for those working in nanotechnology or materials science, providing them with a method of rationally selecting g - quadruplex - forming oligonucleotides . There are a number of other tools that may be used to predict the existence of g - quadruplexes in dna, and links to these are provided from http://www.quadruplex.org/. Bagga and coworkers use a similar algorithm to quadparser called qgrs mapper . It has different default parameters, in particular looking at sequences with fewer consecutive guanines and longer loops, but essentially looks for much the same sequences . Interestingly, it includes a scoring parameter for different possible g - quadruplexes that can be formed . Although this is loosely based on empiric evidence, it is not clear how the g - score produced, which ranges up to a maximum of 105, relates to stability . To the best of our knowledge, no empiric tests have been performed testing the validity of the g - score even as a ranking list, but it is still a useful formulation of established rules of thumb . As well as the qgrs mapper, which also provides the facility to search by genes, they also provide specialised databases, grsdb2 and grs_utrdb, for searching pre - mrnas and utr sequences . Maiti and coworkers offer a site called quadfinder, which implements essentially the same algorithm as quadparser . (at the time of writing it does not appear to be functioning .) At the same institute, chowdhury and coworkers have a site called quadbase, again using essentially the same algorithm . They focus on cross - species analysis, offering an ortholog analysis for finding conserved g - quadruplexes, across either prokaryotes (proquad) (and see) or eukaryotes (euquad). It should be noted that the conservation required is by presence, and no sequence comparison is performed . Lastly in this category is the greglist database of potential g - quadruplex regulated genes, which lists all human genes that have a g - quadruplex in the 1 kb region upstream of the transcription start site . A completely different approach to g - quadruplex prediction is taken by the maizels lab [42, 43]. Whereas other methods aim to predict specific g - quadruplex sequences, largely driven by the desire of structural biologists to have structures to study, and by the desire of medicinal chemists to have a defined form to target, the g4 calculator from eddy and maizels accepts that many of these structures are highly polymorphic in vivo . As a result, they do not aim to predict individual structures but look at the density of sequences likely to lead to g - quadruplex structures . Given that this is an entirely orthogonal approach, it is striking that in many cases, particularly working on the gene functions that are likely to be regulated by g - quadruplexes, very similar conclusions arise from using this approach as the quadparser model . We strongly recommend that for any large - scale genomic studies, both approaches are used to corroborate the results found . Computational methods have been of great use in understanding the role that g - quadruplexes may play in biology, unveiling their function in gene promoters [27, 42] and in regulating translation . They have also revealed that stable g - quadruplexes are generally located in nucleosome - free regions . Stability predictions have been used to develop experimental methods to directly visualise g - quadruplexes using afm . We anticipate that greater availability of ever more reliable tools will both improve the quality of informatic research in this area and make it increasingly easy for experimentalists to access computational results.
While over 90% of vulvar malignancies are squamous cell carcinomas, other histologic types include melanomas, basal cell carcinomas, sarcomas, extramammary paget's disease, and bartholin gland adenocarcinomas . Ectopic breast tissue in the vulva was first described by hartung in 1872, and since that time, several cases of both benign and malignant lesions arising in vulvar mammary tissue have been reported (van der putte, 1994). The histology of the mammary - like tissue has multiple forms, ranging from simple, wide, slightly coiled tubular glands with a smooth outline, to more complicated forms in which the coiled structure has numerous branches and acini forming lobules . Similar to normal breast tissue, both epithelial and myoepithelial cells are present and the glands are subject to both the dysplastic and malignant changes that are seen in normal breast tissue (van der putte, 1994). We report a case of a mammary - like invasive carcinoma presenting as an asymptomatic vulvar nodule . A 65 year old, multiparous, postmenopausal female status - post total hysterectomy with bilateral salpingo - oophorectomy 39 years prior for symptomatic myomatous uterus, presented for her annual well woman exam . She was without complaints and review of systems was negative; specifically, she denied any recent history of unintended weight loss or gain, fever, chills, nausea, melena or hematochezia, vaginal bleeding or discharge, abdominal pain or fullness . Her last mammogram was 12 years prior, after which she was diagnosed with right breast ductal papilloma which was excised . In addition to her prior hysterectomy, gynecologic history was significant for estrogen replacement therapy for 20 years, which she stopped after developing what she reported as two years prior to presentation, she had such a lesion removed from her right labia that had been present for one year and pathology was benign, per patient . On further discussion, she reported that the lesion had returned, was not painful, and was not associated with any further growth for the last six months . Clinical breast examination did not demonstrate evidence of palpable masses, retraction, skin changes, or axillary adenopathy . Genital examination revealed a mobile, soft, non - tender, 2.5 0.75 cm nodule at 11 o'clock on her right labia majora . Excisional biopsy was performed in clinic, which revealed ductal carcinoma in situ with positive margins . The patient then underwent a wide local excision of the area, which pathologically revealed invasive carcinoma with negative margins . The specimen from the wide local excision revealed a surface which was predominantly dark brown and wrinkled with a smooth, white, flat lesion measuring 0.8 0.3 cm; the lesion was 1.2 cm from the 12 o'clock margin and 0.4 cm from the nearest (3 o'clock) margin . On serial sectioning, the lesion was firm and white, and extended to a depth of 0.3 cm . Microscopic examination revealed invasive carcinoma, at least 1.6 mm in greatest dimension (fig . 1), arising from within a background of mammary - like glands of the vulva demonstrating residual focal dcis . The invasive lesion was noted to come within 1.3 mm of the margin, but all margins were negative for invasive disease . Immunohistochemical (ihc) stains including smooth - muscle heavy chain (smm - hc), cd 10, and p63 were performed and while there appeared to be some myoepithelial cells present along the smooth contoured edge by smm - hc only, there were other areas without any definitive basal cell / myoepithelial cell staining (fig . 2). The area of concern demonstrated an irregular growth pattern (no longer lobulocentric) and was not associated with the prior biopsy site changes . The tissue was then tested for hormone receptor status, and was found to be estrogen receptor (er) positive, but progesterone receptor (pr) and her2/cep17 negative . Secondary to narrow margins, re - excision was performed as well as an ipsilateral groin sentinel lymph node dissection, both of which were negative for dcis or malignancy . After referral to a medical oncologist, she was also administered letrozole, an aromatase inhibitor, as adjuvant therapy for the er positive cancer . A relative paucity of reports has documented complications of ectopic breast tissue in the vulva, beginning with hartung in 1872 . Mammary ridges were theorized to have developed in a line from the axilla to the groin, and while the large majority of these ridges would normally regress, it was possible for rudimentary tissue to persist and develop into supernumerary mammary tissue (van der putte, 1994). These mammary - like glands have an unknown function, but were distinguished from eccrine and apocrine glands . The glands are simple tubular structures and are sparsely concentrated; however, in some women they are more numerous and assume more complicated forms that resemble mammary tissue (van der putte, 1994). The more complicated forms contain the basic coiled structure, but are obscured by numerous branches and acini which form lobules . They are distinctly different from eccrine and apocrine glands by multiple factors including: 1) the type of epithelium, 2) the formation of diverticula, branches, and lobuli, 3) the shedding of clusters of epithelium, and 4) the expression of receptors for estrogen and progesterone . Additionally, mammary - like glands are also noted to be different than normal mammary tissue derived from the mammary ridges for multiple reasons as well: 1) their simpler configuration, 2) the higher number of glands than would be expected in rudimentary elements from mammary ridges, and 3) the direct relationship to eccrine glands . Furthermore, based on human embryologic studies, the mammary ridges could not involve the anogenital area, as at the height of the formation of the mammary ridges in 9 mm and 10 mm embryos, the labia majora are still far from their first appearance (van der putte, 1994). However, ectopic breast tissue has been described in the vulva, as well as in any other area of the body . Both ectopic breast tissue and mammary - like glands are susceptible to the physiologic, dysplastic, and malignant changes seen in normal breast parenchyma (castro and deavers, 2001), as there have been reported cases of invasive ductal carcinoma, ductal carcinoma in situ (dcis), lobular carcinoma, mucinous adenocarcinoma, phyllodes tumors, and fibroadenomas in accessory breast tissue in the vulva (lopes et al ., 2006). Although ectopic mammary tissue in the vulva is believed to be present in two to six percent of women, few cases of invasive ductal carcinoma have been reported in the literature (kazakov et al ., 2011). The most common clinical presentation is a painless, solitary nodule, arising most often in the labia majora . Patient age at presentation has been between 45 and 84 years, with most being 60 or older (kazakov et al ., most carcinoma cases are histologically consistent with invasive ductal carcinoma, which is also the most common histology in orthotopic breast tissue (kazakov et al ., 2011). This still leaves a differential diagnosis of primary mammary carcinoma, carcinoma of a skin appendage, or metastatic mammary carcinoma . Recognition of an in situ component is the best clue to establishing the primary origin in the skin, as was seen in our case, rather than a metastatic process (kazakov et al ., 2011). Secondary to the rarity of these lesions, there is no widely accepted recommended management strategy . A recent review only identified approximately 25 cases reported in the literature (benito et al ., 2013). Management strategies have ranged from wide local excision for dcis to radical vulvectomy with bilateral inguinofemoral nodal dissection for invasive carcinomas (irvin et al ., 1999). Adjuvant therapies have included radiation, anthracycline - based chemotherapy, and hormonal therapy, in an attempt to decrease recurrence and improve survival . Patients that did not receive adjuvant therapy had a median survival of 4 months (lopes et al ., 2006). Only 1 patient out of 8 who received adjuvant therapy had died at the time of the previous publications, and the median survival was greater than 22 month (lopes et al ., 2006, irvin et al ., 1999). Not surprisingly, there have been no clinical trials to evaluate the effectiveness of chemotherapy and radiation in such a small number of patients, so information on treatment has been, and should continue to be, extrapolated from treatment of cancers in the breast i.e. Excisional procedure with or without lymph node dissection, followed by other adjuvant therapies as needed . There is little to no data reporting on the utility of sentinel lymph node biopsy in this clinical scenario . Breast carcinoma is almost exclusively managed using sentinel lymph node mapping (krag et al ., 2010), while promising data support the use of this technique for vulvar cancer (levenback et al ., 2012). Vulvar cancer is ideally suited for sentinel lymph node mapping secondary to the predictable lymphatic drainage of the vulva, the low false negative rate, and the substantially reduced morbidity (e.g. Lymphedema) compared to a full groin lymph node dissection (levenback et al ., 2012). We demonstrate this to be a potentially feasible technique for primary breast adenocarcinomas arising within the vulva in mammary - like glands, and in the context of such a rare histology, may be included in the management options of this disease.
The 25 years millennium development goals target number 4 (mdg4) of the united nations required the reduction of the mortality rate of children below 5 years of age (u5mr) by a factor of 67% by the end of 2015 . This translated to the saving of extra 520 babies per day out of the estimated 781 that died every day in nigeria . This was a huge and difficult task that required proactive thinking and development and implementation of revolutionary ideas given the antecedents of such a struggling low - resource country as nigeria . All kinds of money - guzzling programmes that were based on foreign but internationally standardised methods have been executed in nigeria from the inception of the mdg4 . Various international donor agencies identified with a number of paediatrics healthcare needs donated money and collaborated with nigerian ministries of health at federal (fmoh) and state levels for the eradication of diseases and epidemics and general improvement of child health . Large sums of money have been spent in a wide spread recruitment and engagement and reengagement of almost the same sets of expertise in a variety of changing patterns by fmoh . Unfortunately the u5mr has remained high as all these efforts have made little progress towards this ultimate goal . The literature has revealed that a large number of babies die within the first 28 days of life (neonatal period) in many tropical countries . This includes nigeria where over 660 newborn babies were estimated to die every day according to a more recently published report . It has also been demonstrated that neonatal mortality rate (nnmr) accounted for up to 40% of the total u5 mortality . This was quite significant than to be ignored as was realised by some latin american countries . By simple mathematics, it could be explained that any measures taken to reduce nnmr would contribute significantly towards the realisation of mdg4 . However, most of the international and national collaborative programmes repeatedly targeted older children without significant impact on the neonates . Unfortunately, even if the rest of older u5 children were all saved without a significant reduction in nnmr, the mdg4 target of 67% would still not be realised . Many years on from inception, nigeria could not demonstrate significant mdg4 progress going by the ten - year timeline between ibe's increasing admission delivery and ogunlesi et al . Kangaroo - mother - care (kmc) techniques of keeping babies warm may scarcely work for big neonates . However extreme preterm babies (<29 weeks ga) or extreme low - birth - weight babies (<1000 g) might likely die without proper incubator intervention . A typical nigerian special - care - baby - unit (scbu) could have a daily census of up to 45 neonates on admission at the same time of which 15 or more would be incubator - dependent very - preterm or extreme - preterm babies [7, 10]. Such common scenario implied a basic requirement of minimum 20 functional incubators at any given time for a standard nigerian scbu . Sadly no referral centre could demonstrate the availability of up to 4 functional incubators for a consecutive time period of two years . It was rather common to see a large number of dysfunctional and obsolete incubators littering the hospital walkways, workshops, dump sites, and scrap yards whilst the scbus remained empty . The unaffordability of reliable incubator systems in low - income settings was a well - known fact as a modern incubator sold in excess of 25,000 . Poor spare parts supply chain and unstable and erratic power supply constituted some of the harsh operating conditions for the nondurability of these systems whenever any few could be procured . Since such large number of functional incubators must be available on a continuous long term bases to guarantee improved and sustainable neonatal survivability, nigeria should have reconsidered other options in order to save its babies . Individual referral centres in nigeria could have adopted nonconventional measures to create new standards that might save more babies and hence enable the realisation of mdg4 target . The aim of this work was to investigate how remedies to some identified issues of concern might have changed practice and overall neonatal survival rate in few hospitals in nigeria . A collaborative medical outreach was launched in 2003 as a private research initiative to apply some willing nigerian tertiary / referral hospital neonatal centres to develop affordable incubators and techniques that could tackle lack of functional systems at the centres . The application of recycled incubator technology (rit) developed gradually and became popular among a number of leading teaching hospitals scattered across the landscape of nigeria . In a resolution, the committee of chief executives of federal tertiary health institutions (ccefthi) of nigeria endorsed the new application and moved into collaboration in using this to save more babies in a few prospective special care baby centres in nigeria . The new application, though in insufficient number in these centres, provided continuous services that soon revealed the other practice deficiencies that contributed to poor outcome other than lack of incubators . These were individually experimented upon by the provision of prospective solutions before adoption as standard remedies . All participating centres readily adopted the proposed concern remedies, albeit with varying degrees of implementation with which the participating hospitals distinguished themselves based on their various outcomes . In order to investigate how these remedies might have corroborated with the level of practice outcomes from each centre a centre could score a maximum of 5 points on each of the concerns remedies if fully implemented or nothing if completely ignored . The recruitment of hospital centres into this scholarly project followed a natural desire of the hospital management of each prospective centre to reduce their prevailing high neonatal mortality rate (nnmr). Earlier publications demonstrated the trial, introduction, and standardisation of an incubator recycling technique that has safely been applied with high reliability and safety records for up to ten years [1012]. The systems were often fully restored at costs that were smaller than 25% of the costs of modern incubators . The recycled - incubator - technology (rit) systems were constructed using internet - sourced generic components arranged by design and applied to the old casings . The new low - cost systems so - produced were capable of 10 years of life expectancy and easily maintainable by locals [10, 12]. Finished products have been applied in the main referral hospitals that presently serve up to 20 states in nigeria (figure 1). The rit was applied to extensively improve the capacity of outreach centres in terms of the number of functional incubators available to handle the high volume of admission delivery that was on the increase . A good example was the lagos university teaching hospital (luth) that grew from no available functional incubator in january 2007 to become nigeria's largest centre with 38 units of functional incubators and 8 units of neonatal resuscitaires (total 46 units) by december 2013 . These systems were distributed within 3 large hospital wards that looked after babies born within the hospital (inborn), babies referred from outside (outborn), and paediatrics surgical ward, respectively . The concern of incubator availability in each centre was scored as follows: 04 functional incubators received zero (0 pt), 59 (1 pt), 1014 (2 pts), 1519 (3 pts), 2024 (4 pts), and 25 or more (5 pts). The introduction of rit systems and the associated outreach amongst nigerian hospitals particularly popularised attainment of sustainable and consistent presence of functional incubators in any nigerian centre . Prior to this, the use of the very old and crude technique of hot - water - bottles was the most popular method of providing warmth to the neonate at many premier teaching hospitals in nigeria . The sustained presence of functional incubators at the centres soon exposed the other deficiencies of nursing and clinical skills associated with incubator care . It became necessary to develop two levels of elective courses for clinicians and nurses in order to instruct on the various unacceptable practices being exposed . Most of these awful practices ultimately led to infections and cross - infections, as primarily witnessed and evidenced from patient's case - notes, thereby impoverishing overall outcome (figure 2). At the time of preparing this paper, over 1600 candidates had passed through level-1 course and this equipped candidates with a more advanced skill of patient - specific thermoneutral control in cases of very prematurity and very low birth - weight . A perceived improvement on overall outcome through these courses in some centres made the training become an important component for career progress for the staff of the centres . Courses were executed as frequently as the hospitals called for this and up to two streams in a year . Centres were given 1 pt for each course organised between april 2011 and march 2013 up to a maximum of 5 pts . Many centres accepted rit as affordable option of restoring proper neonatal incubation in the scbus . This led to the desire to increase their incubator capacities beyond what the available old / abandoned systems could number to adequately accommodate the increasing admission of incubator - dependent neonates . Hospitals were assisted to obtain casings of used or obsolete models at give - away prices through some foreign agencies . The obtained systems were then restored to functionality using rit components and methods, hence making more incubators available for the babies (figure 3). No assessment score was assigned to this as this application reflected in the scoring of concern-1 . Academics within individual centres were invited to form local research groups and supported to carry out investigative studies of observable phenomena relating to temperature control in the neonate (figure 4). A minimum of one possible research project was proposed to each participating centre accompanied with the provision of scholarly encouragements and affordable material support . This was a difficult concern as the culture of this kind of investigative research was unpopular amongst nigerian clinicians and nurses . Only two individual centres were able to complete and publish any studies [9, 16]. Some of the successful efforts resulted in multicentre publications because the national outlook of this outreach made it possible to extract data on the same phenomenon across centres from different regions of nigeria for comparison [11, 14]. Each centre was awarded 1 pt for every journal publication up to a maximum of 5 pts . Sustainability of the neonatal incubator practice amongst nigerian scbus would be a difficult one if the question of maintenance of spare parts was not addressed . Local independent artisans were assessed and recruited for training wherever available within the cities where the participating hospitals were located . These included welders, painter, electricians, perspex - craftsmen (figure 5(a)), and tailors . These were trained on how to use their own skills and tools at the comfort of their own workshops to reproduce samples of various items of parts they were given . This approach made these parts affordable and readily available whilst creating jobs for the other members of the public . This method had initially been used to assemble a full locally made incubator that has been in use for over 5 years at the federal medical centres (fmc) owerri (figure 5(b)). This was not assigned any centre scoring point because it was not based on how well a hospital performed . Neonatal apnoea episodes were highly frequent conditions in every nigerian scbus corroborating with the country's well - known high neonatal mortality rate . Apnoea might happen as a result of various physiological and clinically diagnosed or undiagnosed factors wilting the neonate . There was no presence of apnoea monitors in any of the centres at the inception of the present outreach . The neonates were so many but attending nurses so few per rota shift, so eye balling was inherently inefficient . Nursing staff strength of the centres was so poor that none of the centres could demonstrate any better nurse - to - neonate ratio of 1: 10 . A typical nigerian busy centre could witness persistent apnoeic attacks on up to 5 babies at the same time . This real life scenario suggested a possibility of losing up to 4 or all of the 5 . This was because at the average of 10-neonate - workload, a nurse attending to the first apnoeic baby might not be aware of the other 4 early enough to save them . Therefore the hospitals were encouraged to (1) work towards 1: 4 ratio as a minimum operational standard by employing more nurses and (2) install individualised cot / incubator fixable apnoea monitors . Market research for an appropriate design and affordable baby breathing / motion monitor was carried out . This yielded a recommendation to apply the bm02 apnoea monitoring system (jablotron, czech republic) on all cots and incubators as minimum standard . Nurse - to - neonate ratio of up to 1: 4 was given the full 5 pts, up to 1: 6 (4 pts), 1: 7 (3 pts), 1: 8 (2 pts), and 1: 9 (1 pt). Apnoea monitors installation up to 3 nos received 1 pt, up to 5 nos (2 pts), up to 10 nos (3 pts), up to 15 nos (4 pts), and up to 20 nos (5 pts). The introduction of rit - incubator and its subsequent application in expanding the capacity of nigerian scbus was intended to ensure availability of adequate number of functional incubators to save more babies . This aim soon began to be frustrated by impoverished mains power supply to operate the incubators when needed . Neonatal incubation being a life support intervention ought not to be switched off to preserve the neonate's life . Unannounced power cuts in nigeria were very frequent and rampant and could last longer than 10 hours for each episode . Hence, neonatal hypothermia defined as body temperature below the physiological range of 36.5c37.4c continued to be a serious concern even when baby managed to secure accommodation in available incubator . The idle period in - between power availability was hence narrowed by the introduction of power - banking technique as a practice standard . 3.5 kva/48 amps inverter - battery system (fussion series, su - kam industries india) for every group of up to 8 incubators and resuscitaires . This was capable of sustaining these systems for up to 10 hours after power failure . If power - bank capacity was increased to power up to 16 or more incubators then the full 5 pts were awarded . Incubators were operated nonstop, day - in day - out, so long as there was an occupant neonate . Hence, this needed to be regularly serviced to guarantee the prevention of avoidable breakdowns . Nigeria had a well - known culture of running systems and appliances to destruction due to neglecting professional maintenance . This often led to long periods of system out - of - use whilst management scrambled all over the place in search of repair or replacement . Fac was introduced as a service of professional advice when needed accompanied with on - site routine system assessment / maintenance . This was a collaboration of professional care that managements were encouraged to enter into via the signing of agreements or memorandum - of - understanding . This automatically empowered a highly reputable third party dedication that ensured the sustainability of the centre's incubator capacity . Professional advice was executed whenever this was called for via electronic communication throughout the year . A score of 5 pts was assigned to centres with unbroken fac service within the last 3 years leading up to march 2013 . Annual performance competition was introduced in 2010 to award prizes to (1) the nation's overall best scbu manager (normally the chief nursing officer of the winning scbu) and (2) the hospital with the best managed collective group of scbus . Each centre was assessed twice a year at 6-month interval for an average annual score but without prior notification of when this would happen in order to capture the actual practice standards of the centre . The 1st and 2nd best hospitals and the best overall manager each received a trophy and various amounts in cash reward . The announcement of the worst scbu of the year was introduced in 2013 . The quest to be announced as best scbu or best manager soon triggered competitiveness that seemed to have positively affected overall practice outcome across the participating centres . Heads of hospital managements were also taking pride in showcasing their strengths in different aspects of the concerns pursuit . The full 5 pts down to 1 pt were given to any centre that achieved 1st up to 5th position in each of the inclusive 3 years in this analysis (i.e., 20112013); average of scores for the three years was awarded . It was expected that positive impact of these concern remedies over time could alter prevailing status of indices such as neonatal mortality rate (nnmr), length of hospitalisation of surviving neonates, and patient influx . The centres were assessed through retrospective data collected from their patient admission / discharge registers . By standards this was supposed to be a summary note - book that captured information such as dates of admission and discharge, birth - weight, gestation age, and overall outcome (dead or alive). Assessment did not require patient's identity to be revealed; however, all participating hospitals were invited to follow their institutional ethical rules to submit their data . Three other big referral hospital centres that were not participating in the outreach were also invited to submit assessment data as the invited control centres represented the common practice status of a typical nigerian scbu and very similar to the states of the outreach centres before intervention began . Submitted sets of data were used to measure three basic indices of clinical success, that is, overall average neonatal mortality rate (nnmr), average length of hospitalisation for the surviving neonates, and the associated patient - traffic (influx). The class of babies that might require incubator intervention for survival, incubator - dependent - neonates (idn), was defined based on the nigerian standards of neonatal classification . This included babies born earlier than 37 weeks of gestation (preterm) or those of birth - weight less than 2500 g (low birth weight). Intrauterine growth restricted (iugr) babies within this boundary were also included provided birth - weight was less than 2500 g. other quantified parameters were the fractional size of idn babies in the presented population and the mortality rate of the idns (idnmr). The total number of idns that died within 48 hours of presentation (d48 babies) was calculated and used to assess how overall nnmr was affected by these . One set of data was to be submitted by each participating centre to measure these performance indicators for the last 24 months leading up to march 2013, tagged post - rit period . Outcomes were compared amongst outreach centres and also against outcomes from control centres . The idn class was further examined to identify the very low birth parameter cases; that is, very - lbw (1500 g) and very - preterm (32 weeks ga) cases . This was applied to investigate how very - low birth parameter cases might be currently affecting overall idn survival statistics at the hospitals . The managements of a very few tertiary hospitals in nigeria accepted to try out the new ideas of recycled incubator technology (rit) initially . All the neonatal care centres responded positively with enthusiasm as the various procedures were being initiated in each centre . The present coverage of the use of rit systems or procedures across hospital centres in the states of nigeria is shown in figure 6 . Many doctors and nurses enrolled for the training courses at various times; some repeated the courses and the associated examinations until they passed them . A good number of the hospitals signed the agreements and operated the failure - preventive audit culture (fac) component of the outreach (figure 7). The managements of few hospitals ensured unbroken fac services, some lasting up to 8 years to the time of this report . Fac was broken a number of times in other hospitals due to administrative successions that never considered this very important but restarted after it became obvious that stoppage had resulted in many scbu disasters . We found that centres with less unbroken fac operated with more consistent or increasing incubator capacities (figures 7 and 8). We observed that there was better structural transformation in hospitals where the management showed higher level of commitment to the entire outreach projects by strictly following their 6-month pac reports and implementing them . Periodic interdepartmental reshovelling of nursing staff as practiced in nigeria no longer affected scbu trained nurses in hospitals where management mandated the proactive participation of the nursing department in the outreach project . There was better improvement of nurse - to - neonate ratio in these hospitals too . We observed that the best performing matrons / nurses were the most regular on courses and seminar attendance . The poorly performing centres in the national competition were mainly those that paid little attention to training courses irrespective of the growth of their incubator capacity . Only two individual centre groups were able to fully complete an investigative research and publish the original journal articles [9, 16]. Only 5 of 15 invited centres submitted their data for inclusion in the present analysis . These were from tertiary hospitals located in the southern nigeria (c1 and c2), middle - belt (c3), and north (c4). Centre (cc), a tertiary hospital in southern nigeria (figure 9). Average neonatal mortality (nnmr) across the outreach centres was computed at 114 deaths per 1000 neonatal admissions (114/1000). When d48 mortality was excluded, death rates were twice higher than these at the control centre (figure 9). Typically, we computed that up to 64% of overall deaths of incubator - dependent - neonates (idn) at the respective centres occurred within 48 hours of presentation . There were very few survivals amongst babies born before 30 weeks gestation or below a birth weight of 1000 g. data also revealed that nearly all of the babies dying within the 48 hour window were either average length of surviving idn hospitalisation at the outreach centres was 20.5 days (average centre range: 832 days). This was not quantified at the control as supplied data lacked such details . The recycled incubator technology (rit) and associated outreach concerns in this study have demonstrated the significant capability of nigerian special care baby units (scbus) in achieving improved newborn survival . An earlier publication to assess the impact of this outreach in 2009 studied performances of the same group of hospitals that were involved in the present analyses . This revealed that facility - based average nnmr had dropped from 254/1000 to 198/1000 babies . This figure was only a reflection of what the country could have achieved if all the nigerian hospital scbus adopted the same techniques as those analysed in the study . The nnmr of our control centre in the present study (250/1000) was very similar to the 254/1000 that was quantified as national average at the inception of the present outreach project as reported by amadi et al . . This is a validation that seemed to suggest that facility - based nnmr within a typical tertiary centre in nigeria might remain within this high figure until the statuses of the concerns that have been identified in this study were positively altered . At the inception of invitation to submit data for a retrospective assessment of the present outreach, all the hospitals invited to submit raw - data accepted with enthusiasm promising to keep up with the provided deadline . However, many of these either failed to comply or out rightly excused themselves from the study for various weak reasons, some of which might have stemmed from the fear of exposing suspected relative poor performance . The few submissions however had the advantage of coming from hospitals in the southern, northern, and middle - belt of nigeria, giving a good national coverage . Our present study has shown that there has been further improvement on the overall outcomes of facility - based performance indices of the outreach centres . Notably, the average nnmr within the facilities has dropped from the figure of 198/1000 as reported by amadi et al . To 114/1000 in the present study . This significant leap must have been a result of the unprecedented dedication to capacity expansion, training, and implementation of fac audit reports by the centres that have been assessed . Analysis was initially designed to use a scoring technique to comparatively measure the performances of entire centres in each of the concerns and to relate these to their respective outcomes . Unfortunately, the analysed data came from just a few of the participating hospitals for which very strong conclusions might not be drawn . However these were the top performing hospitals in the use of the concern remedies (figure 11), and clinical and nursing practices in the centres had not been altered in any other direction since the 2009 assessments of amadi et al . . Hence result could only demonstrate that these concerns were the very vital components that brought about a significant reduction in nnmr as compared to the control . We are not able to accurately quantify the relative contribution of each concern remedy towards the overall lowering of nnmr as these top centres applied varying levels of priority to different concerns . For example, the fmc owerri was enabled to execute up to 7 concerns but particularly favoured continuous retraining of their scbu staff while luth lagos maximised 5 concerns and particularly favoured incubator capacity expansion up to 38 units of functional incubators and 8 units of resuscitaires . This boosted luth's overall baby influx, a turnover of over 150 neonates in some months between patients being admitted in the out - born, in - born . And paediatric - surgery the admission registers of these high performing centres revealed higher influx of neonates as compared to situations prior to the present outreach . Overall baby admission at our control was comparable to other centres and had the third largest neonatal admissions (figure 12). This suggested that the population within the catchment zone of this hospital had significant willingness to seek specialist intervention . However our control recorded the least relative number of idn presentations amongst the admitted babies . This indicates a higher hesitation in the presentation of idn babies to the control centre despite the people's good level of willingness to seek the hospital, perhaps as a result of very low success rate (figure 13). The control centre (cc) saved the least number of idns (74 neonates) amongst the 5 individual centres over the 2-year period that was analysed . This was far smaller than the worst record from any of our outreach centres despite the control's locational and academic advantages as a teaching hospital (figure 14). The clinical team at our control centre argued that the reasons for the poor outcomes might be due to depletion of both infrastructure and skilled manpower as most of the incubators in our scbu were broken down and the few functioning are in serious need of maintenance; also skilled nurses who have training in neonatal care were seldom given the opportunity to remain in the neonatal ward due to reshovelling of nurses around departments irrespective of the special training a nurse might have . Our outreach centre (c4) was probably the most remotely located federal referral hospital in nigeria being situated at extreme northeastern region that was well known for higher infant mortality rates and people's unwillingness to seek hospital care . This centre was highly disadvantaged in terms of its very poor relative number of qualified doctors and nurses, without any fulltime consultant paediatrician . Illiteracy was relatively higher amongst mothers; hence these were very unlikely to seek early medical intervention, orchestrating the common situations of very poor neonatal vital - signs at the point - of - admission . Despite all these c4's index in terms of idnmr was below average rate and was only outperformed by one centre (figure 13). It was our opinion that the overall reduction of average nnmr from 254/1000 to the present 114/1000 amongst our outreach centres was a demonstration of the effectiveness of the provided concern remedies . The present study has also demonstrated the information regarding the neonatal deaths occurring within 48 hours (d48) of presentation . We would suggest that our analysed outreach centres had almost pushed the boundaries of neonatal mortality to the limits except for the d48 babies which constituted up to half of the total mortality in some centres . Most (83%) of the d48 babies were either very lbw or very preterm cases . A further investigation is hence recommended to urgently study the manner of care given to this class of patients, especially in the first 7 days of life, in order to synthesise any possible techniques that might guarantee better survival rate . The corporate nigeria might not have achieved the mdg4 target; however our study has shown that there were pockets of individual hospital centres that have already surpassed this target . For example, our outreach centre c3 has achieved overall nnmr of 89/1000, a reduction of 65% in terms of the national facility - based average as computed in 2009 . It would have been a possibility for nigeria to corporately attain the mdg4 goal had the country paid more attention to the very simple but high impact approaches that were implemented at these few centres.
Short - term intrathecal triamcinolone - acetonide (tca) treatment significantly influences levels of two proteins expressed by astroglia: s-100b and tau protein, suggesting reduction of astrocytic activationin this study, short - term intrathecal tca treatment did not change neurofilament heavy - chain levels, suggesting the absence of a major neuroprotective treatment effectfuture treatment trials may investigate whether hyperacute treatment with intrathecal tca may reduce the inflammatory component of glia in the setting of severe transverse myelitis in multiple sclerosis and possibly also neuromyelitis optica spectrum disorders, and thus improve the clinical outcome multiple sclerosis (ms) is the most common inflammatory disease of the central nervous system in young adults ., the disease disability accumulates, and most patients may enter ongoing progression (progressive ms), whereas acute exacerbation may become less likely . With accumulating disability, patients may suffer from spasms . Consequently, patients report impaired walking and may experience other symptoms, such as unpleasant sensations, pain, and incontinence . A study in sweden revealed that annual costs are 2.4 times higher in patients with severe spasticity than in those with only mild spasticity [57]. Spasmolytic therapeutics taken orally may have systemic side effects, and thus their usage is limited . Intrathecal baclofen therapy and baclofen pumps offer a way to deal with spasticity, but besides the cost factors, systemic side effects and infections are common . Intrathecally applied glucocorticosteroids (gcs) have been used since the 1950s . As therapeutic agents, hydrocortisone and methylprednisolone acetonide side effects such as urinary incontinence, constrictive arachnoiditis, aseptic meningitis, and spinal cord lesions have been reported . However, positive effects on spasticity, with improvement of walking, have also been reported . A number of monocentric trials have shown positive effects of intrathecal gcs application on spasticity [912]. Intrathecal therapy with triamcinolone - acetonide (tca), a derivative of gcs, has been shown to be safe, and no severe side effects have occurred . Intrathecally applied gcs seems to have positive effects on spasticity and may spare ms patients the discomfort of wearing a corset . Nevertheless, in vitro studies have shown that gcs may induce apoptosis in neuronal cells [14, 15]. There is therefore a need to investigate the effects of intrathecal gcs on neurodegenerative markers in the cerebrospinal fluid (csf) to address this issue . Neurofilaments are a highly specific component of the axoskeleton and are released during axonal damage . Neurofilament heavy - chain (nfh) levels in body fluids are an established marker for estimation of axonal damage in a range of neurological disorders . Tau protein, as a phosphorylated microtubule - associated protein, is known as a component of neuronal axons . Released into the csf, it may serve as a biomarker of axonal damage, as shown by previous studies [1921]. Several studies have suggested that these proteins might play a certain role in the regulation of effector proteins and may influence specific parts of signaling pathways or cellular functions . S-100b proteins seem to possess both neuroprotective and neurotoxic properties, and have been used as a marker of astrocytic proliferation and gliosis [2123]. The effects of tca treatment are complex, and its pharmacodynamic effects on tau protein, s-100b, and nfh, as potential predictors of axonal degeneration, glial activation, and astrogliosis in ms patients, have not been extensively characterized . This prospective study aimed to investigate the effects of repeated tca applications on the csf markers suggestive of neurodegeneration: tau protein, s-100b, and nfh in ms patients with severe spasticity . Fifty - four patients suffering from ms, according to the mcdonald criteria, were consecutively recruited to participate in this multicentre study (see table 1). All patients were clinically stable (i.e. They had experienced no relapse or recent progression over the previous 3 months) and were not receiving other immunomodulating or immunosuppressive treatment . Two thirds of the included patients were female and were in the progressive stage of ms . Smirnov test (see table 1).table 1patient characteristicscharacteristicall patientsfemale patientsmale patientspatients (n)543618age (years sd)47.9 9.748.7 9.746.2 9.9course of disease (n) relapsing ms remitting ms463313 primary progressive ms835age at onset of disease (years sd)35.1 8.735.5 8.834.4 8.8disease duration (years sd)12.4 8.413.0 8.511.3 8.1 ms multiple sclerosis, sd standard deviation patient characteristics ms multiple sclerosis, sd standard deviation all patients gave written and informed consent for participation in the trial, which was approved by the local ethics committee at the university of rostock (rostock, germany) and registered with the german clinical trials register (drks; registration no . Depending on the spasticity, the dosage ranged from 40 mg to 80 mg . Furthermore, the applications were done three times or, in cases with severe spasticity, five times over the course of a week . Tca treatments were performed on days 0, 2, and 4 (in patients receiving three applications) and additionally on days 6 and 8 in those receiving five applications . In 26 patients, tca was administered five times, whereas treatment was terminated after three applications in 28 patients because a sufficient antispastic effect had been achieved . The mean dosages over the cycle were 127.4 mg (standard deviation [sd] 22.3) in patients receiving three applications and 205.9 mg (sd 37.3) in those receiving five applications . All lumbar punctures were performed as per routine clinical practice for therapeutic purposes only . Because of the invasiveness of the therapy, no csf from control subjects was obtained . Sprotte cannulas (pajunk medizintechnologie gmbh, geisingen, germany) were used for obtaining csf . After each lumbar puncture, csf was obtained in 10 ml polypropylene tubes and stored within 30 min in 1 ml aliquots at 70 c . The routine parameters of the csf, including the cell count, total protein, albumin, lactate, quantitative immunoglobulin (ig)-g, and oligoclonal igg bands (ocb), were quantified in the local laboratories at each centre . Csf protein biomarkers were quantified in dedicated laboratories (tau protein and s-100b in ulm, germany; nfh in london, uk). Cell - free csf was shipped on dry ice and kept frozen on arrival without interruption of the cooling chain . Mouse monoclonal anti - nfh antibody smi35r was used for capture (covance, princeton, nj, usa), rabbit polyclonal anti - nfh was used as the detecting antibody (sigma - aldrich, st . Louis, mo, usa), and horseradish - labeled swine polyclonal anti - rabbit antibody was used as the indicator antibody (dako, copenhagen, denmark). Chemicals were obtained from sigma - aldrich (ethylenediaminetetraacetic disodium salt [edta], sodium barbitone, sodium carbonate [na2co3], sodium hydrogen carbonate [nahco3], tween 20 [soap], bovine serum albumin [bsa]), from merck (darmstadt, germany; hydrochloric acid [hcl]), and from dako (tetramethylbenzidine [tmb]). For analysis, tau protein levels were determined using a commercial sandwich enzyme - linked immunosorbent assay (elisa) [innotest htau; innogenetics, zwijndrecht, belgium] based on the original method of vandermeeren . The assay was performed according to the protocol supplied with the kit, and csf tau protein levels in the samples were estimated from standard curves made for each assay . S-100b protein levels were measured by means of an immunoluminometric assay kit (lia - mat sangtec 100; ab sangtec, bromma, sweden), which measures the b subunit of s-100b protein . Statistical analyses were performed with spss version 15.0 (chicago, il, usa) and ibm spss statistics version 20 software (armonk, ny, usa). The kolmogorov csf parameters, socio - demographic parameters, and clinical parameters were analyzed with the kruskal for analysis over the course of time, the friedman test and the wilcoxon t test were used . Statistical significance was set at the 95% confidence level (p <0.05). Fifty - four patients suffering from ms, according to the mcdonald criteria, were consecutively recruited to participate in this multicentre study (see table 1). All patients were clinically stable (i.e. They had experienced no relapse or recent progression over the previous 3 months) and were not receiving other immunomodulating or immunosuppressive treatment . Two thirds of the included patients were female and were in the progressive stage of ms . Smirnov test (see table 1).table 1patient characteristicscharacteristicall patientsfemale patientsmale patientspatients (n)543618age (years sd)47.9 9.748.7 9.746.2 9.9course of disease (n) relapsing ms remitting ms463313 primary progressive ms835age at onset of disease (years sd)35.1 8.735.5 8.834.4 8.8disease duration (years sd)12.4 8.413.0 8.511.3 8.1 ms multiple sclerosis, sd standard deviation patient characteristics ms multiple sclerosis, sd standard deviation all patients gave written and informed consent for participation in the trial, which was approved by the local ethics committee at the university of rostock (rostock, germany) and registered with the german clinical trials register (drks; registration no . The dosage ranged from 40 mg to 80 mg . Furthermore, the applications were done three times or, in cases with severe spasticity, five times over the course of a week . Tca treatments were performed on days 0, 2, and 4 (in patients receiving three applications) and additionally on days 6 and 8 in those receiving five applications . In 26 patients, tca was administered five times, whereas treatment was terminated after three applications in 28 patients because a sufficient antispastic effect had been achieved . The mean dosages over the cycle were 127.4 mg (standard deviation [sd] 22.3) in patients receiving three applications and 205.9 mg (sd 37.3) in those receiving five applications . All lumbar punctures were performed as per routine clinical practice for therapeutic purposes only . Because of the invasiveness of the therapy, no csf from control subjects was obtained . Sprotte cannulas (pajunk medizintechnologie gmbh, geisingen, germany) were used for obtaining csf . After each lumbar puncture, csf was obtained in 10 ml polypropylene tubes and stored within 30 min in 1 ml aliquots at 70 c . The routine parameters of the csf, including the cell count, total protein, albumin, lactate, quantitative immunoglobulin (ig)-g, and oligoclonal igg bands (ocb), were quantified in the local laboratories at each centre . Csf protein biomarkers were quantified in dedicated laboratories (tau protein and s-100b in ulm, germany; nfh in london, uk). Cell - free csf was shipped on dry ice and kept frozen on arrival without interruption of the cooling chain . The original method has previously been described in detail . In brief, immunoassays were performed for quantification of nfh . Mouse monoclonal anti - nfh antibody smi35r was used for capture (covance, princeton, nj, usa), rabbit polyclonal anti - nfh was used as the detecting antibody (sigma - aldrich, st . Louis, mo, usa), and horseradish - labeled swine polyclonal anti - rabbit antibody was used as the indicator antibody (dako, copenhagen, denmark). Chemicals were obtained from sigma - aldrich (ethylenediaminetetraacetic disodium salt [edta], sodium barbitone, sodium carbonate [na2co3], sodium hydrogen carbonate [nahco3], tween 20 [soap], bovine serum albumin [bsa]), from merck (darmstadt, germany; hydrochloric acid [hcl]), and from dako (tetramethylbenzidine [tmb]). For analysis, tau protein levels were determined using a commercial sandwich enzyme - linked immunosorbent assay (elisa) [innotest htau; innogenetics, zwijndrecht, belgium] based on the original method of vandermeeren . The assay was performed according to the protocol supplied with the kit, and csf tau protein levels in the samples were estimated from standard curves made for each assay . S-100b protein levels were measured by means of an immunoluminometric assay kit (lia - mat sangtec 100; ab sangtec, bromma, sweden), which measures the b subunit of s-100b protein . The assay was performed according to the manufacturer s protocol . The original method has previously been described in detail . In brief, immunoassays were performed for quantification of nfh . Mouse monoclonal anti - nfh antibody smi35r was used for capture (covance, princeton, nj, usa), rabbit polyclonal anti - nfh was used as the detecting antibody (sigma - aldrich, st . Louis, mo, usa), and horseradish - labeled swine polyclonal anti - rabbit antibody was used as the indicator antibody (dako, copenhagen, denmark). Chemicals were obtained from sigma - aldrich (ethylenediaminetetraacetic disodium salt [edta], sodium barbitone, sodium carbonate [na2co3], sodium hydrogen carbonate [nahco3], tween 20 [soap], bovine serum albumin [bsa]), from merck (darmstadt, germany; hydrochloric acid [hcl]), and from dako (tetramethylbenzidine [tmb]). For analysis, tau protein levels were determined using a commercial sandwich enzyme - linked immunosorbent assay (elisa) [innotest htau; innogenetics, zwijndrecht, belgium] based on the original method of vandermeeren . The assay was performed according to the protocol supplied with the kit, and csf tau protein levels in the samples were estimated from standard curves made for each assay . S-100b protein levels were measured by means of an immunoluminometric assay kit (lia - mat sangtec 100; ab sangtec, bromma, sweden), which measures the b subunit of s-100b protein . The assay was performed according to the manufacturer s protocol . Statistical analyses were performed with spss version 15.0 (chicago, il, usa) and ibm spss statistics version 20 software (armonk, ny, usa). The kolmogorov csf parameters, socio - demographic parameters, and clinical parameters were analyzed with the kruskal for analysis over the course of time, the friedman test and the wilcoxon t test were used . Statistical significance was set at the 95% confidence level (p <0.05). The low rate of headache may have been due to the fact that tca suspension dissolved in saline was administered . The clinical outcomes showed a significantly improved (p 0.001) walking distance . On average, the walking distance improved in all patients from 247 m (sd 302) to 322 m (sd 346). Spasticity was measured on the ashworth scale and improved significantly (p 0.001) during tca applications from 2.2 (sd 1.3) at baseline to 1.6 (sd 1.1) at the end of the cycle . Csf markers were determined during application of tca at baseline (the first application of tca), on day 4 (the third application of tca), and on day 8 (the fifth application of tca). Patients were given tca three times (n = 54), though in cases of severe spasticity (n = 26), tca was applied five times . Besides the aforementioned markers of neurodegeneration, the cell count was determined as part of the routine assessment . Further csf analysis revealed a decreasing cell count from 10.8 (sd 18.9) at baseline to 6.3 (sd 6.2) on day 8 . Nfh levels in the csf of patients treated with tca intrathecally did not increase significantly (baseline level: 0.11 ng / ml [sd 0.11]; level at third application [after two tca applications]: 0.14 ng / ml [sd 0.15], p = 0.053; level at fifth application [after four tca applications]: 0.16 ng / ml [sd 0.15], p = 0.068) during the treatment (see fig . 1). In all patients, csf nfh levels remained within the normal range (maximum value 0.58 ng / ml, below the cut - off of 0.73 ng / ml).fig . 1concentration of nfh during the first cycle of triamcinolone - acetonide (tca) therapy . Nfh neurofilament heavy - chain concentration of nfh during the first cycle of triamcinolone - acetonide (tca) therapy . Nfh neurofilament heavy - chain after application of tca, tau protein levels were increased significantly on day 4 (from 194.07 pg / ml [sd 82.47] at baseline to 250.31 pg / ml [sd 168.84], p = 0.03) and on day 8 (245.54 pg / ml [sd 121.79], p 0.001) [see fig . 2concentration of tau protein during the first cycle of triamcinolone - acetonide (tca) therapy . Statistical analysis: friedman test, wilcoxon test concentration of tau protein during the first cycle of triamcinolone - acetonide (tca) therapy . Statistical analysis: friedman test, wilcoxon test s-100b protein levels decreased significantly over the complete cycle (from 1.18 mg / l [sd 0.66] at baseline to 1.00 mg / l [sd 0.44] on day 8, p 0.05). The effect could already be seen by day 4 (1.01 [sd 0.46], p 0.05) [see fig . 3concentration of s-100b protein during the first cycle of triamcinolone - acetonide (tca) therapy . Statistical analysis: friedman test, wilcoxon test concentration of s-100b protein during the first cycle of triamcinolone - acetonide (tca) therapy . Statistical analysis: friedman test, wilcoxon test the results for nfh, tau protein, and s-100b did not differ significantly between the relapsing ms remitting ms patient group and the primary progressive ms patient group . Intrathecal tca is an efficient therapeutic way to treat spasticity and spare ms patients the discomfort of wearing a corset . It has proven its efficacy in a number of trials [2426]. In vitro experiments have suggested that gcs has neurotoxic properties [14, 15, 17]. In our trial, we measured the levels of nfh, tau protein, and s-100b protein, which are well accepted as surrogate markers of axonal injury and degeneration, neuronal damage [20, 28], and activated astroglia, mainly reflecting parenchymal damage [22, 29]. Nfh, as a surrogate marker of axonal damage, was measured for the first time in patients receiving tca treatment . The results did not reach significance and remained below the assumed critical value of 0.73 ng / ml . Neurofilament activity varies between the different stages of disease and is influenced by clinical activity . In respect thereof, all of our patients were stable with no relapse for 3 months prior to the first application of tca and, although the patients were recruited at different stages of the disease (relapsing ms and progressive ms), with differing expanded disability status scale (edss) scores, the results with regard to nfh did not vary significantly in the intergroup comparison . Another limitation might be the fact that only patients with low nfh levels at baseline were included in the trial . It would have been interesting to see what would have happened to patients with pathological nfh levels at baseline . However, the mean value did not reach the critical value of 300 pg / ml . The question of to what extent such an increase has clinical implications remains open . In a study by hoffmann et al . In 27 patients, tca was applied six times, and the levels of tau protein and s-100b did not increase significantly . Research into the long - term effects of tca on tau protein levels may elucidate whether increased levels have further clinical implications . Currently, the importance of tau protein in ms patients is not completely understood . To what extent this increase could have been explained by release of tau protein from the cytoskeleton of neurons or from glial cells in our patients remains open . The importance of tau protein in neuroinflammation and neurodegeneration in ms is not fully understood . Whereas animal models have suggested a link between axonal damage and tau pathology, findings elucidating tau protein levels in the csf of ms patients have been partly contradictory [3133]. Besides the increases in tau protein levels (significant) and nfh levels (non - significant), a decrease in s-100b levels reduced astrocytic activity during tca treatment may be due to corticosteroid - mediated immunosuppressive effects on microglial activation . As a result, the levels of interleukin-1 might decrease and lead to minor astroglial activation . However, in vitro studies have shown that adrenocorticotropic hormone is responsible for increased secretion of s-100b . At present, this phenomenon is not understood in detail . The neurotoxic and neuroprotective effects of s-100b protein have been discussed previously . To understand the effects of tca on astrocytic proliferation, further assessments are necessary . A dose - dependent excitatory effect of methylprednisolone on cultured neuronal networks could be demonstrable [37, 38]. Intrathecal tca therapy seems to be efficient and well tolerated for the treatment of symptoms in ms patients . Future treatment trials may investigate whether, in addition to symptomatic effects, tca can reduce the inflammatory component of glia in the setting of severe transverse myelitis in ms and possibly also neuromyelitis optica spectrum disorders, and thus improve the clinical outcome . The results of our study did not show neurotoxic effects; the levels of tau protein increased significantly but remained within the reference range . Future long - term studies are needed to clarify the neurotoxic effects of tca treatment.
A total of 3,316 patients, who were referred for coronary angiography to ludwigshafen heart center in southwest germany, were recruited between july 1997 and january 2000 . Inclusion criteria were german ancestry, clinical stability except for acute coronary syndromes, and the availability of a coronary angiogram . One of the original purposes of the study also was the identification of genetic components of coronary artery disease (cad). To avoid population admixture, we aimed at including caucasians only . For pragmatic reasons, we therefore only included patients the indications for angiography in individuals in clinically stable condition were chest pain and/or noninvasive test results consistent with myocardial ischemia . Individuals suffering from any acute illness other than acute coronary syndromes, chronic noncardiac diseases, or malignancy within the 5 past years and those unable to understand the purpose of the study were excluded . People with known diabetes were excluded because c - peptide levels change over the course of diabetes with a high interindividual variability and are in addition altered by different antidiabetic drugs . Moreover, a long duration of type 2 diabetes confers increased cardiovascular risk, thus being an additional potential confounder . Moreover, subjects with missing data on fasting c - peptide were additionally ruled out, resulting in a subgroup of 2,306 (69.5%) of 3,316 ludwigshafen risk and cardiovascular health (luric) study participants for the present analyses . These subjects were excluded when the associations of c - peptide with cardiovascular mortality were analyzed . The study was approved by the ethics committee at the rztekammer rheinland - pfalz and was conducted in accordance with the declaration of helsinki . Diabetes was diagnosed according to the 2010 guidelines of the american diabetes association (15). Fasting glucose, postchallenge glucose (performed in 1,524 of 2,096 participants with fasting glucose <126 mg / dl), and glycated hemoglobin were used to categorize diabetes . Hypertension was diagnosed if the systolic and/or diastolic blood pressure exceeded 140 and/or 90 mmhg or if there was a history of hypertension, evident through the use of antihypertensive drugs . Acute myocardial infarction (mi) was defined as an mi that had occurred within the 4 weeks prior to enrollment . A definite st elevation mi (stemi) was diagnosed if typical electrocardiogram changes were present along with prolonged chest pain, refractory to sublingual nitrates, and/or enzyme or troponin t elevations (> 0.1 g / l). Non - stemi was diagnosed if symptoms and/or troponin t criteria, but not the electrocardiogram criteria for stemi, were met . Cerebrovascular disease was defined clinically by documented history of a previous cerebrovascular disease event (transient ischemic attack, prolonged ischemic neurologic deficit, and cerebral infarction with or without a remaining neurologic deficit) or by documented carotid plaques (50% luminal obstruction). Peripheral vascular disease was defined by a history of intermittent claudication, angiographic documentation of atherosclerotic luminal obstruction of the peripheral arteries, or a history of a peripheral arterial intervention for atherosclerotic disease (angioplasty or surgery). We formed three groups: group 0 (0 score points), group 1 (1 score point), and group 2 (2 score points) (17). A questionnaire with a scoring system ranging from 1, sedentary (avoid walking or exertion), to 11, regular heavy exercise, was used to classify the mean physical activity . The study participants were grouped into the following three categories of physical activity: below average (scores 13), average (scores 47), and above average (scores 811). There was a follow - up for all - cause and cardiovascular mortality; the latter defined as mortality due to cardiovascular diseases (icd-9 codes 390448 and icd-10 codes i00i79). The mean (sd) duration of the follow - up was 7.6 2.1 years . They were blinded to any data of the study participants . In cases of a disagreement or uncertainty concerning the coding of a specific cause of death, classification was made by a principal investigator of the luric study (w.m . ). C - peptide was measured with an enzyme immunoassay (aia - pack c - peptide) on an aia 1200 analyzer (eurogenetics, eschborn, germany). Glucose was measured enzymatically on a hitachi 717 analyzer (roche, mannheim, germany). Insulin was analyzed with an immunoenzymometric assay (aia pack iri) on an aia 1200 analyzer (eurogenetics). Glycated hemoglobin was measured with an immunoassay (hemoglobin a1c unimate 5; hoffmann - laroche, grenzach - whylen, germany). Precipitation method and measured on a wako 30 r analyzer (wako chemicals gmbh, neuss, germany). Triglycerides were quantified with an enzymatic assay on a hitachi 717 analyzer (roche). Creatinine was measured with the jaff method on a hitachi 717 analyzer (roche) (18). The baseline characteristics are reported as numbers and percentages in cases of categorical variables and as means with sd or medians with interquartile ranges in cases of continuous variables . Comparisons among the three groups were made with the test and with anova for categorical and continuous data, respectively . Triglyceride and insulin levels (shapiro - wilk w test) were transformed logarithmically before being used in parametric statistical procedures . Kaplan - meier curves were plotted for the relationships of c - peptide with all - cause and cardiovascular mortality . The cox proportional hazards model was used to perform multivariate analyses for the associations among the tertiles of c - peptide and mortality data . Three predefined models of adjustment (model 1: adjusted for sex and age; model 2: adjusted for sex, age, bmi, hypertension, smoking, glomerular filtration rate [gfr], triglycerides, ldl cholesterol, and hdl cholesterol; and model 3: model 2 with additional adjustment for fasting glucose, fasting insulin, hba1c, newly diagnosed diabetes, proinsulin, free fatty acids, free glycerol, as well as c - reactive protein) were used . Z - transformation was performed to investigate the increase in risk of all - cause and cardiovascular mortality conferred by sd of c - peptide levels included as a continuous variable . All statistical tests were two - sided, and p values <0.05 were considered significant . The spss 15.0 statistical package (spss inc ., a total of 3,316 patients, who were referred for coronary angiography to ludwigshafen heart center in southwest germany, were recruited between july 1997 and january 2000 . Inclusion criteria were german ancestry, clinical stability except for acute coronary syndromes, and the availability of a coronary angiogram . One of the original purposes of the study also was the identification of genetic components of coronary artery disease (cad). To avoid population admixture, we aimed at including caucasians only . For pragmatic reasons, we therefore only included patients the indications for angiography in individuals in clinically stable condition were chest pain and/or noninvasive test results consistent with myocardial ischemia . Individuals suffering from any acute illness other than acute coronary syndromes, chronic noncardiac diseases, or malignancy within the 5 past years and those unable to understand the purpose of the study were excluded . People with known diabetes were excluded because c - peptide levels change over the course of diabetes with a high interindividual variability and are in addition altered by different antidiabetic drugs . Moreover, a long duration of type 2 diabetes confers increased cardiovascular risk, thus being an additional potential confounder . Moreover, subjects with missing data on fasting c - peptide were additionally ruled out, resulting in a subgroup of 2,306 (69.5%) of 3,316 ludwigshafen risk and cardiovascular health (luric) study participants for the present analyses . These subjects were excluded when the associations of c - peptide with cardiovascular mortality were analyzed . The study was approved by the ethics committee at the rztekammer rheinland - pfalz and was conducted in accordance with the declaration of helsinki . Diabetes was diagnosed according to the 2010 guidelines of the american diabetes association (15). Fasting glucose, postchallenge glucose (performed in 1,524 of 2,096 participants with fasting glucose <126 mg / dl), and glycated hemoglobin were used to categorize diabetes . Hypertension was diagnosed if the systolic and/or diastolic blood pressure exceeded 140 and/or 90 mmhg or if there was a history of hypertension, evident through the use of antihypertensive drugs . Acute myocardial infarction (mi) was defined as an mi that had occurred within the 4 weeks prior to enrollment . A definite st elevation mi (stemi) was diagnosed if typical electrocardiogram changes were present along with prolonged chest pain, refractory to sublingual nitrates, and/or enzyme or troponin t elevations (> 0.1 g / l). Non - stemi was diagnosed if symptoms and/or troponin t criteria, but not the electrocardiogram criteria for stemi, were met . Cerebrovascular disease was defined clinically by documented history of a previous cerebrovascular disease event (transient ischemic attack, prolonged ischemic neurologic deficit, and cerebral infarction with or without a remaining neurologic deficit) or by documented carotid plaques (50% luminal obstruction). Peripheral vascular disease was defined by a history of intermittent claudication, angiographic documentation of atherosclerotic luminal obstruction of the peripheral arteries, or a history of a peripheral arterial intervention for atherosclerotic disease (angioplasty or surgery). We formed three groups: group 0 (0 score points), group 1 (1 score point), and group 2 (2 score points) (17). A questionnaire with a scoring system ranging from 1, sedentary (avoid walking or exertion), to 11, regular heavy exercise, was used to classify the mean physical activity . The study participants were grouped into the following three categories of physical activity: below average (scores 13), average (scores 47), and above average (scores 811). A questionnaire with a scoring system ranging from 1, sedentary (avoid walking or exertion), to 11, regular heavy exercise, was used to classify the mean physical activity . The study participants were grouped into the following three categories of physical activity: below average (scores 13), average (scores 47), and above average (scores 811). There was a follow - up for all - cause and cardiovascular mortality; the latter defined as mortality due to cardiovascular diseases (icd-9 codes 390448 and icd-10 codes i00i79). The mean (sd) duration of the follow - up was 7.6 2.1 years . They were blinded to any data of the study participants . In cases of a disagreement or uncertainty concerning the coding of a specific cause of death, classification was made by a principal investigator of the luric study (w.m . ). C - peptide was measured with an enzyme immunoassay (aia - pack c - peptide) on an aia 1200 analyzer (eurogenetics, eschborn, germany). Glucose was measured enzymatically on a hitachi 717 analyzer (roche, mannheim, germany). Insulin was analyzed with an immunoenzymometric assay (aia pack iri) on an aia 1200 analyzer (eurogenetics). Glycated hemoglobin was measured with an immunoassay (hemoglobin a1c unimate 5; hoffmann - laroche, grenzach - whylen, germany). Lipoproteins were separated using a combined ultracentrifugation precipitation method and measured on a wako 30 r analyzer (wako chemicals gmbh, neuss, germany). Triglycerides were quantified with an enzymatic assay on a hitachi 717 analyzer (roche). Creatinine was measured with the jaff method on a hitachi 717 analyzer (roche) (18). The baseline characteristics are reported as numbers and percentages in cases of categorical variables and as means with sd or medians with interquartile ranges in cases of continuous variables . Comparisons among the three groups were made with the test and with anova for categorical and continuous data, respectively . Triglyceride and insulin levels (shapiro - wilk w test) were transformed logarithmically before being used in parametric statistical procedures . Kaplan - meier curves were plotted for the relationships of c - peptide with all - cause and cardiovascular mortality . The cox proportional hazards model was used to perform multivariate analyses for the associations among the tertiles of c - peptide and mortality data . Three predefined models of adjustment (model 1: adjusted for sex and age; model 2: adjusted for sex, age, bmi, hypertension, smoking, glomerular filtration rate [gfr], triglycerides, ldl cholesterol, and hdl cholesterol; and model 3: model 2 with additional adjustment for fasting glucose, fasting insulin, hba1c, newly diagnosed diabetes, proinsulin, free fatty acids, free glycerol, as well as c - reactive protein) were used . Z - transformation was performed to investigate the increase in risk of all - cause and cardiovascular mortality conferred by sd of c - peptide levels included as a continuous variable . All statistical tests were two - sided, and p values <0.05 were considered significant . The spss 15.0 statistical package (spss inc ., chicago, il) was used . Clinical and laboratory baseline characteristics according to c - peptide tertiles are shown in table 1 . In the overall population analyzed, persons in the highest c - peptide tertile had a significantly higher prevalence of cad than subjects in the lower tertiles (71.8 vs. 63.1 vs. 63.3%, respectively). In addition, persons in the highest c - peptide tertile exhibited significantly higher bmi, waist circumference, fasting glucose, fasting insulin, triglycerides, c - reactive protein, and had lower ldl cholesterol levels as well as a lower gfr . Moreover, subjects in the highest tertile had more often newly diagnosed type 2 diabetes, systemic hypertension, high comorbidity, and a lower level of physical activity . The proportion of past and current smokers and the use of ace inhibitors, calcium antagonists, and diuretics were significantly higher in patients with higher c - peptide levels (table 1). Baseline characteristics according to tertile of fasting c - peptide because experimental data suggest that c - peptide may be causally involved in early atherogenesis, we analyzed the association of c - peptide with biochemical markers of endothelial dysfunction and atherosclerosis . Subjects in the highest c - peptide tertile exhibited significantly higher levels of intracellular adhesion molecule (icam)-1, vascular cell adhesion molecule (vcam)-1, p - selectin, and e - selectin, as well as lipoprotein - associated phospholipase a2 (lppla2) (table 2). Moreover, history of mi, peripheral vascular disease, and cerebrovascular disease were more frequent in subjects with high c - peptide levels (table 1). In accordance, the proportion of subjects with prevalent clinical and angiographic cad (table 1) as well as lesion extension as assessed by the friesinger score increased in parallel with the c - peptide tertiles (table 2). Furthermore, cardiac insufficiency as reflected by high new york heart association functional class was more frequent in subjects with high c - peptide levels (table 1). Taken together, these data underscore the hypothesis that c - peptide could play a direct role in the development of arteriosclerosis . Markers of atherosclerosis and friesinger score according to tertile of fasting c - peptide all - cause mortality and causes of death are shown in supplementary table 1 . In 14 subjects, we did not obtain sufficient information to classify their cause of death and therefore only included them in the analyses of all - cause mortality and excluded them from further analyses . Among the 2,306 patients without known diabetes and available c - peptide levels, 440 deaths (19.1%) occurred during a mean follow - up of 7.6 years . Compared with subjects in the lowest tertile, the age- and sex - adjusted hazard ratio (hr) for death was 1.46 (95% ci 1.151.85; p = 0.002) in the highest tertile (table 3). Further adjustment for bmi, hypertension, smoking, gfr, triglycerides, ldl cholesterol, and hdl cholesterol did not influence the prognostic value of c - peptide in the highest tertile, resulting in an hr of 1.49 (1.161.91; p = 0.002) compared with the lowest tertile (table 3, model 2). Additional adjustment for fasting glucose, fasting insulin, hba1c, newly diagnosed diabetes, proinsulin, free fatty acids, free glycerol, as well as c - reactive protein had only minor influence on the hr of 1.46 (1.101.93; p = 0.008) (table 3, model 3). C - peptide levels included as continuous variable increased the risk in all - cause mortality per sd by hr 1.12 (1.051.20, p = 0.001; model 1). The association remained unchanged after adjustment for cardiovascular risk factors, hr 1.12 (1.041.20; p = 0.002; model 2), and after full adjustment in model 3, hr 1.13 (1.041.22; p = 0.005). Mortality according to tertile of fasting c - peptide among the 2,292 subjects with known causes of death, 252 (10.9%) died of cardiovascular causes, 26 (1.1%) of infection, 76 (3.3%) of cancer, and 72 (3.1%) of miscellaneous causes . Compared with the lowest c - peptide tertile, the age- and sex - adjusted hr for cardiovascular death was 1.58 (95% ci 1.152.18; p = 0.005) (table 3). After full adjustment (model 3), the prognostic value of c - peptide in the highest compared with the lowest tertile was still significant, with an hr of 1.55 (1.072.24; p = 0.022). C - peptide levels included as continuous variable increased the risk in cardiovascular mortality per sd by hr 1.16 (95% ci 1.071.26; p <0.001; model 1). The association remained unchanged after adjustment for cardiovascular risk factors, hr 1.15 (1.051.25; p = 0.002; model 2), and after full adjustment in model 3, hr 1.16 (1.051.28; p = 0.004). Kaplan - meier curves followed by a log - rank test showed that all - cause mortality (fig . 1b) significantly increased in the higher c - peptide tertiles (p = 0.006 and p = 0.012, respectively). A: kaplan - meier curves for all - cause mortality according to tertile (13) of fasting c - peptide b: kaplan - meier curves for cardiovascular mortality according to tertile (13) of fasting c - peptide . Clinical and laboratory baseline characteristics according to c - peptide tertiles are shown in table 1 . In the overall population analyzed, persons in the highest c - peptide tertile had a significantly higher prevalence of cad than subjects in the lower tertiles (71.8 vs. 63.1 vs. 63.3%, respectively). In addition, persons in the highest c - peptide tertile exhibited significantly higher bmi, waist circumference, fasting glucose, fasting insulin, triglycerides, c - reactive protein, and had lower ldl cholesterol levels as well as a lower gfr . Moreover, subjects in the highest tertile had more often newly diagnosed type 2 diabetes, systemic hypertension, high comorbidity, and a lower level of physical activity . The proportion of past and current smokers and the use of ace inhibitors, calcium antagonists, and diuretics were significantly higher in patients with higher c - peptide levels (table 1). Because experimental data suggest that c - peptide may be causally involved in early atherogenesis, we analyzed the association of c - peptide with biochemical markers of endothelial dysfunction and atherosclerosis . Subjects in the highest c - peptide tertile exhibited significantly higher levels of intracellular adhesion molecule (icam)-1, vascular cell adhesion molecule (vcam)-1, p - selectin, and e - selectin, as well as lipoprotein - associated phospholipase a2 (lppla2) (table 2). Moreover, history of mi, peripheral vascular disease, and cerebrovascular disease were more frequent in subjects with high c - peptide levels (table 1). In accordance, the proportion of subjects with prevalent clinical and angiographic cad (table 1) as well as lesion extension as assessed by the friesinger score increased in parallel with the c - peptide tertiles (table 2). Furthermore, cardiac insufficiency as reflected by high new york heart association functional class was more frequent in subjects with high c - peptide levels (table 1). Taken together, these data underscore the hypothesis that c - peptide could play a direct role in the development of arteriosclerosis . All - cause mortality and causes of death are shown in supplementary table 1 . In 14 subjects, we did not obtain sufficient information to classify their cause of death and therefore only included them in the analyses of all - cause mortality and excluded them from further analyses . Among the 2,306 patients without known diabetes and available c - peptide levels, 440 deaths (19.1%) occurred during a mean follow - up of 7.6 years . Compared with subjects in the lowest tertile, the age- and sex - adjusted hazard ratio (hr) for death was 1.46 (95% ci 1.151.85; p = 0.002) in the highest tertile (table 3). Further adjustment for bmi, hypertension, smoking, gfr, triglycerides, ldl cholesterol, and hdl cholesterol did not influence the prognostic value of c - peptide in the highest tertile, resulting in an hr of 1.49 (1.161.91; p = 0.002) compared with the lowest tertile (table 3, model 2). Additional adjustment for fasting glucose, fasting insulin, hba1c, newly diagnosed diabetes, proinsulin, free fatty acids, free glycerol, as well as c - reactive protein had only minor influence on the hr of 1.46 (1.101.93; p = 0.008) (table 3, model 3). C - peptide levels included as continuous variable increased the risk in all - cause mortality per sd by hr 1.12 (1.051.20, p = 0.001; model 1). The association remained unchanged after adjustment for cardiovascular risk factors, hr 1.12 (1.041.20; p = 0.002; model 2), and after full adjustment in model 3, hr 1.13 (1.041.22; p = 0.005). Among the 2,292 subjects with known causes of death, 252 (10.9%) died of cardiovascular causes, 26 (1.1%) of infection, 76 (3.3%) of cancer, and 72 (3.1%) of miscellaneous causes . Compared with the lowest c - peptide tertile, the age- and sex - adjusted hr for cardiovascular death was 1.58 (95% ci 1.152.18; p = 0.005) (table 3). After full adjustment (model 3), the prognostic value of c - peptide in the highest compared with the lowest tertile was still significant, with an hr of 1.55 (1.072.24; p = 0.022). C - peptide levels included as continuous variable increased the risk in cardiovascular mortality per sd by hr 1.16 (95% ci 1.071.26; p <0.001; model 1). The association remained unchanged after adjustment for cardiovascular risk factors, hr 1.15 (1.051.25; p = 0.002; model 2), and after full adjustment in model 3, hr 1.16 (1.051.28; p = 0.004). Kaplan - meier curves followed by a log - rank test showed that all - cause mortality (fig . 1b) significantly increased in the higher c - peptide tertiles (p = 0.006 and p = 0.012, respectively). A: kaplan - meier curves for all - cause mortality according to tertile (13) of fasting c - peptide . B: kaplan - meier curves for cardiovascular mortality according to tertile (13) of fasting c - peptide . The current study demonstrates that levels of the proinsulin cleavage product c - peptide are independently associated with all - cause mortality and cardiovascular mortality in a large cohort of patients referred to coronary angiography . Moreover, c - peptide was associated with prevalent cad, and the severity of angiographic changes increased with c - peptide levels . The association of c - peptide with mortality as well as cardiovascular mortality remained significant even after full adjustment including bmi, fasting glucose, fasting insulin, hba1c, proinsulin, free fatty acids, free glycerol, high - sensitivity c - reactive protein, and gfr, as well as newly diagnosed diabetes . Elevated levels of the proinsulin cleavage product c - peptide have been found in patients with insulin resistance, early type 2 diabetes, as well as those with chronic kidney disease . Measurement of c - peptide levels has been used as a surrogate for insulin secretion (e.g., in obese patients with insulin resistance) (19). In this context, the association of c - peptide with cardiovascular mortality could simply reflect the increased risk of patients with insulin resistance and early type 2 diabetes in our population . Indeed, subjects in the highest tertile of c - peptide had higher fasting glucose, higher fasting insulin, a higher bmi, as well as a higher prevalence of newly diagnosed diabetes . However, adjustment for all of these factors did basically not change the hr for cardiovascular mortality as well as overall mortality, suggesting that c - peptide is an independent risk factor in our study population . To the best of our knowledge, this is the first study to demonstrate that c - peptide is associated with mortality and cardiovascular mortality in patients without previously known diabetes . Various studies have shown that elevated c - peptide levels are associated with mortality as well as cardiovascular mortality in patients with diabetes and nondiabetic cancer patients, most likely serving as an indirect marker for obesity, insulin resistance, and undiagnosed diabetes (12,13). Various data from in vitro as well as animal models have shown proatherogenic effects of c - peptide (1,7,20). Our data, demonstrating an association of c - peptide levels with markers of endothelial activation such as icam-1, vcam-1, and e - selectin, as well as a highly significant association with levels of lppla2, a novel marker of arteriosclerosis, raise the question if c - peptide could potentially also directly influence atherogenesis in humans, but our data cannot prove a causal relationship . Furthermore, patients in the highest tertile of c - peptide had the highest incidence of cad and the most severe lesion extension as assessed by the friesinger score, underscoring the hypothesis that c - peptide may play a role in the pathophysiology of cad . Still, our study cannot prove causality, and further research is warranted to address this question . Of note, therapeutic application of c - peptide in patients with type 1 diabetes, lacking endogenous c - peptide, exhibits beneficial effects on microvascular complications such as neuropathy (4), suggesting different mechanisms of action of exogenous c - peptide in c - peptide deprived patients and those with elevated endogenous c - peptide levels . Our study has some limitations: the results obtained in this study apply only to a highly selected caucasian patient population undergoing coronary angiography without other major noncardiac diseases or malignancies . Therefore, the results may not be generalizable to other populations with younger age, other ethnicity, or other underlying diseases . Moreover, each of the study participants coronary angiography was clinically indicated, which may have produced referral bias . Finally, despite the fact that we adjusted our results for multiple potential confounders, we cannot rule out additional factors . Taken together, our study suggests that elevated levels of c - peptide are predictive of both all - cause as well as cardiovascular mortality in patients undergoing coronary angiography and raises the hypothesis that c - peptide could potentially play a causal role in atherogenesis in humans . However, future research has to prove causality and examine the underlying mechanism for the findings of this study.
Spastic torticollis, also known as stiff neck or wryneck, is a condition in which the head is laterally flexed and rotated on the neck, secondary to painful spasm of the neck muscles . There may be a report of the patient having slept in a draft from a fan, open window, or air conditioner . The patient is generally afebrile, and if there is any history of trauma, it is of the mildest variety . Chloroethane (ethyl chloride) spray is a vapocoolant applied topically in the treatment of athletic injuries . The manufacturer indicates that it may be used as a counterirritant in the management of myofascial pain and muscle spasm . We reviewed the literature and could find no large study on pediatric patients regarding this usage . We report our experience with the use of chloroethane spray treating torticollis in 66 patients ranging in age from 1 to 16 years . Patients presenting to the pediatric emergency service of a 600-bed inner - city hospital with the compliant of stiff neck were selected for treatment . An initial evaluation was performed, which included a medical and family history, a physical examination, and x - ray study of the cervical spine (a - p, lateral, and open mouth views). The patients were enrolled into either group 1, usage of ethyl chloride spray, or group 2, usage of normal spray . In the patients enrolled in group 1 (after explaining the procedure to the parent and child and obtaining informed consent), the chloroethane spray bottle was held inverted 12 inches directly over the point of maximal neck tenderness . The valve of the bottle was opened, allowing the liquid to leave as a fine jet stream . The stream was left in contact with the skin for 4 to 5 seconds or until frosting of the skin occurred . The procedure was considered successful if, after a 5-minute waiting period, the patient was able to freely and painlessly move his or her head and neck in the opposite side . For the control group, normal saline is applied as a spray using a syringe with plastic catheter over the point of maximum tenderness and asked to move the neck after 5 minutes and note if patient can move the head and neck freely in the opposite side . A total of 132 children with a mean age of 8.97 years (range = 1 - 16 years) presented to the emergency room with torticollis over a period of 3 years and were enrolled in the study . Of the 132, patients in group 1 were treated with chloroethane spray and those in group 2 were treated with normal saline spray . Of the 66 patients treated with chloromethane spray, 63 (95%) responded to the treatment as evidenced by their subsequent ability to move the head and neck freely (see table 1). No patients returned for additional treatment . Of the patients in group 2, who treated with normal saline spray, no patient responded to saline spray with ability to move the head freely in the opposite side (see figure 1). Series 1: patients rxed with chloroethane spray (63/66 positive response and 3/66 negative response). The torticollis was in several cases associated with a local head or neck condition, but 46.97% of the cases involved no underlying pathology (table 2). Of the 3 treatment failures, 2 occurred in patients with neck trauma and 1 failure occurred in a patient with cystic hygroma . Conditions associated with spastic torticollis . Abbreviation: uri, upper respiratory infection . To test the null hypothesis that the percentages improved in the 2 groups equally, we calculated the z statistic . The z statistic of 24 corresponded to a p value <.0001, whether the test is 1-sided or 2-sided . To test the null hypothesis that the percentages improved in the 2 groups equally, we calculated the z statistic . The z statistic of 24 corresponded to a p value <.0001, whether the test is 1-sided or 2-sided . Torticollis is a condition in which the head is laterally flexed and rotated on the neck due to shortening or spasm of the sternocleidomastoid muscle . Acquired torticollis can be caused by a variety of problems including cold exposure, minor neck muscle trauma, and any inflammatory condition of the neck such as cervical lymphadenitis, or retropharyngeal abscess . Less common causes include drug ingestion (eg, phenothiazines) and a variety of neurological conditions such as dystonia musculorum deformans, brain stem, or posterior fossa tumor or cyst . We focus on describing chloroethane treatment for spastic torticollis caused by sternomastiod spasm that may be primary or secondary to a local inflammatory condition . This form of torticollis is most often treated with warm compresses, analgesics, and muscle relaxants . It is thought that the relief is the result of the wrong acting as a counterirritant or a local anesthetic or by a placebo effect . Similarly, a vapocoolant therapy has been described to successfully relieve acute myofascial trigger with point pain in children . In conclusion, from our study, for children and adolescents with spastic torticollis, chloroethane spray was more superior to normal saline in the pediatric emergency room.
Thyroid lesions containing gross fat include heterotopic fat nests, thyrolipoma, teratoma, liposarcoma, and thyroid cancers of the papillary or follicular type (234567). Most of these cases are only described upon obtaining pathological data after surgery; radiologic methods are of limited use (89). With the approval of the institutional review board of our hospital, we reported the case of an intrathyroid nodule composed mainly of gross fat detected by computed tomography (ct) and confirmed by lobectomy as a follicular variant of papillary thyroid cancer (fvptc) with mature fat . A 58-year - old woman presented with an incidentally detected mass in the thyroid on routine ultrasonography (us) at a primary clinic . The patient had a 10-year history of diabetes mellitus and hypertension, and was currently taking medication . Physical examination revealed a firm mass of approximately 3 cm in size in the right anterior neck . Laboratory tests revealed that serum anti - microsomal antibody, anti - thyroglobulin antibody, thyroglobulin, calcitonin, t3, free t4, and thyroid - stimulating hormone levels were within normal ranges . Ultrasonography revealed an ovoid lesion with a smooth margin of approximately 2.9 2 1.3 cm in size . The nodule was generally hyperechoic, with the peripheral portion more hyperechoic than the central area . The perithyroidal fat, muscle, and vertebral bodies were therefore not clearly visualized (fig . Doppler us did not show any vascular signals in the nodule . Based on these findings a board - certified radiologist performed us - guided fine needle aspiration using a freehand technique with a 23-gauge needle and a 5-ml disposable plastic syringe . Cytologic smears were made on glass slides and immediately fixed in 95% alcohol for both papanicolaou staining and may - grunwald - giemsa staining . Cytology revealed atypia of undetermined significance or a follicular lesion of undetermined significance with negative for braf mutations and positive for an nras 61 mutation . The thyroid ct protocol at our hospital includes pre - contrast, early, and delayed phases . Early phase images (40 seconds) were scanned from the hyoid bone to the thoracic inlet to cover the entire thyroid and level iii - vi lymph nodes . Delayed scans (90 seconds) covered the area from the skull base to the upper mediastinum . All scans were acquired with a slice thickness of 2.5 mm and reconstruction increment of 2.5 mm . Pre - contrast ct showed markedly low attenuation (mean ct number, -80 hounsfield units [hu]), in a well - defined mass with fine reticular and thick septa - like structures in the parenchyma of the right mid - to - upper portion of the thyroid (fig . 1b). To measure the enhancement pattern of the tumor, 25 mm of the region of interest was placed in the area of the fatty mass, including the reticular and thick septa - like soft tissue lesions . The tumor was enhanced in the early phase (mean ct number, 16 hu) and was washed out in the delayed phase (mean ct number, -13 hu) (fig . The tumor was ovoid in the longitudinal direction, had a lobular appearance in the axial plane, and was completely encapsulated in the thyroid parenchyma, with no evidence of extrathyroidal extension . Based on these findings, we suspected a fat - containing, neoplastic lesion of the thyroid gland, such as thyrolipoma, teratoma, lipoma, low - grade liposarcoma, or a follicular tumor with stromal fat . Considering the results from cytopathology and the gene mutation analysis it showed a yellowish, homogeneous, and well - demarcated nodular lesion measuring 2.5 2.2 1.5 cm at the upper - to - mid pole . Microscopic examination revealed a well - circumscribed, thin, fibrous, and capsulated nodule with mature fat . There were atypical follicular cells with nuclear enlargement, nuclear overlapping, chromatin clearing, and nuclear grooves without papillary structures in the nodule (fig . On immunohistochemistry, the tumor cells stained positive for hbme-1 (dako, glostrup, denmark, 1:100) and galectin-3 (novocastra, newcastle upon tyne, uk, 1:200). Finally, we tested for the v600e and k601e mutations in exon 15 of the braf gene and codons 12, 13, and 61 of the kras and nras genes . In our case, the tumor was a yellowish, homogeneous, and well - demarcated nodular lesion on gross examination, with negative value of attenuation on ct, suggesting fat . However, it was difficult to recognize the tumor as a fat containing mass on us examination . The hyperechoic nature of the mass and the curtain - like hyperechoic shadowing posterior to the mass may suggest rich fatty component . We suspected that the hyperechoic shadowing might have been due to a reverberating artifact caused by innumerable fat globules and septa of the tumor, similar to the artifact frequently observed along the thick abdominal walls that consist of several layers of fat globules surrounded by connective tissues, skin, and fascia . Computed tomography and gross specimen findings of our case were very similar to those of thyrolipoma, a benign fat - containing tumor (810). Thyrolipoma is considered a variant of follicular adenoma, which is characterized by a well - circumscribed and encapsulated nodule resulting from the proliferation of thyroid follicles admixed with mature fat (1011). This case could have been misdiagnosed as a follicular neoplasm or thyrolipoma, as the tumor cells did not show a papillary growth pattern upon microscopic examination and the gross features were very similar to thyrolipoma . Two reports provided the ct and magnetic resonance imaging findings of thyrolipoma (812). The mass predominantly had fat attenuation and distinct margins in the thyroid gland . However, the authors only performed pre - contrast ct on the lesion . Therefore, it is uncertain whether thyrolipoma and thyroid cancer with massive stromal fat can be differentiated by ct with contrast enhancement . In our case, the tumor was enhanced in the early phase and slightly washed out in the delayed phase . Fat - containing thyroid cancer of the papillary or follicular type, have only been reported from pathological studies (2456). Fat tissue was observed only by microscopy, so it was assumed that small amounts of fat tissue could not be detected by ct . We also assumed that the detectability of the fat component is variable, as it depends on the total number of fat cells in the mass . Mature teratoma can be recognized if the fat - containing mass contains calcification, bone or cartilage, and cystic components . However, this is not possible, as radiological images do not always exhibit all these attributes (7). Ct images of liposarcoma (3) in the thyroid gland are very similar to those in our case, and differentiating between teratoma, thyrolipoma, liposarcoma, and papillary thyroid cancer with massive fat may be impossible using radiology alone . Some investigators have insisted that fat cells are derived from displaced remnants of embryonic structures in the thyroid . In amyloid goiter, schrder and bcker (1) postulated that adipose tissue may be derived from metaplasia of stromal fibroblasts due to either impaired circulation and diffusion or tissue hypoxia caused by amyloid deposition . Derienzo and truong (5) suggested that the fat cells in thyrolipoma and follicular carcinoma might be neoplastic tissue because 1) fat did not exist in the normal thyroid, 2) fat was a major component of the tumor, 3) there was no evidence of degenerative changes such as dystrophic calcification, cystic or hemorrhagic degeneration, or amyloid deposition, 4) fat was widely distributed in the tumor, and 5) there were similar fat - containing tumors in other organs such as thymolipoma, parathyroid adenoma, and fat - containing fibroadenoma of the breast . Our case showed similar microscopic features to theirs in agreement with the suggestion that the fat cells might be neoplastic cells themselves . In summary these findings included a hyperechoic mass with a smooth margin with a reverberating artifact and no detectable doppler signal on us . Ct revealed a well - encapsulated fat - attenuation mass with enhancing septa - like structures . Fvptc should be included in the differential diagnosis of fat - containing focal lesions in the thyroid, in addition to thyrolipoma, teratoma, liposarcoma, and small focal fat nests near the capsule . Moreover, fine needle aspiration cytology with gene analysis may be helpful in determining the nature of fatty tumors before surgery.
A 65-year - old male patient visited the department of oral medicine of the institute for getting his missing teeth replaced . On intraoral clinical examination of the patient, a deep red - colored multinodular but small swelling (measuring 1.5 cm 2 cm 2 cm) was observed in the left posterior palatal region (missing 26, 27, 28) extending toward the midline [figure 1]. As per the patient's information, the swelling was observed nearly 6 months back and was found to be gradually increasing . On palpation, the mass was firm and fixed to the underlying tissues . Neither loosening of teeth nor tenderness on percussion was noted . Orthopantomograph and occlusal radiograph did not reveal any significant bone changes, suggesting that the lesion was still superficial [figure 2]. The patient was diabetic for the last 22 years and was on medication for the same . The solitary, firm, asymptomatic, and nonulcerated mass seen on the palate instantly suggested a benign salivary gland tumor . Clinical differential diagnoses included benign lymphoepithelial lesion or mucus extravasation phenomena . However, we also considered malignancies such as malignant salivary gland tumor, lymphoma, and neoplasm of the maxillary sinus as the possible diagnoses . Incidentally, our patient did not complain of swelling or discomfort; in fact, the patient's visit to the hospital was aimed at getting his missing teeth replaced . The most commonly observed benign salivary gland tumors on the palate include pleomorphic adenoma (pa) and basal cell adenoma . Pa, also known as mixed tumor, is the most common tumor of the major and minor salivary glands, more prevalent in elderly males . Intraorally, palate is the most common site where they appear as firm, painless swellings, and in the vast majority of cases, they do not cause ulceration of the overlying epithelium . Pa is typically lobulated and enclosed within a connective tissue pseudocapsule that varies in thickness within the major salivary glands, whereas in minor salivary glands, the capsule is poorly defined to absent in areas . . They account for approximately 34% of parotid malignancies, 20% of submandibular gland malignancies, and 30% of minor salivary gland malignancies, with palate as the most common intraoral site . Since there was no pain, paresthesia, or ulceration, the possibility of mucoepidermoid carcinoma was ruled out . Lymphoma was considered plausible as the swelling was asymptomatic, showing spongy to firm tumescence; these features simulated our case . The clinical presentation of lymphomas of the oral region varies with their site of origin and tumor type, but most present as a mass or an ulcerated mass and resemble squamous cell carcinoma or salivary neoplasm . Within the oral cavity, lymphoid tissue is chiefly represented in waldeyer's ring; elsewhere within the oral cavity, it appears as unencapsulated lymphoid tissue within the base of the tongue and soft palate, as well as within the major and minor salivary glands . Non - hodgkin's lymphoma (nhl) is very often seen on the hard palate, primarily in elderly men and women with an average age of 70 years . Oral lymphomas are relatively rare and often difficult to diagnose in a clinical setting, and most often mimic other pathological entities, such as dentoalveolar abscess, periodontal abscess, infected dental cyst, or benign jaw tumors . It typically is characterized by swollen and nonpainful enlargement of the lymph nodes, especially at the junction of hard and soft palates . Although lymphoid lesions in the hard palate are very likely to be lymphomas, follicular lymphoid hyperplasia can present in this anatomic location; hence, a thorough histologic examination is of utmost significance . Histologic differentiation must be made among lymphoid hyperplasia, nhl, and benign lymphoepithelial lesion, when present in the hard palate . Since it was a nodular and nonulcerated swelling, trauma could have also caused the nodule formation because of mucus extravasation caused by the severance of salivary gland excretory duct . Although palate is not the common site of mucus extravasation phenomenon, it usually presents as a relatively painless smooth - surfaced mass ranging in size from a few millimeters to 2 cm in size, consistent with our case . However, our patient reported that the mass was persistent for the last few months, which negated the possibility of a mucocele . Last but not least, even neoplasm of maxillary sinus could be a reason for the palatal swelling . Occasional maxillary sinus cancers may present as a palatal ulcer and mass representing extension through the bone and soft tissue of the palate . Fine needle aspiration cytology of the lesion was done and the smear stained with papanicolaou stain revealed mucin and keratin, which was nonspecific . Histopathology of the specimen revealed parakeratinized stratified squamous epithelium which was hyperplastic at areas with an underlying fibrocellular connective tissue stroma . The stroma showed unencapsulated, circumscribed tumor mass composed of homogeneous population of cuboidal to columnar isomorphic cells with prominent, bland, often vesicular ovoid to spindle - shaped nuclei and minimal eosinophilic cytoplasm . The tumor cells were arranged as solid nests, lobules, and glandular patterns extending till the entire depth of the received specimen [figure 3a and b]. Few areas of intraluminal mucin and hyalinization of stroma surrounding the tumor cells were evident . (a) low power magnification demonstrates parakeratinized stratified squamous epithelium and multiple lobules showing glandular and ductular patterns of tumor cells (h and e, 40). (b) tumor cells display prominent vesiculated appearance of the ovoid nuclei arranged in glandular patterns . Cellular atypia and multiple mitotic figures are also observed (h and e, 400). (c) perineural invasion by tumor cells is evident in focal areas (400) patient is on a regular follow - up and there is no recurrence reported even after 1 year of follow - up . Histopathologically, the most difficult neoplasms to differentiate from plga are benign mixed tumor and adenoid cystic carcinoma (acc). However, furthermore, neither benign mixed tumors do show neurotropism, nor they do demonstrate perivascular, osseous, or cartilaginous infiltration . Possibly, acc is the most difficult tumor to differentiate histopathologically from plga . They are also unencapsulated, infiltrating tumors that have a strong tendency for neural invasion . Some other features accs have in common with plgas include the histologic patterns such as cribriform, solid, and tubular . Nevertheless, the distinction between acc and plga can be done based on histologic examination . For example, the nuclei of acc are more hyperchromatic and more angular than those of plga . Cribriform areas with accumulation of basophilic glycosaminoglycans are observed frequently in acc, not in plga . Moreover, the cytoplasmic staining of plga is eosinophilic to amphophilic, whereas that of acc is very pale to clear staining . The distinction between acc and plga is important because acc is more aggressive and relentless clinically with multiple recurrences . On the other hand, plga is an indolent neoplasm where patients are free of the tumor after conservative wide surgical excision, with least requirement of postoperative radiotherapy and chemotherapy . Although considered to be a low - grade malignancy, a case of de novo plga was described which was observed in a minor salivary gland with a large radiographic extent . Plga was earlier referred to as terminal duct carcinoma owing to its probable origin in the ductal system of the salivary glands . Plga is formed by luminal epithelial, myoepithelial, and basal epithelial cells simulating terminal duct carcinoma . Plga is a low - grade malignant neoplasm because of rare occurrence of regional lymph node metastasis, with even extremely rare manifestation of distant metastasis . Microscopic or histopathologic observation of perineural invasion does not appear to affect the prognosis . This neoplasm manifests almost exclusively in minor salivary glands, with a high predilection for the palate . However, some cases have been reported in major salivary glands, for example, in the parotid gland, where most cases have been diagnosed as part of a carcinoma ex pa, such as those in the case series reported by kemp et al ., who found that only two out of twenty cases arose de novo, and the rest showed remnants of pa . Plgas arising in major salivary glands have clinicopathological and immunohistochemical features similar to those of plgas originating in minor salivary glands . The tumor, histopathologically, shows a uniform and bland cytological picture, a diverse yet characteristic growth pattern and a prominent neurotropism . This clinicopathologic entity is, sometimes, a diagnostic dilemma due to its multiple histomorphologic patterns and its cytologic uniformity . Immunohistochemical studies comparing pa and plga have shown that glial fibrillary acidic protein is negative in plga and positive in pa . However, plga differs from pa because it is characterized by infiltrative margins and an absence of chondromyxoid stroma . Plga presenting as palatal lesion is not so rare and should be included in the differential diagnosis of palatal lesions, presenting as nonulcerated nodular swellings . Other salivary gland malignancies commonly seen on the palate, including mucoepidermoid carcinoma and acc, should also be considered in the differential diagnosis . It is of prime importance to differentiate among these malignant lesions and reach a conclusive diagnosis as the management and prognosis varies for each of them . While acc is associated with low long - term survival rates, plga is a low - grade malignancy, and its biologic behavior is apparently not influenced by the different morphologic and cell differentiation patterns that it may reveal . It is of prime importance for a pathologist to inform the predominant histologic pattern in plga, the presence of perineural invasion and vascular permeation, so as to affirm if these criteria are helpful to identify its biologic behavior.
Malignant melanoma is a malignant tumor of the melanocytes or the cells that develop from melanocytes . Histologically, malignant melanomas are asymmetrical and poorly circumscribed lesions with architectural disturbance and usually marked cytological atypia . The major histologic subtypes of melanoma are superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma and acral lentiginous melanoma, which show a variety of cytomorphological features, architectural patterns and stromal changes that may be observed in malignant melanomas . Moreover, there are numerous uncommon histological variants, including clear, signet ring, pseudolipoblastic, rhabdoid, plasmacytoid and balloon cell melanomas . Among them, balloon cell malignant melanoma (bcmm) is the rarest variant composed of large, polygonal, foamy cells with abundant cytoplasm . These features can also be seen in balloon cell nevus (bcn), but the presence of nuclear pleomorphism, atypia, mitoses and a lack of maturation of melanocytes with descent into the dermis help to differentiate bcmm . A balloon cell change may occasionally be observed in conventional melanomas, but it mostly remains in focal areas . Malignant melanoma accounts for only 35% of primary cutaneous malignancies and it mostly develops in people over 50 years . This report presents a young patient of primary bcmm with near - complete effacement by foamy cells . A 32-year - old female presented to our hospital with a tumor on the left lumbar region . The lesion of concern was an erythematous and focal eccentric pigmented nodule of 15 mm in diameter (fig . The clinical appearance of the lesion raised a suspicion of malignancy, and surgical resection was performed . The surgically resected specimen showed a nodule with focally pigmented areas, and the cut surface of the nodule was asymmetrical and not ulcerated . Histological examinations showed a skin fragment with nearly complete effacement by foamy cells and asymmetry (fig . The foamy cells in the superficial dermis extended to the fatty tissue to a depth of 10 mm . 1c). The intraepidermal component was composed of both nested and single atypical melanocytes with areas of intraepidermal pagetoid spread (fig . The pathological diagnosis was of bcmm (breslow depth 10 mm, clark level v) without ulcer . A month after the excision, the excised bilateral inguinal lymph nodes showed diffuse replacement of the lymph nodes by foamy cells (fig . Bcmm is an uncommon histopathological subtype of malignant melanoma that was first described by gardner and vazquez in 1970 ., balloon cell changes of melanocytes have been noted in numerous neoplasms, including melanoma . Histologically differential diagnosis of bcmm includes bcn, clear cell sarcoma, clear cell metastatic renal cell carcinoma, basal cell carcinoma, squamous cell carcinoma and dermatofibroma . The clinical appearance could also be interpreted as that of spitz nevus, basal cell carcinoma, eccrine poroma or eccrine porocarcinoma . A microscopic examination showed a dominant nodular component comprised exclusively of markedly distended epithelioid melanocytes showing abundant vacuolated cytoplasm . Bcmm differs from bcn in its nuclear pleomorphism, atypia and the lack of melanocyte maturation with a decent proliferation into the dermis . While fontana - masson staining of melanoma cells is generally positive, balloon cells in bcmm are generally negative in this stain [4, 5, 6, 7] the balloon cell component of our current case was also stained negative with fontana - masson . The occurrence of the foamy cells has been suggested to be related to an enlarged defective melanosome formation . They are likely developed by the transformation of epithelioid cells, because balloon cells are mainly observed at metastatic or recurring lesions . Previous case reports suggested that patients with bcmm may have longer survivals than those without balloon cell changes . However, the actual mortality rate of bcmm tends to be high because bcmm are thick at the time of presentation . In fact, there was no apparent difference in a more recently reported survival rate between bcmm and other variants of melanoma . Malignant melanoma may affect people of all ages but is mostly observed in people over the age of 50 years . The case reported here is a young case of primary bcmm with near - complete effacement by foamy cells.
They give rise to all the other major brain cell types, which include oligodendrocytes, astrocytes, and neurons . However, molecular pathways, which are complicated and control the proliferation and differentiation of nscs, have been characterized incompletely to date . There is multiple indirect evidence suggesting a critical role of mirnas in nscs [47]. Post - transcriptional control mediated by mirnas was identified as a further important regulation level, especially in neural proliferation where the action of these negative modulators of gene expression is pervasive . Mirnas are small single - stranded non - coding rnas molecules which participate in the transcriptional regulation of eukaryotic genes and produce biological effects through inhibiting translation or destabilizing target mrnas [1012]. Mirnas act as vital adjusters in multiple biological processes, including cell metabolism, differentiation, proliferation, and apoptosis . Mir-126 is highly enriched in endothelial cells and previous studies found that mir-126 plays critical roles in vascular integrity and can promote angiogenesis during embryonic development [1518]. Reported that the expression level of mir-126 was downregulated after spinal cord injury and that it can promote angiogenesis and attenuate inflammation in rats . However, the effects of mir-126 in nscs is still unclear; therefore, we intended to study its role in nscs proliferation and survival . The immortalized human nsc lines hb1.f3 and hb1.a4 were maintained with dulbecco s modified eagle s medium (dmem, sigma) containing 10% fetal bovine serum (fbs). Total rna was extracted from cell lines with a mirvanamirna isolation kit (applied biosystems, foster city, ca, usa) according to the manufacturer s instructions . The expression level of mir-126 was detected by qrt - pcr according to the taqman mirna assays protocol (applied biosystems) and normalized by u6 small nuclear rna (rnu6b; applied biosystems) with the 2 method . Lv3-pglv - h1-gfp+puro plasmids with mir-126 mimics and negative control (lv - mir-126 and lv - mir - nc) were purchased from genepharma (shanghai, china). Hb1.f3 and hb1.a4 stably expressing mir-126 were established by transfecting lentivirus according to the manufacturer s instructions . Cells were plated in 96-well plates in triplicate at approximately 1000 cells per well and cultured in the growth medium . Cells were then treated with the indicated reagent and the numbers of cells per well were measured by the absorbance (450 nm) of reduced water - soluble tetrazolium salt (wst) at the indicated time points . Cells were plated into 6-well plates at a density of 1000 cells / well in 2 ml medium . The colonies were observed using a phase - contrast microscope at a magnification of 4 (we counted the colonies containing at least 50 cells). Cells were transfected with mir-126 mimics or their respective controls and were harvested after transfection at 48 hours and then marked with the annexinv / pi double staining kit (bd biosciences, usa) according to the manufacturer s instructions . Flow cytometry was used to assess the apoptotic cells in triplicates and all assays were repeated at least 3 times . Student s t test or one - way anova were used for analysis when appropriate . The immortalized human nsc lines hb1.f3 and hb1.a4 were maintained with dulbecco s modified eagle s medium (dmem, sigma) containing 10% fetal bovine serum (fbs). Total rna was extracted from cell lines with a mirvanamirna isolation kit (applied biosystems, foster city, ca, usa) according to the manufacturer s instructions . The expression level of mir-126 was detected by qrt - pcr according to the taqman mirna assays protocol (applied biosystems) and normalized by u6 small nuclear rna (rnu6b; applied biosystems) with the 2 method . Lv3-pglv - h1-gfp+puro plasmids with mir-126 mimics and negative control (lv - mir-126 and lv - mir - nc) were purchased from genepharma (shanghai, china). Hb1.f3 and hb1.a4 stably expressing mir-126 were established by transfecting lentivirus according to the manufacturer s instructions . Cells were plated in 96-well plates in triplicate at approximately 1000 cells per well and cultured in the growth medium . Cells were then treated with the indicated reagent and the numbers of cells per well were measured by the absorbance (450 nm) of reduced water - soluble tetrazolium salt (wst) at the indicated time points . Cells were plated into 6-well plates at a density of 1000 cells / well in 2 ml medium . The colonies were observed using a phase - contrast microscope at a magnification of 4 (we counted the colonies containing at least 50 cells). Cells were transfected with mir-126 mimics or their respective controls and were harvested after transfection at 48 hours and then marked with the annexinv / pi double staining kit (bd biosciences, usa) according to the manufacturer s instructions . Flow cytometry was used to assess the apoptotic cells in triplicates and all assays were repeated at least 3 times . Ibm spss 19.0 statistical software was used to analyze the data . Student s t test or one - way anova were used for analysis when appropriate . To further study the potential function of mir-126 in nscs, hb1.f3 and hb1.a4 were stably transfected with mimics or negative control (nc). As indicated in figure 1a, mir-126 mimics showed good overexpression effects both in hb1.f3 and hb1.a4 and both cell lines overexpressing mir-126 showed marked cell growth increase (figure 1b, 1c). To further confirm the function of mir-126 on cell proliferation and growth, soft agar colony formation assay was performed . As shown in figure 2a, 2b, the colony formation was significantly increased by mir-126 compared with the negative control group . We next investigated the function of mir-126 in the regulation of cell apoptosis by flow cytometry . Cells overexpressing mir-126 displayed a significant decrease in the apoptotic rate when compared with the control group (figure 3a, 3b). These results suggest that mir-126 may inhibit apoptosis in nscs, which may contribute to the growth induction features of mir-126 . To further study the potential function of mir-126 in nscs, hb1.f3 and hb1.a4 were stably transfected with mimics or negative control (nc). As indicated in figure 1a, mir-126 mimics showed good overexpression effects both in hb1.f3 and hb1.a4 and both cell lines overexpressing mir-126 showed marked cell growth increase (figure 1b, 1c). To further confirm the function of mir-126 on cell proliferation and growth, soft agar colony formation assay was performed . As shown in figure 2a, 2b, the colony formation was significantly increased by mir-126 compared with the negative control group . We next investigated the function of mir-126 in the regulation of cell apoptosis by flow cytometry . Cells overexpressing mir-126 displayed a significant decrease in the apoptotic rate when compared with the control group (figure 3a, 3b). These results suggest that mir-126 may inhibit apoptosis in nscs, which may contribute to the growth induction features of mir-126 . They can profoundly regulate the expression of massive target genes that encode proteins, which may finally lead to the change of biological function . Mir-126, which is located in intron 7 of the egf - like domain 7 (egfl7) gene, is a microrna that is highly enriched in endothelial cells and regulated by the transcription factors ets-1 and ets-2 in endothelial cells . Previous studies found that mir-126 promoted angiogenesis during embryonic development and after injury by targeting spred1 and pik3r2 . Also, knockdown of mir-126 resulted in delayed angiogenic sprouting, collapsed blood vessels, widespread hemorrhages, and partial embryonic lethality during zebrafish and mouse embryogenesis . It is also involved in cell growth regulation in several organs, such as colorectal cancer, gastric cancer, and liver carcinoma, by regulating multiple target genes, including insulin receptor substrate, p85, pi3k, akt, and crk [2629]. Reported that mir-126 plays an important role in angiogenesis and inflammation after contusion spinal cord injury in rats . In the current study, we verified that when the expression level of mir-126 was overexpressed by mimics transfection, the proliferation and survival of nscs were both significantly promoted . Cck8 assay and colony formation assay both demonstrated that mir-126 can induce the proliferation of nscs . Although the mechanism is not yet completely understood, our study provides further evidence in this area.
The a: icr (crl: cd1(icr)) and b: icr (tac: icr) mice were purchased from two different commercial laboratory animal breeding company in korea . The mice were housed in a pathogene - free animal facility under a standard 12 h light / dark cycle, maintained in conventional housing conditions at 221 and 5010% relative humidity . All mice were provided with ad libitum access to standard irradiated chow diet (purina rodent chow 38057) consisting of moisture (9.03%), crude protein (21.52%), crude fat (5.03%), crude fiber (4.33%), crude ash (6.31%), calcium (1.18%), and phosphorus (0.35%) and drinking water . All animal procedures were conducted in accordance with guidelines of the institutional animal care and use committee, national institute of food and drug safety evaluation (nifds, cheongju, korea). Phylogenetic trees were constructed using the dnapars program of phylip (phylogeny inference package) version 3.695 (http://evolution.genetics.washington.edu/phylip.html) based on alignment of snps . Seqboot and condense programs of the phylip package were used in bootstrap analysis of 100 resamplings of the data sets . Measurement of body and organ weights was made based on the methods of the international mouse phenotyping consortium . Hematological analysis was determined using by advia 2010i hematology system with autoslide (siemens, germany). Clinical chemical parameters were measured from a single serum sample using a hitachi 7100 automatic analyzer (hitachi, japan). We noticed that korl: icr had a significant increase in the litter size of the second litter as compared with the first litter . Therefore, the number of offspring of the first litter was taken as to litter size . Time to delivery represents the time period from mating to birth and fertility rate represents a ration between number of female mice giving birth and those used for mating . Cages were observed three times per week and the number of pups born dead or alive and the number of pups weaned in each cage was recorded . Statistical changes in all data were determined using one - way analysis of variance (anova). The a: icr (crl: cd1(icr)) and b: icr (tac: icr) mice were purchased from two different commercial laboratory animal breeding company in korea . The mice were housed in a pathogene - free animal facility under a standard 12 h light / dark cycle, maintained in conventional housing conditions at 221 and 5010% relative humidity . All mice were provided with ad libitum access to standard irradiated chow diet (purina rodent chow 38057) consisting of moisture (9.03%), crude protein (21.52%), crude fat (5.03%), crude fiber (4.33%), crude ash (6.31%), calcium (1.18%), and phosphorus (0.35%) and drinking water . All animal procedures were conducted in accordance with guidelines of the institutional animal care and use committee, national institute of food and drug safety evaluation (nifds, cheongju, korea). Phylogenetic trees were constructed using the dnapars program of phylip (phylogeny inference package) version 3.695 (http://evolution.genetics.washington.edu/phylip.html) based on alignment of snps . Seqboot and condense programs of the phylip package were used in bootstrap analysis of 100 resamplings of the data sets . The neighbor program of phylip measurement of body and organ weights was made based on the methods of the international mouse phenotyping consortium . Hematological analysis was determined using by advia 2010i hematology system with autoslide (siemens, germany). Clinical chemical parameters were measured from a single serum sample using a hitachi 7100 automatic analyzer (hitachi, japan). We noticed that korl: icr had a significant increase in the litter size of the second litter as compared with the first litter . Therefore, the number of offspring of the first litter was taken as to litter size . Time to delivery represents the time period from mating to birth and fertility rate represents a ration between number of female mice giving birth and those used for mating . Cages were observed three times per week and the number of pups born dead or alive and the number of pups weaned in each cage was recorded . Statistical changes in all data were determined using one - way analysis of variance (anova). The phylogenetic trees based on distance, the minimum genetic distance of korl: icr and other icr showed a genetic structure with three main clusters (figure 1). From these distance matrices, one can infer the nature of the identified stock differences and the genetic relationships between them . This indicated that b stock has small genetic diversity compare with a stock and korl stock, and there is large genetic diversity between a stock and b stock . We measured litter size, body weight, body length, various organ weight, hematology and clinical blood chemistry of the korl: icr compared to other icr . Mouse phenotype analysis, body weight of korl: icr, a: icr and b: icr has been measured during the designated developmental periods (3 weeks to 12 weeks). Korl: icr and other icr mice experienced similar in body length as well (figure 2b). Female korl: icr mice were experiencing a significant increase in liver, heart and kidney weight compared to a: icr, but no difference with b: icr was observed (figure 2c). Male korl: icr mice had a significant increase in liver, spleen and testis weight compared to a: icr (figure 2d). The mean litter size of korl: icr mice was 14.52.9 which was higher than commercial icrs' (table 1). Otherwise, there are no significant differences among the biological phenotypes of korl: icr and other icr . As summarized in table 2, 3, some hematological and biochemical parameters have been observed with sporadically significant differences . Laboratory mice are the essential resource to identify the potency and safety of the drugs and estimate the potency and adverse effects of drugs during the developing stage . According to the effectuation of the nagoya protocol, laboratory mice are not only indispensable for life science studies but also are being considered as important resource . Among laboratory mice, icr mice are most popular outbred mice broadly used in various fields like toxicology, oncology, pharmacology, pharmaceutical products safety test, and etc . . Swiss mice, that were crossed in 1926 and then adopted by the institute of cancer research in 1974 (subsequently merged with commercial production of icr by companies began in 1959, and the strain was made available to academic and commercial breeders . Currently, many breeders worldwide have possessed their own icr stocks and the icr stocks have been reproduced for their academic or commercial purpose . The opinions that there might be possible to have differences among icr mice already ensured in various regions of world which have been raised for long term and among mice of the identical stock bred by different breeders have been proposed in some point of view, but there are not sufficient evidences to support these yet . Even though outbred mice are hard to realize the reliability and the repeatability, they are consider to be worked as better resource when the test result is applied to human due to the genetic diversity if compared to inbred mice . In addition, the advantage of using the icr stocks is extremely high in reproduction rate than the other outbred mice so that it may help the study cost effective . Every icr breeder has adopted and being bred these following common protocols: maintain icr stocks as long as possible, maintain genetic diversity as possible by composing multiple colonies, and minimize the genetic drift . This study is carried out to establish scientific supports how different in aspect of normal growth curve, breed grad, and clinical blood chemical analysis to ensure the characterization data of icr stock mouse which have been bred by circulation breed approach for more than 50 years . The breed capacity of both korl: icr mice and commercial icr mice were identified . The mean litter size of the korl: icr mice were higher than other icr mice . Between monthly litter size and litter size per number of pregnancy of the korl: icr mice did not experience a significant difference, but the size of second and third litter were higher than first and fourth litter (data not shown). The korl: icr mice were having median value of between a: icr and b: icr in a basic phenotype analysis . To tests evaluation of reactivity drugs, their behavioral changes were observed after administering caffeine and chlorpromazine which activates the central nervous system; it was possible to identify that the recovery rate of the korl: icr was similar to other icr mice (data not shown). Korl: icr was deduced that there was no significant difference with commercial icrs, and some weight gaps of the organs might be dependent the breeding condition (feeds, the breeding place). Since the comparison studies on laboratory mice have not been remarkably activated, we have faced the difficulty of comparing the study result at this moment . (2016) suggested that three icr groups, the korl: icr, a: icr and b: icr derived from different sources have an overall similar response of body weight, histopathological structure and inflammation to gastric ulcer inducers . Alcohol induced ulcer model comparison assessment for reviewing the characteristics of the korl: icr were having similar result . However, the result would be more appropriate to compare with control group rather than the comparison of the absolute value because of a significant difference in certain enzymes . Another implication of the study is that references data (breeding rate and basic phenotype) regarding the korl: icr are secured and can be shared with the other researchers . The korl: icr stocks have been used for decades in terms of conducting lot release project and more, in the national institute of food and drug safety evaluation . In conclusion, we will study secure general documents needed to have as mice resource through this accumulation of various experimental data.
With development of thoracic diseases and thoracic surgery skills, spinal posterior pedicle internal fixation has been clinically used to treat thoracic fractures and tumors, according to the spinal 3-column theory of denis . It has become an important spinal fixation operation due to its slight operational trauma, short fixed segments, and high fixed strength . Boucher first reported a successful pedicle internal fixation in 1959, but it has not been applied widely until 1986, after roy - camille reported excellent clinical efficacy of pedicle fixation in treating spinal fractures . However, the vertebral arch in the mid - upper thoracic vertebrates is relatively thinner and has more malformations, making screw placement difficult high risk . Complications of failed placement, such as spinal cord damage, injuries of great vessels around the vertebral bodies, fracture of pedicles, and thoracic nerve root injury, happen occasionally, posing a technical difficulty that needs to be solved . Although intra - operative imaging positioning and screw placement under navigation have been popular research topics in recent years, and accuracy of screw placement has been improved, the high expense, complex process, and increased radiation risks during the operation have limited its adoption . This article was based on, and made some improvements of, lu s method, which applied 3-d printing technique in thoracic pedicle screw placement . Medical mimics software, combined with 3-d laser printing technique, was used in manufacturing individualized thoracic pedicle screw guide plates, which could assist in accurate insertions . There were 2 males and 1 female, with han ethnicity and an average death age of 52.3 years (all provided by the anatomy dept . The t1 to t12 vertebral bodies were taken out of each body, with peripheral soft tissues wiped out and intervertebral discs preserved . A thin - layer 3-d ct scan was performed (siemens, germany) to exclude the samples with bone - structure damage such as fractures, occupation, or malformation of vertebral pedicles . Samples were scanned with 3-d ct, and images with a dicom format were obtained (the layer and scan thickness were all <1.25 mm). 3-d images of spines were produced by medical mimics software and corresponding 3-d images of each vertebral body were designed with 3-d data from ptc creo 3.0 (http://www.ptc.com/product/creo) and geomagic (http://www.geomagic.com/en/products/design/overview) software . Ptc creo was used to design models and geomagic was used in reverse engineering technique . A virtual screw panel was established on a vertebral model by medcad, which was parallel with the long axis of the pedicle (figure 1). After the virtual screw was placed, an entry point and an axial leaning angle and sagittal dip angle of the screw were confirmed on 3-d reconstructive images . The screw panels were hollowed out and a 3.04.0 cylindrical panel formed reversely, which made a guide plate leading channel (figure 2). In the manufacture of guide plates, the inner sides of guide plates and the bony structures of the backsides of vertebral bodies were made to completely fit with each other . Then the models were saved in stl format and transmitted to a sps600 3-d printer (provided by xian jiaotong university). Posterior soft tissues of vertebral bodies were cleared, and normal bony structure damage was avoided . The operator held the corresponding individualized guide plate in one hand, matched it up with the vertebral body, and kept it stable while the surgeon held the screw panel alongside the leading panel on the plate with the pedicle instrument open in the other hand . After the guide plate was removed, screws were placed alongside bilateral panels (figure 5). X - ray assistance or screw panels correction by exploration during placement should be avoided to reflect true assistant effects of guide plates . After placement of screws, the positional relation between the screw and the neighboring structures, such as vertebral body and spinal canal (figure 6), were first directly observed, then all the samples for examination were dissected to assess the positional relationship between the screw and the neighboring structures (figure 7). There were 2 males and 1 female, with han ethnicity and an average death age of 52.3 years (all provided by the anatomy dept . The t1 to t12 vertebral bodies were taken out of each body, with peripheral soft tissues wiped out and intervertebral discs preserved . A thin - layer 3-d ct scan was performed (siemens, germany) to exclude the samples with bone - structure damage such as fractures, occupation, or malformation of vertebral pedicles . Samples were scanned with 3-d ct, and images with a dicom format were obtained (the layer and scan thickness were all <1.25 mm). 3-d images of spines were produced by medical mimics software and corresponding 3-d images of each vertebral body were designed with 3-d data from ptc creo 3.0 (http://www.ptc.com/product/creo) and geomagic (http://www.geomagic.com/en/products/design/overview) software . Ptc creo was used to design models and geomagic was used in reverse engineering technique . A virtual screw panel was established on a vertebral model by medcad, which was parallel with the long axis of the pedicle (figure 1). After the virtual screw was placed, an entry point and an axial leaning angle and sagittal dip angle of the screw were confirmed on 3-d reconstructive images . The screw panels were hollowed out and a 3.04.0 cylindrical panel formed reversely, which made a guide plate leading channel (figure 2). In the manufacture of guide plates, the inner sides of guide plates and the bony structures of the backsides of vertebral bodies were made to completely fit with each other . Then the models were saved in stl format and transmitted to a sps600 3-d printer (provided by xian jiaotong university). Posterior soft tissues of vertebral bodies were cleared, and normal bony structure damage was avoided . The operator held the corresponding individualized guide plate in one hand, matched it up with the vertebral body, and kept it stable while the surgeon held the screw panel alongside the leading panel on the plate with the pedicle instrument open in the other hand . After the guide plate was removed, screws were placed alongside bilateral panels (figure 5). X - ray assistance or screw panels correction by exploration during placement should be avoided to reflect true assistant effects of guide plates . After placement of screws, the positional relation between the screw and the neighboring structures, such as vertebral body and spinal canal (figure 6), were first directly observed, then all the samples for examination were dissected to assess the positional relationship between the screw and the neighboring structures (figure 7). We measured 25 vertebral samples through 3-d ct scans and precisely rebuilt them into 3-d models using medical mimics software . The corresponding individualized guide plates were established by medical mimics software . Under assistance of guide plates, the operative time of placement was 50 to 62 seconds, with an average time of 56.2 seconds . Post - operative direct observation showed a good positional relationship between screws and neighboring structures, such as vertebral bodies and vertebral canals, and no bony cortex was penetrated (figure 6). The dissection showed that the 50 screws were all in pedicles and vertebral bodies, with proper length and angles (figure 7). With development of spinal surgery, internal fixation of posterior thoracic pedicle screw has grown increasingly important . A key point of this operation is the one - time accurate placement of pedicle screw, but this has been very difficult and has safety concerns because of anatomic characteristics of thoracic pedicles . According to the spinal 3-column theory, placement of thoracic pedicle screw through pedicle bony channel better complied with the thoracic anatomic mechanical properties, which could improve spine stability compared with other internal fixations . With less trauma and a shorter fixed segment, a maximum number of active segments of spine can be maintained . However, according to kothe, thoracic pedicles were thin, short, and narrow, and the cortex was thin and fragile; therefore, thoracic pedicles easily broke . In addition, the angles of thoracic pedicles were often different from each other, which made the error rate of one - time placement of thoracic pedicle screws very high, thereby damaging peripheral tissues and leading to severe consequences . In complex thoracic fractures or vertebral malformation, . The penetration rate of free - hand insertion of thoracic pedicle screw can be 30% to 40% . Physicians started to consider safety of screw insertion as the most important factor in this operation . Some experts thought that thoracic pedicle screw fixation must be limited to spinal posterior column damage and 3-column damage with complete spinal cord injury . Some even thought that other thoracic screw fixations must replace thoracic pedicle screw placement . In order to remedy a lack of precision, many researches relied on imaging techniques in spinal surgeries, using intra - operative c - arm or ct imaging navigation to assist accurate placement of pedicle screw placement, and achieved great improvement . The intra - operative imaging navigation screw placement technique played an important role in accurate placement of thoracic pedicle screws, which was considered as the best way to assess accuracy of pedicle screw placement . But with errors brought by intra - operative position changes and spinal instability, and disadvantages such as a lack of real - time navigation, operating complexity, and high cost, this technique was not suitable for wide use . Therefore, a practical, less - expensive, and easy - spreading assistant screw placement method is necessary for clinical work . With application of 3-d printing in orthopedic spinal surgeries, individualized production of guide plates the relatively simpler operation processes and higher safety of guide plates remedied limitations of imaging navigation . Some researchers have applied this technique in assisting cervical pedicle or vertebral plate screw placement, and proved that it could ensure accurate placement of screws . This study aimed to improve lu s method: to select experimental thoracic samples, to design and produce individualized thoracic pedicle screw guide plates, and perform assistant screw placement . The pilot study showed that 3-d models of thoracic pedicle screw placement guide plates built using medical mimics software could be used for establishing screw placement parameters by a surgeon on the basis of accurate assessment of thoracic pedicle - related anatomic structures . The individualized screw placement guide plates could be used for assisting screw placement and to ensure accurate insertion of screws . Medical mimics software used for guide plate designation could set accurate parameters and reduce data lost during process exchange between software, through which models had a higher compliance . The process of guide plate designation has been used in daily education and research, which more closely resembled clinical work, and could be used in demonstrative teaching . While producing the guide plate, a 3-d printer could print melted materials into formation layer by layer, with shorter time and lower cost, making it possible to use guide plates in emergent surgeries . Printing materials are made of medical biodegradable polylactic acid, which could be used in operations after sterilization by ethylene oxide at a low temperature, making it more convenient and less risky for clinical application . Radiation damage by c - arm or ct imaging navigation in traditional operations and high expense were reduced . Data transformation between 3-d ct scans and printing might have some errors or defects, which make models different from actual structures . Materials for printing are still limited and might have morphological changes from formation to usage, which could lead to small errors when plates are applied to vertebral bodies . But with development and perfection of 3-d printing technique and material science, these limitations could be overcome and clinical advantages would be predominant . In this study, vertebral bodies were removed from corpses, followed by thoracic pedicle screw placement, which might cause deviations when compared with placement during surgeries in real life . However, crew placement should also be done after complete exposure of vertebral plate in lumbar posterior approaches, which is not significantly different from the method of this study . Another limitation is that there was no comparison between actual surgeries and experiments on corpses in this study . With development of spinal surgery, internal fixation of posterior thoracic pedicle screw has grown increasingly important . A key point of this operation is the one - time accurate placement of pedicle screw, but this has been very difficult and has safety concerns because of anatomic characteristics of thoracic pedicles . According to the spinal 3-column theory, placement of thoracic pedicle screw through pedicle bony channel better complied with the thoracic anatomic mechanical properties, which could improve spine stability compared with other internal fixations . With less trauma and a shorter fixed segment, a maximum number of active segments of spine can be maintained . However, according to kothe, thoracic pedicles were thin, short, and narrow, and the cortex was thin and fragile; therefore, thoracic pedicles easily broke . In addition, the angles of thoracic pedicles were often different from each other, which made the error rate of one - time placement of thoracic pedicle screws very high, thereby damaging peripheral tissues and leading to severe consequences . In complex thoracic fractures or vertebral malformation, . The penetration rate of free - hand insertion of thoracic pedicle screw can be 30% to 40% . Physicians started to consider safety of screw insertion as the most important factor in this operation . Some experts thought that thoracic pedicle screw fixation must be limited to spinal posterior column damage and 3-column damage with complete spinal cord injury . Some even thought that other thoracic screw fixations must replace thoracic pedicle screw placement . In order to remedy a lack of precision, many researches relied on imaging techniques in spinal surgeries, using intra - operative c - arm or ct imaging navigation to assist accurate placement of pedicle screw placement, and achieved great improvement . The intra - operative imaging navigation screw placement technique played an important role in accurate placement of thoracic pedicle screws, which was considered as the best way to assess accuracy of pedicle screw placement . But with errors brought by intra - operative position changes and spinal instability, and disadvantages such as a lack of real - time navigation, operating complexity, and high cost, this technique was not suitable for wide use . Therefore, a practical, less - expensive, and easy - spreading assistant screw placement method is necessary for clinical work . With application of 3-d printing in orthopedic spinal surgeries, individualized production of guide plates improved accuracy of operations . The relatively simpler operation processes and higher safety of guide plates remedied limitations of imaging navigation . Some researchers have applied this technique in assisting cervical pedicle or vertebral plate screw placement, and proved that it could ensure accurate placement of screws . This study aimed to improve lu s method: to select experimental thoracic samples, to design and produce individualized thoracic pedicle screw guide plates, and perform assistant screw placement . The pilot study showed that 3-d models of thoracic pedicle screw placement guide plates built using medical mimics software could be used for establishing screw placement parameters by a surgeon on the basis of accurate assessment of thoracic pedicle - related anatomic structures . The individualized screw placement guide plates could be used for assisting screw placement and to ensure accurate insertion of screws . Medical mimics software used for guide plate designation could set accurate parameters and reduce data lost during process exchange between software, through which models had a higher compliance . The process of guide plate designation has been used in daily education and research, which more closely resembled clinical work, and could be used in demonstrative teaching . While producing the guide plate, a 3-d printer could print melted materials into formation layer by layer, with shorter time and lower cost, making it possible to use guide plates in emergent surgeries . Printing materials are made of medical biodegradable polylactic acid, which could be used in operations after sterilization by ethylene oxide at a low temperature, making it more convenient and less risky for clinical application . Radiation damage by c - arm or ct imaging navigation in traditional operations and high expense were reduced . Data transformation between 3-d ct scans and printing might have some errors or defects, which make models different from actual structures . Materials for printing are still limited and might have morphological changes from formation to usage, which could lead to small errors when plates are applied to vertebral bodies . But with development and perfection of 3-d printing technique and material science, these limitations could be overcome and clinical advantages would be predominant . In this study, vertebral bodies were removed from corpses, followed by thoracic pedicle screw placement, which might cause deviations when compared with placement during surgeries in real life . However, crew placement should also be done after complete exposure of vertebral plate in lumbar posterior approaches, which is not significantly different from the method of this study . Another limitation is that there was no comparison between actual surgeries and experiments on corpses in this study . Thoracic pedicle screw placement guide plates can be produced by 3-d printing . With high accuracy in placement and convenient operation, it provides a new method for accurate placement of thoracic pedicle screws.
A sound, agreeable or disagreeable, is a stimulus discerned by the sense of hearing . Disagreeable or undesired sounds were described as noises, which may cause undesirable masking of sounds, may interfere with speech and communication, may produce pain, injury and brief or perpetual loss of hearing . The acoustic environment of learning - teaching activities at a dental college is characterized by high noise levels in relation to other teaching areas, due to the exaggerated noise produced by the use of dental equipment's by many users at the same time . The sources of dental sounds that can be treated as potentially damaging to hearing are high - speed turbine handpieces, low - speed handpieces, high - velocity suction, ultrasonic instruments and cleaners, vibrators and other mixing devices, model trimmers and also worth mentioning are air conditioners . In dental learning areas, teachers and students exposure to noise constitutes a health risk and has both auditory and non - auditory effects . The non - auditory effects include hypertension, sleep disturbance, decreased learning performance, stress reactions, interference with communication and concentration, annoyance, mental fatigue and a reduction in efficiency . While auditory effects may include permanent hearing loss, and short term exposure to loud noise can cause a temporary change in hearing or a tinnitus . Hearing loss caused by noise is referred to as noise induced hearing loss . According to the national institute for occupational safety and health (niosh), exposure to noise levels above 80 db is associated with these consequences, which depends on the intensity of the noise, distance to the source, total duration of noise and the individual's age, physical condition and sensitivity . Furthermore, noise has an adverse impact on patients as a factor that may cause fear . Exposure to noise is measured in units of sound pressure levels called decibels, named after alexander graham bell, using a - weighted sound levels [db(a)]. The a - weighted sound levels closely match the perception of loudness by the human ear . Niosh has recommended that all worker exposures to noise should be controlled below a level equivalent to 85 db(a) for 8 h / day to minimize occupational noise induced hearing loss . Noise pollution is one of the most important situations requiring a solution by the contemporary world . Niosh has recognized noise as 1 of the 10 leading causes of work related diseases and injuries . In dental learning areas, teachers and students are vulnerable to different noise levels while working and teaching in dental clinics and laboratories . The aims of this study were to measure noise levels produced during the different dental learning clinics, by equipment's used in dental learning areas under different working conditions and by used and brand new handpieces under different working conditions . In pre - clinical and clinical areas, the microphone was placed at ear level at a distance of 15 cm from a main noise source to simulate the auditory position of the operator . The noise levels of the equipment's were measured at the corner of the learning area, which may consider the less noisy part of the learning area to eliminate as much as possible the interference with the external noise . The noise levels were measured over about 20 s interval and the maximum and minimum intensities in decibel was recorded . This was repeated three times sequentially in the same day, so we recorded 6 measurements for each equipment, one maximum and one minimum for each time interval . The noise levels of equipment's used in laboratories, pre - clinical and clinical areas at the dental college of damascus university were measured under different working conditions . The laboratories, pre - clinical and clinical areas will be henceforth referred to as dental learning areas in this study . The equipment's of which the noise levels were measured in clinical areas were: ultrasonic scaler, turbine, contra angle handpiece, micro motor handpiece, low volume suction pump, high volume suction pump and amalgamator (capsule). The measurements were taken with the equipment only turned on (without cutting) and during cutting operations . Ultrasonic scalers with or without suction pump and suction pumps running free and when they touch mucosa were measured for noise levels . The noise level of micro motor handpiece was measured by setting it at 35.000 rpm . The noise levels of different dental learning clinics was measured by placing the noise level meter at the center of the clinic during the middle - third of working time (between 11 a.m. and 1 p.m.), which nearly represents the highest noise hours . The number of equipments, which were used at the same time, was 20 equipments . Noise levels were measured in seven clinics: operative, fixed prosthodontics, removable prosthodontics, endodontics, pedodontics, oral surgery and periodontics . The sound levels were measured with a precision sound level meter (beha unitest 93517, germany) with a microphone in dental learning areas . The sound level is measured on the a scale, which was designed to mimic the response of the human ear . At the dental laboratories, the microphone was placed near the technician's ear at a distance of 15 cm from a main noise source to simulate the noise intensity reaching the eardrum and another reading was taken 2 m away . This was to simulate the person within a 2 m radius of the operator who is also exposed to the same noise . The equipments of which the noise levels were measured in the dental laboratories were: stone trimmer, automatic molding machine, manual molding machine and sandblaster . The data were collected, tabulated and statistically analyzed using t - tests with significance level set at 5% using the statistical package for the social sciences program version 13(spss inc ., chicago, il, usa). The results of the noise levels of equipments measured in dental laboratories at two distances of 15 cm and 2 m are shown in table 1 . Noise levels [db(a)] of dental laboratory engines the noisiest laboratory equipment recorded in this study was by the sandblaster with an la(eq) of 93.32 1.99 at 15 cm distance . The results of the noise levels of the equipments measured in pre - clinical and clinical areas are shown in table 2 . Noise levels [db(a)] of the equipments measured in pre - clinical and clinical areas the results indicated that the maximum sound levels of equipments in dental clinics and laboratories were 92.2 db and 96 db, respectively . In dental clinics, the highest noise was produced by the micro motor handpiece while cutting on acrylic (92.2 db) and the lowest noise (51.7 db) was created by the ultrasonic scaler without suction pump . The highest noise in laboratories was caused by the sandblaster (96 db at a distance of 15 cm) and the lowest noise by the stone trimmer while only turned on (61.8 db at a distance of 2 m). There was significant differences in noise levels of the equipments used in dental laboratories and dental learning clinics (p = 0.007). The mean noise levels of the equipments used in dental laboratories were much higher than those used in dental clinics . The mean noise level for dental laboratories engines at a distance of 15 cm from a main noise source was 81.62 db, compared with the mean value of 73.75 db for dental clinic equipments (p = 0.009). The laboratory engines had the highest noise levels (at a distance of 15 cm from a main noise source-81.62 db), whereas the noise levels in high - speed turbine handpieces (75.44 db) (p = 0.003) and the low - speed contra angle handpieces (68.31 db) (p <0.01) were decreased . The noise level of a contra angle handpiece at the clinical areas was significantly lower than at the pre - clinical areas (p = 0.019). Noise levels of turbine in cutting activities compared to non - cutting showed that the noise levels measured during the cutting activities were significantly higher to those found when only turned on (p <0.01). The average value of the difference was equal to 5.38 db(a) and 19.45 db(a) for brand new and used respectively . There was no significant difference in noise levels of contra angle handpiece in cutting activities compared to non - cutting activities in both pre - clinical and clinical areas for brand new and used ones . The average value of difference for turbine and contra angle handpieces (only turned on) was equal to 6.09 db(a). Furthermore, there was no significant difference in noise levels of micro motor handpiece in cutting activities compared to non - cutting activities . There was no significant difference in noise levels between low and high volume suction pump . Furthermore, there was no significant difference in noise levels of ultrasonic scaler without suction pump and with suction pump . Noise levels of used equipments compared with brand new equipments in the clinics showed that the noise levels produced by used turbine were significantly higher than those produced by the brand new turbine (p = 0.045) [figure 1]. There was no significant difference in noise levels between brand new and used contra angle handpieces in both pre - clinical and clinical areas . Average noise levels of used and brand new dental turbine compared in this study, the high - speed turbine was significantly noisier than low - speed contra angle (p = 0.001). The average value of difference for the high - speed turbine against low - speed contra angle handpiece was equal to 7.14 db(a) [figure 2]. Mean noise levels of high - speed turbine and contra angle handpieces the results of the noise levels at the center of the dental learning clinics are shown in table 3 . The highest noise level for all dental clinics was at the pedodontic clinic (67.37 5.56 db(a)) (p = 0.019). In this study, noise levels of the equipments used in dental learning areas under different working conditions were measured . The noise levels measured in this study were similar to that measured in other international studies of noise in dentistry . Noise levels for suction pump were 69.41 - 81.51 db(a) in this study, whereas in the united kingdom they were 68 - 70 db(a), in portugal they were 70 - 74 db(a) and in india they were 79 - 81 db(a). Noise levels for the turbine were 66.72 - 84.16 db(a) in this study, whereas in the united kingdom, portugal, india and saudi arabia were 70 - 75 db(a), 68 - 76 db(a), 75 - 81 db(a) and 69 - 76d b(a), respectively; for contra angle handpiece they were 66.12 - 70.5 db(a) in this study, whereas in united kingdom, portugal, india and saudi arabia were 72 - 75 db(a), 69 - 75 db(a), 70 - 76 db(a) and 65 - 71 db(a), respectively . The noise levels of turbine measured during the cutting activities were significantly higher to those found when only turned on . Presented average values of + 10 db(a), in similar conditions of measurement in portugal and saudi arabia, respectively . The significant level of difference in noise levels of used turbines compared to brand new turbines could be an indication of bearing failure . The bearing resistance is affected by wear, not only of the metal surfaces, but also of the ball - cages, when roughness contributes to friction . In general, the used turbine was noisier at an average of about 9.11 db(a) difference more than the brand new one [figure 1]; therefore, the hearing damage risk may be lesser among dentists who use brand new turbine . In this study, the high - speed turbine was the noisiest equipment compared to low - speed contra angle . This is concordant with antecedent studies mentioning that the high - speed turbine handpiece generates a higher noise level than the low - speed handpiece . Maximum sound pressure levels of the noise created by the dental drill was 91.9 db by the brand used dental turbine while cutting on a tooth, which has a risk of damage to the dentists hearing . The noise level of a contra angle handpiece at the clinical areas was lower than at the pre - clinical areas, which may have been because students rarely used the maximum speed of the air contra angle handpiece during dental treatment, while in the pre - clinical area it was always used at the higher speeds . . Dental laboratories in dental teaching institutions were the areas of highest noise levels when compared to other dental learning areas . The effect of noise on learner comfort affecting the work performance and mental efficiency has been researched . Noise can induce learned helplessness, increase arousal, alter the choice of task strategy and decrease attention to the task . Suggest the classification given by cavanaugh to set a limit value in places of learning in dental teaching institutions . Accordingly, 56 db(a) could be adequate as the upper limit value for a relaxed communication at a normal tone at 3 m. all the evaluated areas presented a value higher than this maximum . The highest noise level recorded for all dental clinics was at the pedodontic clinic . This may be due to the children who are normally crying during the oral health treatment . Comparison of noise levels in this study with some european limits indicated that they did not comply with these laws . Some international legal limits for equipment noise levels in la(eq) (db[a]) are: italy 40, france 38, sweden 35, portugal 46 and india 50 . In this study, there were some high recorded measurements of noise levels, which have a risk of damage to the dentists hearing (exceeding the limit of risk of hearing loss of 85 db[a]) such as stone trimmer, manual molding machine, sandblaster (at distance 15 cm and 2 m), low volume suction pump, turbine (brand used) and micro motor handpiece . Therefore, a necessary reduction of exposure in sound levels is required for acoustic comfort . Based on the above study it can be concluded that the noise levels detected in this study were considered to be close to the limit of risk of hearing loss (85 db(a)); a necessary reduction of exposure in sound levels is required for acoustic comfort.
The growing resistance of pathogens is one of the biggest public problems worldwide.1 multidrug - resistant bacterial strains can cause severe infections as they are no longer responsive to most conventional antibiotics.2,3 to combat these pathogens, efforts have been extended to develop a new generation of antibiotics . Host defense peptides for their immunomodulatory properties, are cationic amphiphilic peptides, which are the first line of defense to protect organisms from microbial infection.48 it has been demonstrated that naturally occurring or synthetic amps can be a new functional class of antibiotics.9,10 amps are antimicrobial agents based on their activity against the prokaryotic membrane . These agents adopt globally amphipathic conformations upon initial contact with bacterial membranes rich in anionic phospholipids . The conformations, which resemble detergent - induced micelle formation, result in total membrane disintegration in which their cationic and hydrophobic side groups segregate into distinct regions . This finding indicates that amps are potential antibiotic agents with a different antimicrobial mechanism, and that this activity mainly depends on their physical mechanism.11 the structural and sequence diversity of amps include amphipathic -helices (eg, cathelicidins), -sheets with 24 disulfide bridges (defensins and protegrins), extended conformation (indolicidin), and beta - loop peptides (brevinin).1215 among the amps, human defensins and cathelicidins play an important role, linking innate and acquired immunities (figure 1).8 importantly, amps are therapeutic agents with a lower tendency to elicit antibiotic resistance than conventional antibiotics . Currently, the main reasons for the limited practical application of amps include their very high susceptibility to proteolytic degradation by microbial enzymes, toxicity due to high amounts of drug needed for therapy, relatively short half - life, and their high production cost.16 the design and synthesis of peptide mimics (peptidomimetics) have been developed to mimic the structure, function, and mode of action of host - defense amps, which act on bacterial cell walls or membranes and can potentially circumvent those obstacles . Antimicrobial peptidomimetics display antibacterial activity against a broad - spectrum of bacteria, including drug - resistant strains, and are less susceptible to resistance development in bacteria . A number of antimicrobial peptidomimetics have been developed in the last decade, such as -peptides,1719 peptoids,2025 arylamide oligomers,26,27 and -turn mimetics.28,29 recently, a new class of antimicrobial peptidomimetics termed aapeptides because they contain n - acylated - n - aminoethyl amino acid units derived from chiral peptide nucleic acid backbones have been developed . They are highly resistant to proteolytic degradation and their amphipathic structures can mimic the bactericidal mechanism of amps.3032 currently, different antimicrobial aapeptides have been developed, such as -aapeptides and -aapeptides.33,34 this review aims to describe recent progress in the discovery of peptidomimetics as new generation antimicrobial agents and discusses future directions for antimicrobial peptidomimetics in the emergence of multidrug - resistant bacteria . To improve the antimicrobial activity of peptidomimetics, the relationship between the structure and function of these peptides must be considered . Interestingly, antimicrobial peptidomimetics may be designed by joining amphiphilic peptide building blocks . In this regard, a potent and broad - spectrum antimicrobial activity can be fine - tuned by changing the ratio of cationic / hydrophobic groups via the introduction of hydrophobic building blocks, suggesting that the structure activity relationships in antimicrobial peptidomimetics indicate the balance of forces required for bactericidal activity.3538 to date, peptidomimetics have been designed by cyclization of linear peptides or coupling of stable unnatural amino acids . In addition, hu et al,30 and niu et al31,32 reported the development of a new class of peptidomimetics termed aapeptides, and depending on the position of the side chain (connected to either the -c or -c in relation to the carbonyl group), two subclasses of these peptides (-aapeptides and -aapeptides, respectively) have been designed (figure 2). Previous studies have indicated that focused libraries of linear aapeptide (including both -aapeptides and -aapeptides) sequences have been developed so that these sequences might mimic natural linear amps and adopt globally amphipathic conformations upon initial contact with bacterial membranes.39,40 moreover, it has been reported that the antimicrobial activity of some aapeptides is still generally comparable, or even superior, to the amp magainin as well as a previously reported linear -aapeptide 1 against several bacterial strains.31 interestingly, padhee et al39 reported that a focused library of different linear -aapeptide sequences such as 1 and 2 has been prepared to minimize the hemolytic activity, but with a potent and broad - spectrum antimicrobial activity to arrest the growth of both gram - positive and gram - negative bacterial pathogens . In addition, these authors showed that 1 and 2 are the most potent antimicrobial -aapeptide peptidomimetics with broad - spectrum activity, especially toward clinically relevant strains including the multidrug - resistant strains vancomycin - resistant enterococcus faecalis and methicillin - resistant staphylococcus aureus (mrsa).39 furthermore, a focused library of linear -aapeptide sequences containing -1, -2, -3, and -4 has been prepared.40 in this context, it has also been demonstrated that compared with -1, -2 and -3, -4 contains enhanced bactericidal activity against gram - positive strains, indicating that some -aapeptides are very potent and supporting their potential development as antimicrobial agents to treat gram - positive bacterial infections.40 however, they are quite toxic to blood cells as well as other mammalian cells . In fact, the antimicrobial activity of -aapeptides is likely to be enhanced if the overall hydrophobicity increases, which at the same time also leads to increased hemolytic activity and cytotoxicity . Interestingly, the hemolytic activity and cytotoxicity can be minimized by introducing more cationic residues . On the other hand, focused libraries of lipo aapeptides (including both -aapeptides and -aapeptides) sequences have been developed, and the lipid tails of these lipopeptides are important for biological activity to facilitate bacterial membrane interaction, giving them broad- spectrum activity against both gram - positive and gram - negative bacteria.30,31 a focused library of lipo antimicrobial -aapeptides sequences has been prepared, including 3 and 4.32 interestingly, the development of cyclic -aapeptides that mimic function of amps has been reported.41 these cyclic peptides have enhanced antimicrobial activity compared with their linear antimicrobial aapeptides, as their structures adopt a semirigid backbone conformation, resulting in a more stable amphipathic structure . Structure activity relationships of cyclic antimicrobial -aapeptides incorporating a global distribution of cationic and hydrophobic residues are in development . In this context, group amphiphilic building blocks can be joined, and the resulting oligomers are cyclized.42 since peptidomimetics interact nonspecifically with their target membranes, the addition of a positive charge by adding arginine, lysine, or histidine residues to the peptide sequence is required for initial electrostatic attraction with negatively charged bacterial membranes, whereas hydrophobic bulk guides insertion into the bacterial membrane . Recent developments in peptidomimetics that are formed through insertion into the amino acid backbone or heteroatom replacement indicate that several peptidomimetics form structural designs such as helices, sheets, turns, and loops via noncovalent interactions . To prepare aapeptides, the current literature indicates that different approaches have been developed.32,43 originally, the synthesis of these peptides was achieved using the building block strategy (figure 3).3032 in this approach, 9-fluorenylmethyloxycarbonyl - protected peptide building blocks were prepared and then assembled on a solid support to provide the desired peptide sequences.4447 another approach termed this approach is a solid - phase synthesis of the peptides, which eliminates the need of building block preparation; thus, chemically diverse functional groups can be conveniently introduced into the desired peptide sequences.43 at present, it has also been reported that preorganized secondary structures including helical or sheet - like conformations within peptidomimetics are unnecessary in the antibacterial activities of these peptides.39,40,4850 in contrast, the presence of backbones with certain flexibility can lead to a potent and broad - spectrum antimicrobial activity, indicating the importance of the conformational rigidity in the molecular design of antimicrobial peptidomimetics.5153 in this context, peptidomimetics have more dihedral angles compared to canonical peptides, and this molecular design induces high flexibility . Previous studies have indicated that in the molecular design of the amphipathic helical 21-mer peptide ([kaakkaa]3; amino acid sequence: k a a k k a a k a a k k a a k a a k k a a), the peptide s cytotoxic activity is highly dependent upon the spatial positions of tryptophan and cationic residues within the hydrophobic sector of an -helix.54 more recently, the synthesis of enzymatically resistant versions of amps by partial substitution of l - residues with nonnatural d- or b - residues has been developed . In this regard, mcgrath et al55 synthesized a lysine - leucine or klotho peptide known as (klaklak)2, which had low toxicity toward mammalian cells, with high antimicrobial activity . Furthermore, these authors demonstrated that d(klaklak)2, a variant of this molecule, is bactericidal against several gram - negative species, including escherichia coli, klebsiella pneumoniae, and acinetobacter baumannii . Interestingly, a strain of k. pneumoniae, which was resistant to conventional antibiotics, was susceptible to this peptide with the minimal inhibitory concentration of 75 g / ml . In addition, this peptide has stronger fungicidal activity.55 in the remainder of this review, the discussion will highlight the discoveries that have led to our current understanding of the development of peptidomimetics in the context of their use as therapeutic agents . Peptidomimetics represent an important field in pharmacology as they circumvent the limitations of amps used in therapy . Therapeutic applications of antimicrobial peptidomimetics have also been considered in regard to their high resistance against enzymatic degradation.5659 regarding antimicrobial peptidomimetics which are currently in phase ii clinical trials, choi et al60 have designed small arylamide foldamers that mimic amps . Importantly, these authors also demonstrated that hydrogen - bonded restraints in the structure of arylamide increase activity toward s. aureus and e. coli . On the other hand, the pharmaceutical company lytix biopharma as (troms, norway) has recently commenced phase i / iia clinical trials with another antimicrobial peptidomimetic known as lytixar tm (also known as ltx-109) for nasal decolonization of mrsa (http://www.lytixbiopharma.com). This peptidomimetic, containing a modified tryptophan derivate as lipophilic bulk, displayed a combination of high antibacterial activity against methicillin - resistant staphylococci and staphylococcal biofilms.57 another antimicrobial peptidomimetic, which is currently in phase ii clinical trials for the broad spectrum treatment of mrsa infections, is brilacidin (also known as pmx-30063).61 this peptidomimetic was synthesized by polymedix (radnor, pa, usa) and was then acquired by cellceutix (beverly, ma, usa) in september 2013 . It is important to consider that this peptide is a potent bactericidal with broad - spectrum activity, not only against gram - positive bacteria such as s. aureus and enterococcus faecium but also against gram - negative species such as e. coli . Interestingly, in a phase ii study involving patients with acute skin infections, 215 patients were treated with three different doses of brilacidin, and brilacidin s favorable profile was noted in 65%87% of patients.61 another antimicrobial peptidomimetic known as pol7080 has also been developed . Researchers at the university of zurich and polyphor ltd (allschwill, switzerland) have developed this peptidomimetic by means of a novel approach to peptide preparation named protein epitope mimetic technology . This peptidomimetic specifically targets pseudomonas aeruginosa through a mode of action that is different from the membrane - disrupting activity of the parent compound.29 currently, pol7080 is being prepared by the company polyphor, and in preclinical studies, this antimicrobial peptidomimetic was highly active on a broad panel of clinical isolates, including multidrug resistant pseudomonas bacteria, with a potent bactericidal activity in a mouse septicemia infection model . P. aeruginosa is an opportunistic bacteria which causes serious infections in patients with reduced immune systems (eg, those having acquired immunodeficiency syndrome or cancer). At present, phase i clinical studies29 of the peptidomimetic pol7080 have been completed in healthy individuals in europe, demonstrating the clinical safety and tolerability of this peptide (http://www.polyphor.com/products/pol7080). Regarding an important role of antimicrobial peptidomimetics in the prevention of virus infections and the treatment of cancer metastasis, antimicrobial peptidomimetics that inhibit virus replication as well as possess antitumor activity against different cancer cell lines have been designed . Importantly, the structure of these peptidomimetics has been explored by using a peptidomimetic library in order to obtain higher plasma and metabolic stabilities . Furthermore, the use of nanotechnology as delivery tool for both classes of peptides will be presented later in the review . As shown in previous reports, some -aapeptides are effective in arresting the growth of both gram - positive and gram - negative drug - resistant bacterial pathogens.50 in addition, it has been demonstrated that -aapeptides can mimic the human immunodeficiency virus (hiv) tat peptide by binding to hiv-1 rna with a high affinity, comparable to their peptide counterparts.44,46 moreover, it is well known that the cxc chemokine receptor 4 (cxcr4) is a coreceptor of hiv-1 infection in human cells.62,63 in this context, development of peptidomimetic ligands for cxcr4 as therapeutic agents for hiv-1 infection and cancer has been reported.6466 in fact, the peptidomimetic arg()-arg - nal(2)-cys(1x)-tyr - gln - lys-(d - pro)-pro - tyr - arg - cit - cys(1x)-arg - gly-(d - pro) () (pol3026) is a novel specific beta - hairpin mimetic cxcr4 antagonist, with potent anti - hiv activity.67 this peptide has a ten - fold increase in potency, with a good bioavailability profile following subcutaneous administration.67 interestingly, ligand binding site mapping using a panel of cxcr4 mutants demonstrated that the new analog d(1 - 10)-vmip - ii-(9 - 68)-sdf-1 (rcp222) share the interactive amino acids on cxcr4 with hiv-1 glycoprotein 120.68,69 to date, peptidomimetic ligands have served as inhibitors of stromal cell - derived factor-1 since these peptidomimetics are involved in the interactions of hiv-1 envelope glycoprotein and stromal cell - derived factor-1 with membrane ligands of cd4 + human cells.7074 another peptide with anti - hiv activity is alx40 - 4c . The structure of this peptide was designed from the basic hiv-1 transactivation domain for the inhibition of tat tar interaction.75 the anti - hiv effects of this peptide are elicited through selective binding to cxcr4.76 in addition, zhou et al77 reported that the human apj, a g protein - coupled seven - transmembrane receptor, is essential for its coreceptor activity for hiv-1; thus, it is an alternative target of alx40 - 4c to block hiv glycoprotein 120 from binding to the cellular membrane . On the other hand, regarding the development and improvement of nanoparticles for stabilization and delivery of antiviral peptides, a nanoformulation of the amphiphatic -helical peptide p41 (a positively - charged analog of c5a peptide, derived from the hepatitis c virus protein) has been designed to treat hiv / hepatitis c virus coinfection, indicating the potential of this nanoformulation for stabilization and delivery of antiviral peptides, while maintaining their functional activity.78 currently, cancer is a major concern in relation to human mortality, and all types of cancer are characterized by irregular cell growth . Antitumor drugs are subject to differences in target tissue and absorption, which can be particular to each patient . In addition, acquired drug resistance is considered the widespread cause for tumor recurrence.79 at present, some radiolabeled -aapeptides have been used as tracers for positron emission tomography, indicating a therapeutic application as anticancer agents.45 in addition, peptidomimetics have been the basis for a number of studies performed to discover new novel anticancer agents.79,80 in this regard, the in vivo inhibitory effects on the growth of tumor cell xenografts in nude mice by the cyclic pentapeptide fc092 ([d - arg2]-fc131), a cxcr4 antagonist, have been reported.81,82 the intrinsic relationship between its structure and its high specificity to tumor cells is likely playing the key role in the cytotoxicity of peptidomimetics . These characteristics allow the peptidomimetics to bind to cancer cells and disrupt the negatively - charged tumor cell membrane, which is derived from a greater than normal expression of anionic molecules such as sialic acid - rich glycoproteins or phosphatidylserine.83 importantly, these chemical differences aid the electrostatic interaction of the positively - charged peptide and the negatively - charged tumor cell membranes.80 studies have reported of amps that are effective against bacteria and cancer cells but not against normal mammalian cells such as cecropins from insects and magainins from amphibians.84,85 on the other hand, signal transducer and activator of transcription (stat) proteins are a family of cytoplasmic transcription factors . Phosphorylation induces their homo- or heterodimerization, and an important function of these dimers is to control gene expression . Stat3 is frequently activated in many human cancer cell lines and is involved in cancer development and progression . Importantly, dysregulation of stat3 can lead to increase in its activity and contribute to tumorigenesis ., it has been reported that an oxazole - based small - molecule stat3 inhibitor, which modulates stat3 stability, induces significant antitumor cellular effects.86 one primary goal of drug delivery for cancer therapy is to increase the amount of drug delivered to the tumor site and decrease its exposure to healthy tissues.87 recent advances in microencapsulation technologies have been used to enhance drug protectivity, availability, and distribution by employing different biodegradable delivery platforms like liposomes, dendrimers, nanoemulsions, polymeric nanocarriers, and nanoparticles . These nanoformulations can be used to control drug / molecule release and enhance targeted delivery and effectiveness.88 in this regard, wang and zhang89 encapsulated a polypeptide isolated from the unicellular green algae chlorella pyrenoidosa, which exhibited the highest inhibitory activity on human liver hepg2 cancer cells (49%), and they named the polypeptide chlorella pyrenoidosa antitumor polypeptide . The main mechanism of action of this peptidomimetic is condensation / fragmentation of nuclear chromatin.89 the in vitro release of this peptide against gastric cancer cells provided a basis for the development of encapsulated antitumor peptides . The peptidomimetics klaklakklaklak and the isoasp - gly - arg (or isodgr) peptides serve as potent tools for developing new antitumor peptides . They can selectively kill cd13/v3 + breast cancer cells in both in vitro and in vivo experiments by inhibiting angiogenesis by binding to v3 +, which is increased on tumor cells.90 currently, the antitumor role of the analgesic - antitumor peptide (agap) isolated from the scorpion buthus martensii has been reported . This protein, consisting of a small ubiquitin - related modifier linked with a hexahistidine tag from e. coli, was used as an antitumor peptide, and the main mechanism of action of this peptidomimetic is through cell cycle arrest.91 the recombinant system agap showed considerable inhibition of lymphoma and glioma propagation.91 interestingly, using sw480 human colon cancer cells, it was proposed that recombinant agap induces cell cycle arrest in the g0/g1 phase, attended by the decrease in the s phase without significant change in the g2/m phase.91 together, these studies strongly suggest that the use of peptidomimetics is a potent tool for developing new antitumor peptides . The main limitations in the use of these peptides are their poor bioavailability due to insolubility related to their intrinsic physicochemical properties, potential toxicity to host cells, tissue distribution, and poor pharmacokinetic issues . Despite these disadvantages, it is important to consider that further studies are needed to investigate the cost of large scale production of peptidomimetics and the transition of these peptides from the laboratory to the clinic to confirm that they provide an effective new class of therapeutic agents . However, the combination of the therapeutic use of peptidomimetics and conventional therapy against cancer (eg, chemotherapy, radiotherapy, or surgical procedures) can help in overcoming drug resistance in cancer cells . Increased funding and innovative research approaches to prepare peptidomimetics are required for practical use of these peptides as therapeutic agents . Substantial progress has been achieved in the past decade with respect to the development of antimicrobial peptidomimetics that mimic the bactericidal activity and mode of action of amps . Since several classes of peptidomimetics have great potential as a new generation of antimicrobial agents due to their low immunogenicity and enhanced stability compared with amps, in the near future, it will be important to resolve issues of hemolytic activity and cytotoxicity of some antimicrobial peptidomimetics . It will also be possible to gain further insight into the development of molecular design in peptidomimetics and to explore its medical and pharmacological applications . In fact, fine - tuning the biological activity of peptidomimetics may be readily achieved with the introduction of a variety of additional hydrophobic building blocks . In addition, mechanistic studies are needed to evaluate the possibility for antimicrobial peptidomimetics to induce drug resistance in bacteria, and research will be focused on the development of antimicrobial peptidomimetics against gram - negative bacteria, as they are generally more difficult to kill than gram - positive bacteria . Finally, an important challenge over the next decade will be to develop new potential drug delivery systems for peptidomimetics . In this context, nanoformulation approaches have emerged as an important tool to improve the delivery and stability of antimicrobial peptidomimetics . In conclusion, peptidomimetics possess significant properties that support their inclusion in the generation of new antimicrobial agents . Moreover, antimicrobial peptidomimetics have several advantages over amps, including enhanced stability, cell specificity, and better tolerability . Furthermore, the synthetic flexibility of these molecules allows fast structure modifications to create novel antimicrobial peptidomimetics, having particular pharmacological properties . Finally, structure activity relationships clear the way to establish peptidomimetic libraries, which can lead to the development of novel antimicrobial agents . A better understanding of the structural properties of peptidomimetics will potentially facilitate the practical use of these peptides as important therapeutic agents.
Chlamydia trachomatis, neisseria gonorrhoeae, mycoplasma genitalium, and trichomonas vaginalis are known pathogens in sexually transmitted infection (sti). However, most of those species present atypical symptoms and some patients have no symptoms [1 - 3]. Therefore, physicians sometimes have difficulty in making a differential diagnosis . Moreover, except for chlamydia trachomatis and mycoplasma genitalium, the treatment regimens between the species differ . In addition, routine bacterial culture may give negatives result for commercial sexual workers or asymptomatic people who recently experienced unprotected sexual contact and have acquired an sti [1 - 3]. Thus, there are neglected asymptomatic patients in the community who can serve as a reservoir of the sti [1 - 4]. Generally, ureaplasma urealyticum is not known as a clinical pathogen but it can be a cause of urethritis, especially in patients resistant to routine treatment . Mycoplasma hominis also does not play a great role in the pathogenesis of urethritis, but it can be a heavy pathogen in immunocompromised patients . Recently, many reports have revealed that microorganisms such as ureaplasma urealyticum and mycoplasma hominis can contribute not only to lower genitourinary infection but also to infertility . Therefore, identification of the incidence of asymptomatic sti such as urethritis or cervicitis and determination of the prevalence of these 6 species in asymptomatic people is very important . Accordingly, we conducted a screening test for sti by using multiplex polymerase chain reaction (pcr) in asymptomatic people . The institutional review board of the catholic university of korea, college of medicine, approved the study protocol, and all patients provided written informed consent to participate in the study (irb no: sc10snsi0091). From july 2010 to december 2010, 802 persons who came to the hospital for a general checkup participated voluntarily and a brief questionnaire was obtained . Sexually active, asymptomatic people aged between 20 and 60 years were enrolled . We defined' sexually active' as having had sexual intercourse in the preceding 3 months . People with genitourinary symptoms such as urethral or cervical discharge, dysuria, or itching in the genital area were also excluded . First - voided urine specimens were collected in sterile 50 ml screw - cap plastic bottles . In women, the specimens from the 709 men and women were equilibrated to room temperature and centrifuged at 5000xg for 15 minutes . The supernatant was discarded, and the pellet was resuspended in 1 ml 1xpbs before dna extraction . Genomic dna was extracted from the pretreated specimens (swab or urine) by using the qiaamp dna mini kit (qiagen, hilden, germany) according to the manufacturer's instructions . Pcr amplification was performed with the seeplex std6 ace detection kit (seegene, seoul, korea) according to the manufacturer's instructions . The kit contains sets of primers that were specifically designed from highly conserved regions of genetic sequences for the 6 organisms (chlamydia trachomatis, neisseria gonorrhoeae, mycoplasma genitalium, ureaplasma urealyticum, mycoplasma hominis, and trichomonas vaginalis) by use of seegene dpo technology . An internal control was included in the pcr mix to detect the presence of pcr inhibitors . For the negative control, sterile deionized water was used as the pcr template instead of nucleic acid . The positive control contained in the kit amplified fragments were separated by use of the labchip dx seeplex assay system(caliper, hopkinton, ma, usa). Amplified pcr products were separated and detected by automated gel electrophoresis with the labchip dx seeplex assay system (caliper, hopkinton, ma, usa). The pcr products from each sample (20 l) were transferred to 96-well plates and placed in the labchip dx instrument . The samples were loaded automatically on the seeplex chip and detected sequentially according to the sample order . Analysis was performed with designated software (seegene viewer) that presents each of the samples and identifies the fragments that yield a positive readout for the bands of interest in the presented results . A tabulated matching matrix provides a simple readout, identifying matching bands to the type of sti pathogens . From july 2010 to december 2010, 802 persons who came to the hospital for a general checkup participated voluntarily and a brief questionnaire was obtained . Sexually active, asymptomatic people aged between 20 and 60 years were enrolled . We defined' sexually active' as having had sexual intercourse in the preceding 3 months . People with genitourinary symptoms such as urethral or cervical discharge, dysuria, or itching in the genital area were also excluded . First - voided urine specimens were collected in sterile 50 ml screw - cap plastic bottles . In women the specimens from the 709 men and women were equilibrated to room temperature and centrifuged at 5000xg for 15 minutes . The supernatant was discarded, and the pellet was resuspended in 1 ml 1xpbs before dna extraction . Genomic dna was extracted from the pretreated specimens (swab or urine) by using the qiaamp dna mini kit (qiagen, hilden, germany) according to the manufacturer's instructions . Pcr amplification was performed with the seeplex std6 ace detection kit (seegene, seoul, korea) according to the manufacturer's instructions . The kit contains sets of primers that were specifically designed from highly conserved regions of genetic sequences for the 6 organisms (chlamydia trachomatis, neisseria gonorrhoeae, mycoplasma genitalium, ureaplasma urealyticum, mycoplasma hominis, and trichomonas vaginalis) by use of seegene dpo technology . An internal control was included in the pcr mix to detect the presence of pcr inhibitors . For the negative control, sterile deionized water was used as the pcr template instead of nucleic acid . The positive control contained in the kit amplified fragments were separated by use of the labchip dx seeplex assay system(caliper, hopkinton, ma, usa). Amplified pcr products were separated and detected by automated gel electrophoresis with the labchip dx seeplex assay system (caliper, hopkinton, ma, usa). The pcr products from each sample (20 l) were transferred to 96-well plates and placed in the labchip dx instrument . The samples were loaded automatically on the seeplex chip and detected sequentially according to the sample order . Analysis was performed with designated software (seegene viewer) that presents each of the samples and identifies the fragments that yield a positive readout for the bands of interest in the presented results . A tabulated matching matrix provides a simple readout, identifying matching bands to the type of sti pathogens . The mean age in this study was 45.48.1 years . Among the 709 persons, 229 (32.3%) had at least one microorganism in his or her genitourinary tract . The prevalences of chlamydia trachomatis, neisseria gonorrhoeae, mycoplasma genitalium, ureaplasma urealyticum, mycoplasma hominis, and trichomonas vaginalis in asymptomatic people were 5.6% (40/709), 0.4% (3/709), 0.3% (2/709), 22.1% (157/709), 11.6% (82/709), and 1.1% (8/709) respectively(table 1). Among all persons, 50 (7.1%) had pathogens of sti in their urinary tract (table 2). Of these patients, the number of patients with a single pathogen was 47 (6.6%), for which the number with isolated chlamydia trachomatis, neisseria gonorrhoeae, mycoplasma genitalium, or trichomonas vaginalis was 37, 1, 1, and 8, respectively . Three double - infections, i.e., samples containing 2 or more pathogens, were identified, and each sample had chlamydia trachomatis . When trichomonas vaginalis was identified, it was either isolated alone or co - infected with mycoplasma hominis only and no other species . The total number of people who had at least two microorganisms in their genitourinary tract was 55 (7.5%). Among these 55 persons, 48 (6.8%) had two different species whether they were pathogens or not, 6 (0.8%) persons had three different species, and 1 patient had four different species . The prevalences of ureaplasma urealyticum and mycoplasma hominis were 22.1% and 11.6%, respectively . In people who were infected with chlamydia trachomatis, the prevalences of ureaplasma urealyticum (35.0%) and mycoplasma hominis (22.5%) were higher than in those not infected with chlamydia trachomatis (table 1, 2). Chlamydia trachomatis infection is the most prevalent sexually transmitted bacterial infection among women and men worldwide . Chlamydia trachomatis is usually described as an obligate intracellular pathogen and accounts for 30% to 40% of the etiopathogenesis of urethritis . However, about half of infected subjects are asymptomatic . Thus, there are many silent infections in the community . Mason et al showed that the prevalence of chlamydia trachomatis in asymptomatic men was 4% (14/349). Takahashi et al reported that chlamydia trachomatis was detected in 6% of samples from healthy people . Similarly, in our study, chlamydia trachomatis was observed in 40 persons (5.6%). Thus, most asymptomatic patients are neglected unless they visit the clinic to be evaluated for sti by chance or face complications such as pelvic inflammatory disease . Neisseria are fastidious gram - negative cocci that require nutrient supplementation to grow in laboratory cultures . Although gonorrhea can be diagnosed by inspection of the yellowish discharge from the urethra, many patients infected with neisseria gonorrhoeae have no discharge . Furthermore, it is known that 10% of infected males and 50% of infected females are asymptomatic . Hein et al studied 2672 sexually active adolescents and reported that prevalence rates of asymptomatic gonorrhea were 1.9% among boys and 7.0% among female adolescents . Regardless of age group, a surveillance study reported prevalence rates of gonorrhea in healthy people of 0.06% to 0.18% . In our study, the prevalence in asymptomatic people was 0.4%, and the result was easily obtained by pcr test with a urine or endocervical swab sample . Mycoplasma genitalium was first isolated in 1981 from two men with nongonococcal urethritis (ngu). Results from a meta - analysis of 19 studies on patients with ngu showed that mycoplasma genitalium was found in 21% compared with 7% of patients without ngu . Ross et al reported a detection rate of 0.6% for mycoplasma genitalium in asymptomatic british persons . Currently, nucleic acid amplification tests (naats) are usually recommended to detect this pathogen . Wikstrm and jensen reported that mycoplasma genitalium is a common cause of persistent or recurrent urethritis among men treated with doxycycline, and erythromycin appears to be less efficient than azithromycin in eradicating the infection . Therefore, when physicians encounter patients who complain of continuous symptoms of urethritis, they should consider drug - refractory urethritis such as that caused by mycoplasma genitalium . It is another pathogen of sti of the urogenital tract, and men with this infection rarely exhibit symptoms . In women, greenish - yellow frothy vaginal discharge and itching trichomoniasis is treated and cured with metronidazole or tinidazole, which should be prescribed to any sexual partners as well because they may be asymptomatic carriers . Sutton et al reported that the prevalence of trichomonas vaginalis in reproductive - aged women was 3.1% . In our study, the total prevalence rate was 1.1% in asymptomatic people, and prevalence rates in asymptomatic men and women were 0.2% and 2.5%, respectively . Screening for these pathogens is important not only to identify infected symptomatic individuals for the diagnosis and management of their infections but also to identify asymptomatic individuals who serve as reservoirs for infection [1 - 4]. In our study, there were 50 (7.1%) infected people among 709 asymptomatic people; thus, this proportion should not be neglected . Among the 50 infected patients, 37 with chlamydia trachomatis, 1 with neisseria gonorrhoeae, 1 with mycoplasma genitalium, 8 with trichomonas vaginalis, 2 with chlamydia trachomatis with neisseria gonorrhoeae, and 1 with chlamydia trachomatis with mycoplasma genitalium were confirmed . Thus, regardless of chlamydia trachomatis, 13 patients should be considered as candidates for antimicrobial treatment . These facts suggest the possibility of prescribing the wrong medicines if practitioners give symptomatic patients some regimens empirically . Therefore, the need to detect multiple species at once should be raised . In this sense, use of the multiplex pcr assay to screen asymptomatic people may be important because the treatment regimen could be optimized for people with positive results, especially for those with multiple organisms . However, ureaplasma urealyticum is frequently isolated from the urethra of healthy men, and some studies have reported that there is no significant difference in its prevalence between men with ngu and men without ngu . Nevertheless, there are some reports that ureaplasma urealyticum serves as a cause of persistent ngu . For that reason, ureaplasma urealyticum therefore, ureaplasma infection must be considered in patients with treatment failure for ngu or patients with multiple sexual partners . If no species except ureaplasma are detected, symptoms and leukocyte numbers in first voided urine samples are helpful for clarifying the diagnosis of ureaplasma infection . Mycoplasma hominis is frequently identified from the genitourinary tract . In general, it is known as a commensal species but it can work as a pathogen in special conditions such as in an immunocompromised state . Generally, the rate of colonization of mycoplasma hominis in the urogenital tract was reported to be between 4% and 13% in men and between 21% an 54% in women . Our study showed that the incidence of this species was 9.1% in men and 15.4% in women . In our study, the incidences of ureaplasma urealyticum and mycoplasma hominis were 22.1% and 11.6% . Nevertheless, those incidences were increased up to 35.0% and 22.5% when the samples had chlamydia trachomatis . The reason for this increase with chlamydial infection is not clear, and a limitation of our study is that we did not include people who had symptoms . Therefore, comparison of the prevalence of these species in symptomatic urethritis patients is necessary . As described previously, most pathogens causing sti as well as commensal microorganisms are difficult to cultivate by routine microbiological diagnosis . However, naats, such as pcr, are useful for the identification of microorganisms that are difficult to cultivate and for those that grow slowly . In another recent study in korea, the multiplex pcr kit (seegene inc ., seoul, korea) was used to detect causative microorganisms of sti in patients with chronic prostatitis and vaginitis . In multiplex pcr, more than one target sequence can be amplified by including more than one pair of primers in the reaction . Multiplex pcr has the potential to produce considerable savings of time and effort within the laboratory without compromising test utility . Furthermore, when the clinical sample amount is limited, multiplexing allows more targets to be analyzed by using a single aliquot of sample material . Although there are some worries that multiplex pcr in the clinical setting may have difficulties such as poor sensitivity or specificity, false - negative results, and nonspecific interactions, horii et al showed that the multiplex pcr assay had an overall sensitivity of 96% and specificity of 100% compared with uniplex pcr assays . Furthermore, there was no cross - reaction with other microorganisms . The multiplex pcr assay has recently made it convenient for clinicians to test for causative organisms simultaneously from many clinical fields . Similarly, we succeeded in easily generating results by use of multiplex pcr with only a single sample per person . Asymptomatic individuals play a role as reservoirs for sti . In our study, there were 50 (7.1%) infected people among 709 asymptomatic people; therefore, this proportion should not be neglected . An sti screening test should be considered as a part of a general checkup, especially in high - risk groups, e.g., commercial sexual workers, people who have multiple sexual partners, and immunocompromised persons [1 - 3,6,25]. By use of multiplex pcr, physicians can easily identify many microorganisms at once, and treating asymptomatic persons contributes to the prevention of sti in the community . The prevalences of chlamydia trachomatis, neisseria gonorrhoeae, mycoplasma genitalium, ureaplasma urealyticum, mycoplasma hominis, and trichomonas vaginalis in asymptomatic people were 5.6%, 0.4%, 0.3%, 22.1%, 11.6%, and 1.1%, respectively . We hope that these results will be used as baseline data for future studies . In our study, about 32.3% of asymptomatic people had at least one microorganism, and excluding ureaplasma urealyticum and mycoplasma hominis from the count, the prevalence rate of silent sti was 7.1% with multiplex pcr therefore, screening for sti should be considered as a part of a general checkup, especially in high - risk groups.
After the implementation of the first prenatal care in the late 1930s in england and northern ireland (1), little by little, it has become recognized that there is a gap in the continuum of maternal care and that preconception care, which is the missing link in this continuum, can improve pregnancy outcomes, parturition, and children s health (2). Pregnancy is one of the most important experiences of a woman s life, and planning for pregnancy is one of the best solutions to solve problems related to pregnancy, to create a safe pregnancy, and to maintain and improve the health of mothers and their children (3). Preconception care, as part of antenatal care, is a golden opportunity that can identify risk factors in pregnancy and conduct any required interventions before pregnancy occurs (4). Various studies have shown that poor access to prenatal care is a major obstacle to improving pregnancy outcomes . Given that a significant number of women in developing countries are receiving prenatal care, there is growing evidence to show that mere access to care is not enough; rather, the quality of that care is a key component to improve health outcomes for both the mother and the baby (5). The quality obstetrics care has a huge impact on the outcome of pregnancy and childbirth (6). During pregnancy, all women experience some mental and physical changes that may affect the outcome of pregnancy . The mother s age is one factor that can, in some cases, increase the risk associated with pregnancy . Today, throughout the world, teenage girls are getting married in increasing numbers, especially in developing countries (7). In iran, teenage girls give birth to 100,000 babies annually . Census data show that in 2011, 1.8% of females between the ages of 10 and 14 were married, and 21% of those between the ages of 15 and 19 were married . Among the provinces, khorasan razavi province has the highest percentage of females between the ages of 10 and 14 who are married, as well as the highest percentage of those between 15 and 19 who are married (8). The young age of females during pregnancy is considered as an important factor of adverse outcomes . Research shows that the majority of teenage females pregnancies are unplanned, and teenagers rarely are referred to preconception counseling . Since the age at marriage is relatively low and pregnancy occurs frequently among these very young females (9), the researchers decided to conduct a study in mashhad, which has the highest population of reproductive age women among the other cities of khorasan razavi province, to evaluate and compare the quality of preconception care provided to reproductive age women in healthcare centers and to identify the strengths and weaknesses of such care in the city s health centers . The donabedian model was chosen to evaluate the quality of such care, because it focuses on the final results of patient care (10). This model is the most extensively used model in the evaluation of healthcare (1), and it was first presented in 1966 to address three areas, i.e., structure, process, and outcome (11). For example, it was used in studies in india (12) and kenya (13) to compare the quality of antenatal care in the public and private sectors of the two countries . This cross sectional study was conducted in healthcare centers covered by five health centers in mashhad, khorasan razavi province, iran . Three hundred and sixty reproductive age women who were referred to 24 healthcare centers covered by five health centers to receive preconception care were selected for study . In addition, 39 health staff who worked in these centers providing preconception care participated in the study . Since our literature review indicated that no study similar to the present investigation had been published, we conducted a pilot study that involved 30 women of reproductive age to calculate the sample size . Then, the sample size was determined by the following formula in all three components, i.e., structure, process, and outcome . Since the estimated sample size was based on knowledge of the consequence component, the same index was considered as the sample size, and the 95% confidence and 5% accuracy levels were calculated for about 360 persons . The five health centers in mashhad were considered categories, and the names of the covered healthcare centers were listed and considered as clusters . Then, by using the following formula and considering the covered population, each of the five health centers was considered to be a quota (105 subjects at health center number one, 75 subjects at health center number two, 90 subjects at health center number three, 15 subjects at health center number four, and 75 subjects at health center number five). With regard to the quota and in proportion to the number of healthcare centers in urban areas covered by the five centers, a number of clusters was selected by lottery (including seven clusters from health center number one, five clusters from health center number two, six clusters from health center number three, one cluster from health center number four, and five clusters from health center number five). Among all of the reproductive age women who were referred to the desired healthcare centers to receive preconception care, the tools used in this study were developed by the researchers, and they included two checklists to examine the structure and the process of care . The forms of care outcomes also included a questionnaire concerning the study units satisfaction with the care that was provided, e, a questionnaire concerning the study units knowledge about preconception care, and a follow - up form of care performed by the healthcare providers . The checklist of used to evaluate quality structure consisted of 65 questions in three parts, which examined the physical infrastructure, the human infrastructure, and organizing the forces . Answers to the checklist were in form of have and not have . (it should be noted that for each question in the checklist of care, levels 1 to 3, was considered, and their priority was determined based on the views of professors, numerous articles in the field of antenatal services, and the standards of care centers according to their priorities in providing preconception care .) Then, the total points were calculated to compare the percentage in three levels, i.e., 0 to 33% as poor quality, 34 to 66% as moderate quality, and 67 to 100% as good quality . The checklist used to evaluate the quality process consisted of 42 questions related to both technical performance and interpersonal care interactions . Each question examined in the checklist was scored in one of three forms, i.e., done (2), incompletely done if, during the viewing, any of the questions did not need to be answered, that option was marked, and the question was not included in calculating of the scores . The data that were from the checklist were calculated to compare with a good level in percentage terms, i.e., 0 to 33% as poor quality, 34 to 66% as moderate quality, and 67 to 100% as good quality . Then satisfaction questionnaire contained 28 questions, four of which dealt with the subjects level of satisfaction, i.e., 1) satisfaction with the care structure, 2) satisfaction with counseling and care, 3) satisfaction with the training that was provided, and 4) satisfaction with the physician . The questions were scored using a five - point likert scale of completely agree (4), agree (3), no idea (2), disagree (1), and strongly disagree (0), and the range of scores was considered between 0 to112 . The data obtained from the questionnaire were estimated in terms of percentage in four levels, i.e., 0 to 25% as strongly dissatisfied and dissatisfied, 26 to 50% as neutral, 51 to 75% as satisfied, and 76 to 100% as strongly satisfied . For correct answer to each question one score and for each wrong answer or not know, the data obtained from the questionnaire were estimated at three levels, i.e., 0 to 33% as poor knowledge, 34 to 66% as moderate knowledge, and 67 to 100% as appropriate knowledge . The follow - up form contained four questions and answers to the given questions in either the data obtained from the form were estimated to compare with the good level in terms of percentage in the three levels of 0 to 33% as poor follow - up, 34 to 66% as average follow - up, and 67 to 100% as desired follow - up . The content validity method was used to validate all of the instruments used in the study . The instruments were given to 10 experts who were professors of mashhad university of medical sciences and research to evaluate . After the tools were revised based on their comments, they were used in the study . To make them reliable, the form of related information to the characteristics of the care provider was confirmed by a test - retest method with the correlation of 0.90, 10 days later . The form of the information related to the characteristics of the healthcare center was confirmed by the equivalent fraction method with the correlation of 0.92 . The reliability of the checklist of structure quality evaluation was confirmed by the equivalent fraction method with the correlation of 0.85 . The reliability of the checklist of process quality evaluation and the reliability of these tools were confirmed by the equivalent fraction method with the correlation of 0.80 . The reliability of knowledge and satisfaction questionnaire was confirmed by chronbach s alpha with alpha score of 0.89 and 0.70, respectively . The reliability of the tracking form was confirmed by chronbach s alpha with an alpha score of 0.89 . After the research was approved by the ethics committee and after an introduction letter was obtained from the nursery and midwifery university of mashhad, these documents were presented to the authorities of the five health centers in mashhad, and the researcher visited the healthcare centers . Generally, this study was performed in the following five steps: after coordinating with the heads of the five health centers and the authorities at the selected healthcare centers, the characteristics form was given to healthcare providers . The researcher also completed the characteristics forms of the healthcare center using the information available in the family planning offices and the statistical forms that were available in the obstetrics and family health units with the help of the personnel in these units . In addition, the researcher completed the structural evaluation form of the care that was provided while the researcher was at the center for observation and conducted interviews with the personnel who were available at the selected healthcare centers.in the later stages, the researcher went to one center every day . The researcher identified the women who met the inclusion criteria of the study, and, after obtaining written consent and emphasizing the confidentiality of information to the research units, the women were given information about the purpose, the procedure, the method, and the way to answer the questionnaires . Then, the researcher completed the personal and obstetric characteristics form by conducting interviews with the appropriate people in the research units.in the third step, the researcher visited the care center with the research unit while care was being provided and wrote his observations related to healthcare provider s function, the relationship between the healthcare provider and the subjects, and the referees in the related checklist.after the caring process was completed by the healthcare provider, the physician, the dentist at the center, and the woman who received the care were asked to answer the satisfaction and awareness questionnaire.in the last step, after completing the period during which care was being provided, the researcher evaluated the follow - up . After coordinating with the heads of the five health centers and the authorities at the selected healthcare centers, the characteristics form was given to healthcare providers . The researcher also completed the characteristics forms of the healthcare center using the information available in the family planning offices and the statistical forms that were available in the obstetrics and family health units with the help of the personnel in these units . In addition, the researcher completed the structural evaluation form of the care that was provided while the researcher was at the center for observation and conducted interviews with the personnel who were available at the selected healthcare centers . In the later stages, the researcher identified the women who met the inclusion criteria of the study, and, after obtaining written consent and emphasizing the confidentiality of information to the research units, the women were given information about the purpose, the procedure, the method, and the way to answer the questionnaires . Then, the researcher completed the personal and obstetric characteristics form by conducting interviews with the appropriate people in the research units . In the third step, the researcher visited the care center with the research unit while care was being provided and wrote his observations related to healthcare provider s function, the relationship between the healthcare provider and the subjects, and the referees in the related checklist . After the caring process was completed by the healthcare provider, the physician, the dentist at the center, and the woman who received the care were asked to answer the satisfaction and awareness questionnaire . In the last step, after completing the period during which care was being provided, the researcher evaluated the follow - up . This study was conducted at the mashhad university of medical sciences with the approval of the university s research ethics committee . The researcher explained the purpose of the study and the procedures that would be used in the research to the healthcare providers and gave them the written informed consent of participation in the study . The data that were collected were deemed to be confidential, and the participants in the study were informed that they could withdraw from the study at any time if they desired to do so . Descriptive and inferential statistics were used to analyze the data . After the data were collected, coded, and entered in the computer, they were analyzed by spss version 11.5 (spss inc . If a variable had a normal distribution, parametric tests were used; otherwise, nonparametric tests were used . Statistical indicators, such as mean, standard deviation, and frequency tables, were used to describe the personal, obstetric information of the healthcare provider and the characteristics of the healthcare center . The spearman correlation coefficient was used to investigate the correlation of the quantitative data if they had an abnormal distribution, and the pearson correlation coefficient was used if they had a normal distribution . The relationship between the qualitative data and the score of the care structure was determined by the statistical parametric independent t - tests and anova, and the relationship of the qualitative data with the score of the care process was determined with the statistical non - parametric mann - whitney and kruskal - wallis statistical tests . The relationship of the qualitative data with the satisfaction and awareness of the participants was determined by the statistical parametric independent t - tests and anova . It is noteworthy that the significance level was considered as =0.05 in the statistical tests . This cross sectional study was conducted in healthcare centers covered by five health centers in mashhad, khorasan razavi province, iran . Three hundred and sixty reproductive age women who were referred to 24 healthcare centers covered by five health centers to receive preconception care were selected for study . In addition, 39 health staff who worked in these centers providing preconception care participated in the study . Since our literature review indicated that no study similar to the present investigation had been published, we conducted a pilot study that involved 30 women of reproductive age to calculate the sample size . Then, the sample size was determined by the following formula in all three components, i.e., structure, process, and outcome . Since the estimated sample size was based on knowledge of the consequence component, the same index was considered as the sample size, and the 95% confidence and 5% accuracy levels were calculated for about 360 persons . The five health centers in mashhad were considered categories, and the names of the covered healthcare centers were listed and considered as clusters . Then, by using the following formula and considering the covered population, each of the five health centers was considered to be a quota (105 subjects at health center number one, 75 subjects at health center number two, 90 subjects at health center number three, 15 subjects at health center number four, and 75 subjects at health center number five). With regard to the quota and in proportion to the number of healthcare centers in urban areas covered by the five centers, a number of clusters was selected by lottery (including seven clusters from health center number one, five clusters from health center number two, six clusters from health center number three, one cluster from health center number four, and five clusters from health center number five). Among all of the reproductive age women who were referred to the desired healthcare centers to receive preconception care, the tools used in this study were developed by the researchers, and they included two checklists to examine the structure and the process of care . The forms of care outcomes also included a questionnaire concerning the study units satisfaction with the care that was provided, e, a questionnaire concerning the study units knowledge about preconception care, and a follow - up form of care performed by the healthcare providers . The checklist of used to evaluate quality structure consisted of 65 questions in three parts, which examined the physical infrastructure, the human infrastructure, and organizing the forces . Answers to the checklist were in form of have and not have . (it should be noted that for each question in the checklist of care, levels 1 to 3, was considered, and their priority was determined based on the views of professors, numerous articles in the field of antenatal services, and the standards of care centers according to their priorities in providing preconception care .) Then, the total points were calculated to compare the percentage in three levels, i.e., 0 to 33% as poor quality, 34 to 66% as moderate quality, and 67 to 100% as good quality . The checklist used to evaluate the quality process consisted of 42 questions related to both technical performance and interpersonal care interactions . Each question examined in the checklist was scored in one of three forms, i.e., done (2), incompletely done (1), and not done if, during the viewing, any of the questions did not need to be answered, that option was marked, and the question was not included in calculating of the scores . The data that were from the checklist were calculated to compare with a good level in percentage terms, i.e., 0 to 33% as poor quality, 34 to 66% as moderate quality, and 67 to 100% as good quality . Then satisfaction questionnaire contained 28 questions, four of which dealt with the subjects level of satisfaction, i.e., 1) satisfaction with the care structure, 2) satisfaction with counseling and care, 3) satisfaction with the training that was provided, and 4) satisfaction with the physician . The questions were scored using a five - point likert scale of completely agree (4), agree (3), no idea (2), disagree (1), and strongly disagree (0), and the range of scores was considered between 0 to112 . The data obtained from the questionnaire were estimated in terms of percentage in four levels, i.e., 0 to 25% as strongly dissatisfied and dissatisfied, 26 to 50% as neutral, 51 to 75% as satisfied, and 76 to 100% as strongly satisfied . For correct answer to each question one score and for each wrong answer or not know, zero score was considered . The data obtained from the questionnaire were estimated at three levels, i.e., 0 to 33% as poor knowledge, 34 to 66% as moderate knowledge, and 67 to 100% as appropriate knowledge . The follow - up form contained four questions and answers to the given questions in either yes (2) or no (0). The data obtained from the form were estimated to compare with the good level in terms of percentage in the three levels of 0 to 33% as poor follow - up, 34 to 66% as average follow - up, and 67 to 100% as desired follow - up . The content validity method was used to validate all of the instruments used in the study . The instruments were given to 10 experts who were professors of mashhad university of medical sciences and research to evaluate . After the tools were revised based on their comments, they were used in the study . To make them reliable, the form of related information to the characteristics of the care provider was confirmed by a test - retest method with the correlation of 0.90, 10 days later . The form of the information related to the characteristics of the healthcare center was confirmed by the equivalent fraction method with the correlation of 0.92 . The reliability of the checklist of structure quality evaluation was confirmed by the equivalent fraction method with the correlation of 0.85 . The reliability of the checklist of process quality evaluation and the reliability of these tools were confirmed by the equivalent fraction method with the correlation of 0.80 . The reliability of knowledge and satisfaction questionnaire was confirmed by chronbach s alpha with alpha score of 0.89 and 0.70, respectively . The reliability of the tracking form was confirmed by chronbach s alpha with an alpha score of 0.89 . After the research was approved by the ethics committee and after an introduction letter was obtained from the nursery and midwifery university of mashhad, these documents were presented to the authorities of the five health centers in mashhad, and the researcher visited the healthcare centers . Generally, this study was performed in the following five steps: after coordinating with the heads of the five health centers and the authorities at the selected healthcare centers, the characteristics form was given to healthcare providers . The researcher also completed the characteristics forms of the healthcare center using the information available in the family planning offices and the statistical forms that were available in the obstetrics and family health units with the help of the personnel in these units . In addition, the researcher completed the structural evaluation form of the care that was provided while the researcher was at the center for observation and conducted interviews with the personnel who were available at the selected healthcare centers.in the later stages, the researcher went to one center every day . The researcher identified the women who met the inclusion criteria of the study, and, after obtaining written consent and emphasizing the confidentiality of information to the research units, the women were given information about the purpose, the procedure, the method, and the way to answer the questionnaires . Then, the researcher completed the personal and obstetric characteristics form by conducting interviews with the appropriate people in the research units.in the third step, the researcher visited the care center with the research unit while care was being provided and wrote his observations related to healthcare provider s function, the relationship between the healthcare provider and the subjects, and the referees in the related checklist.after the caring process was completed by the healthcare provider, the physician, the dentist at the center, and the woman who received the care were asked to answer the satisfaction and awareness questionnaire.in the last step, after completing the period during which care was being provided, the researcher evaluated the follow - up . After coordinating with the heads of the five health centers and the authorities at the selected healthcare centers, the researcher also completed the characteristics forms of the healthcare center using the information available in the family planning offices and the statistical forms that were available in the obstetrics and family health units with the help of the personnel in these units . In addition, the researcher completed the structural evaluation form of the care that was provided while the researcher was at the center for observation and conducted interviews with the personnel who were available at the selected healthcare centers . In the later stages, the researcher identified the women who met the inclusion criteria of the study, and, after obtaining written consent and emphasizing the confidentiality of information to the research units, the women were given information about the purpose, the procedure, the method, and the way to answer the questionnaires . Then, the researcher completed the personal and obstetric characteristics form by conducting interviews with the appropriate people in the research units . In the third step, the researcher visited the care center with the research unit while care was being provided and wrote his observations related to healthcare provider s function, the relationship between the healthcare provider and the subjects, and the referees in the related checklist . After the caring process was completed by the healthcare provider, the physician, the dentist at the center, and the woman who received the care were asked to answer the satisfaction and awareness questionnaire . In the last step, after completing the period during which care was being provided, the researcher evaluated the follow - up . This study was conducted at the mashhad university of medical sciences with the approval of the university s research ethics committee . The researcher explained the purpose of the study and the procedures that would be used in the research to the healthcare providers and gave them the written informed consent of participation in the study . The data that were collected were deemed to be confidential, and the participants in the study were informed that they could withdraw from the study at any time if they desired to do so . Descriptive and inferential statistics were used to analyze the data . After the data were collected, coded, and entered in the computer, they were analyzed by spss version 11.5 (spss inc . If a variable had a normal distribution, parametric tests were used; otherwise, nonparametric tests were used . Statistical indicators, such as mean, standard deviation, and frequency tables, were used to describe the personal, obstetric information of the healthcare provider and the characteristics of the healthcare center . The spearman correlation coefficient was used to investigate the correlation of the quantitative data if they had an abnormal distribution, and the pearson correlation coefficient was used if they had a normal distribution . The relationship between the qualitative data and the score of the care structure was determined by the statistical parametric independent t - tests and anova, and the relationship of the qualitative data with the score of the care process was determined with the statistical non - parametric mann - whitney and kruskal - wallis statistical tests . The relationship of the qualitative data with the satisfaction and awareness of the participants was determined by the statistical parametric independent t - tests and anova . It is noteworthy that the significance level was considered as =0.05 in the statistical tests ., the highest frequencies were related to the two groups of couples at the secondary education level (40 and 44%, respectively), and the lowest frequency was related to ability in reading and writing (which, in the couples, were 1.7 and 2.6%, respectively). The highest frequency of employment status was among women in the housewife group (90.9%), and the lowest frequency was related to the working group (0.6%). The husbands of most of the women in the study were self - employed (59.1%). According to the data, most women (76.6%) indicated that their income level was adequate . In this study, 67.4% of women had been pregnant, and 18.8% of the women reported that they had experienced complications and disorder in their previous pregnancies . Also, 8.3% of women stated that they had an underlying disease . In the study, 53.6% of the women reported that they had information on preconception care, but 61.5% of them reported that they had very little information . The results showed that there were no statistically significant relationships between the mean number of midwives providing healthcare (p=0.179). Twenty - nine of the women who were providers (74.4%) had experienced pregnancy and childbirth . Among the 39 healthcare providers who participated in the study, 23 of them (59%) were formally employed, 12.8% were in their commitment period, 20.5% of the employments were in the form of a treaty, and only 3 people (7.7%) were contractor s employees . The average total service time of the healthcare providers in the healthcare sector was 13.647.26 years . The average number of years that the healthcare providers had been working in mothers unit was 4.301.15 years . Among the healthcare workers 35.9% of them had been transferred from the treatment section to the health section, and 2.6% of them had been working in an office before being employed in the health section, and 12.8% had no work experience and had almost completed their studies . Among the healthcare providers 48.7% reported that they had participated in teaching preconception care classes in 2011, and the other healthcare providers had not been received any special training except for one course concerning the mothers integrated care plan . To compare the quality of preconception care in the five health centers of mashhad, first the general quality of care then, the results of the quality of provided care were graded and divided into three levels, i.e., poor quality, average quality, and favorable quality . Evaluation of the quality level of the care structure dimension showed that 86.4% of the research centers were satisfactory . Evaluation of the quality level of care process dimension showed preconception care was provided in 94% with average quality . The results in examining the dimension of care outcomes also showed that 95.4% of the women who participated in the study were very satisfied by the services that were provided . Sixty - eight percent of them had average awareness of preconception care, and the follow - up of the care provided by health personnel had a poor level of quality in 65.7% of the cases . The kruskal - wallis test showed that there was no difference between the mean score of the care structures in the five centers (p=0.112). However, the results of the care process provided by the health personnel at the centers differed (table 2). The anova showed that there was a significant relationship between the mean score of the care process in the five health centers . The care process provided by the health personnel of healthcare centers covered by the health center number 3 was higher than those of the other centers . In addition, the results of the anova showed that there was no statistically significant difference between the satisfaction mean score of the research units (p=0.151) and the awareness mean score of the research units in the five health centers (p=0.135). However, the anova results elated to the examining follow - up part by the health personnel of the centers showed that there was a statistically significant relationship between the mean care follow - up score in the health centers, and the follow - up of the personnel of health center number 5 was higher in quality . The kruskal - wallis test showed a statistically significant relationship between the mean score of the care process and the healthcare providers attitudes toward the need for providing preconception care (p=0.001). The spearman correlation coefficients also showed a statistically significant relationship between the experience of the healthcare providers in the mothers integrated care unit and the mean score of the care process (p=0.024). In addition, the results showed that there was a statistically significant relationship between the mean score of the care process that was provided with the experience of participating in the training courses in antenatal care that were held in 2011 (p=0.028); however, there was no statistically significant relationship between the mean score of the care process with the participation in the training courses at the beginning of the mothers integrated care plan (p=0.070)., the highest frequencies were related to the two groups of couples at the secondary education level (40 and 44%, respectively), and the lowest frequency was related to ability in reading and writing (which, in the couples, were 1.7 and 2.6%, respectively). The highest frequency of employment status was among women in the housewife group (90.9%), and the lowest frequency was related to the working group (0.6%). The husbands of most of the women in the study were self - employed (59.1%). According to the data, most women (76.6%) indicated that their income level was adequate . In this study, 67.4% of women had been pregnant, and 18.8% of the women reported that they had experienced complications and disorder in their previous pregnancies . Also, 8.3% of women stated that they had an underlying disease . In the study, 53.6% of the women reported that they had information on preconception care, but 61.5% of them reported that they had very little information . The results showed that there were no statistically significant relationships between the mean number of midwives providing healthcare (p=0.179). Twenty - nine of the women who were providers (74.4%) had experienced pregnancy and childbirth . Among the 39 healthcare providers who participated in the study, 23 of them (59%) were formally employed, 12.8% were in their commitment period, 20.5% of the employments were in the form of a treaty, and only 3 people (7.7%) were contractor s employees . The average total service time of the healthcare providers in the healthcare sector was 13.647.26 years . The average number of years that the healthcare providers had been working in mothers unit was 4.301.15 years . Among the healthcare workers 35.9% of them had been transferred from the treatment section to the health section, and 2.6% of them had been working in an office before being employed in the health section, and 12.8% had no work experience and had almost completed their studies . Among the healthcare providers 48.7% reported that they had participated in teaching preconception care classes in 2011, and the other healthcare providers had not been received any special training except for one course concerning the mothers integrated care plan . To compare the quality of preconception care in the five health centers of mashhad, first the general quality of care was calculated in the three dimensions of structure, process, and outcome . Then, the results of the quality of provided care were graded and divided into three levels, i.e., poor quality, average quality, and favorable quality . Evaluation of the quality level of the care structure dimension showed that 86.4% of the research centers were satisfactory . Evaluation of the quality level of care process dimension showed preconception care was provided in 94% with average quality . The results in examining the dimension of care outcomes also showed that 95.4% of the women who participated in the study were very satisfied by the services that were provided . Sixty - eight percent of them had average awareness of preconception care, and the follow - up of the care provided by health personnel had a poor level of quality in 65.7% of the cases . The kruskal - wallis test showed that there was no difference between the mean score of the care structures in the five centers (p=0.112). However, the results of the care process provided by the health personnel at the centers differed (table 2). The anova showed that there was a significant relationship between the mean score of the care process in the five health centers . The care process provided by the health personnel of healthcare centers covered by the health center number 3 was higher than those of the other centers . In addition, the results of the anova showed that there was no statistically significant difference between the satisfaction mean score of the research units (p=0.151) and the awareness mean score of the research units in the five health centers (p=0.135). However, the anova results elated to the examining follow - up part by the health personnel of the centers showed that there was a statistically significant relationship between the mean care follow - up score in the health centers, and the follow - up of the personnel of health center number 5 was higher in quality . The kruskal - wallis test showed a statistically significant relationship between the mean score of the care process and the healthcare providers attitudes toward the need for providing preconception care (p=0.001). The spearman correlation coefficients also showed a statistically significant relationship between the experience of the healthcare providers in the mothers integrated care unit and the mean score of the care process (p=0.024). In addition, the results showed that there was a statistically significant relationship between the mean score of the care process that was provided with the experience of participating in the training courses in antenatal care that were held in 2011 (p=0.028); however, there was no statistically significant relationship between the mean score of the care process with the participation in the training courses at the beginning of the mothers integrated care plan (p=0.070). The results of the study in evaluating and comparing the quality of the care structure showed that the preconception care structures were appropriate in most cases and that there were no differences between the preconception care structures in the centers; in other words, the necessary facilities and equipment for the preconception cares were the same in all of the centers . Despite the appropriateness of the quality of the care structure the defects were associated with the lack of dentists, lack of laboratory technicians, lack of adequate maintenance of the health document files in the human resource section, lack of laboratory space, lack of required facilities (such as telephones), lack of appropriate waiting rooms, lack of adequate seating, lack of a water cooler, lack of toilets with hand - wash liquid in the equipment and administrative sections, lack of availability of dentistry services every day of the week, lack of notification about preconception services in the centers and lack of specification of the services that were provided . Sohail agha, who examined and compared the quality of family planning services in kenya s public and private sectors in his study, reported that the quality of the care structure was just average (13). In this study, the structural problems that had led to a lower level of quality were associated with the personnel s having too little work experience and a lack of training in the field of family planning services . The quality also was diminished by the fact that trained personnel were not available for all working hours of the week, as well as the long time it took to get to the healthcare centers, and the lack of teaching aids . Simbar et al . Also examined the equipment, including the necessary facilities, waiting room, obstetrics unit, and teaching facilities in the investigation of the care structure component . The results showed that the service providers had access to a minimum of the necessary facilities . Simbar reported that the major defects associated with the preconception care structure in tehran were in the media facilities and in the failure to inform the clients (1). The results of evaluating and comparing the quality of preconception care showed that the level of quality of the care process in the health centers was just average and that only 5.2% of the women were offered the desired quality of care . Boller et al . Also reported that the quality of antenatal care provided by the healthcare workers in tanzania as poor (16). The results of the study by simbar et al . Also showed that, despite a relatively favorable offer of obstetrical checkups and paraclinic tests, the other provided care provided and the counseling provided were average or poor . The results of comparing the centers in the process of providing care also showed that the same quality of preconception care was not provided; health center number 3 received higher scores for the process of providing care than the other health centers (table 2). One reason for this was the quality of personnel s attitude concerning the necessity for preconception care . The results showed that the healthcare providers provided higher quality care when they agreed that there was a need to provide more preconception care . In health center number three, about 100% of the healthcare providers who worked in the center reported that the necessity of preconception care was high or very high . The results also showed that healthcare personnel who had worked more years in the mothers integrated care provided higher quality preconception care . The results showed that about 80% of the personnel working in the centers covered by center number three had been working in mothers unit since the mothers integrated care project began . The results also showed 61.5% of healthcare providers who participated in the study had other responsibilities in the center in addition to their responsibility for providing preconception care . Among the personnel working in the centers covered by center number three, 75% also had other responsibilities, and only 25% of them worked fulltime in mothers center . However, in these centers in which the healthcare providers had other responsibilities, the center had either two midwives in the mother s unit or the personnel from the family health unit also helped the midwives in the center in providing preconception care . An exact examination indicated that the average number women who were provided preconception care on a monthly basis in the centers was similar to that in the other centers . It seems that these centers were trying harder to increase the quality of the care they were providing . The results of the evaluation and comparison of the outcomes related to preconception care showed that 95.1% of the women participating in this study were satisfied with the quality of the care they received . The results of the comparison of the clients satisfaction with the various centers showed that there were no differences . The other related results to care outcomes showed that all of the women who participated in the study had the same level of knowledge (intermediate) about preconception care . Since examining the level of knowledge was one of the midterm results in the studies that addressed the quality of care based on the donabedian model, the knowledge of the participants was examined, such as in sharon s examination of the quality of antenatal care (14) and simbar s examination of the quality of family planning services (15). In both studies, the results related to the follow - up component concerning the outcomes of the quality of care, showed that the care that was provided by the healthcare providers at the centers was followed up poorly, with more than 65% of the women receiving no follow - up at all by the healthcare providers . Comparing the follow - up in the centers showed that the level of follow - up was not consistent among them (table2). The follow up provided by the personnel of health center number five was the best among the centers . It seems that one of the reasons could have been the existence of active health liaisons in those centers, since each center had categorized its covered area and assigned one liaison for that area, and the liaisons had followed up as requested . Liaisons working in the centers are volunteer women who cooperate voluntarily in to protect the health of the women to encourage their neighbors to interact with the healthcare centers . Concerning the administrative constraints of the study, we can say that the researcher had no control over some of the variables, such as individual and personality differences or the mental and psychological states of the subjects, which clearly could have influenced how they answered the questions . Another constraint was that it was possible that the subjects did not answer some of the questions truthfully concerning their level of satisfaction with the services . With regards to psychological problems or underlying diseases, the researcher considered the statements of research units to be accurate . In relation to observing the action of the care provider(s), despite what was done to put the subjects at ease when the researcher was present in the centers that were providing care, his presence was considered to be somewhat of a constraint in the present study . The results of the study showed that the qualities of the preconception care process provided in the centers covered by mashhad five health centers were different . In addition, there was a difference between the care that was provided in follow - up by the healthcare personnel, and this was a part of care outcome dimension and the working personnel in the covered centers by the health center number 5 provided higher quality follow up . The functional significance of these findings is that they can be used in areas of education, research, and management services . It is recommended that the quality of preconception care be evaluated from the perspectives of those being served . Further studies of the process of providing preconception care and of the healthcare personnel who work in the healthcare centers should be conducted in the future.
The first pandemic reportedly occurred in 412 bc,, and the first attributed to influenza in 1580 ., since then, 31 influenza pandemics have been described; the five most recent in 1889, 1900, 1918, 1957 and 1968,,,,, were separated respectively by 11, 18, 39 and 11 years . The present threat of a new influenza pandemic is at the origin of renewed interest in the 1918 spanish flu, as it was undoubtedly the most deadly influenza pandemic in modern history . Recently, murray et al . Extrapolated, in their paper published in 2006, the potential global pandemic influenza mortality occurring in 2004, based on data from the 19181920 influenza pandemic . A first american report in 1927 suggested that the main 19181919 wave was responsible for 21 millions of deaths worldwide . A revised estimation of influenza pandemic mortality given in 1991 was 247393 million, and another published in 2002 even set the death toll up to 100 million to take into account the lack of data in a large part of the world . The lack of reliable mortality data in a large part of the world leads to reliance on anecdotal evidence and extrapolation of mortality rates for entire countries from punctual information obtained in highly specific settings ., concerning europe, we are aware of only three published papers reporting estimates of the mortality burden .,, herein, we analyzed the mortality data obtained during the pandemic, in 14 european countries, which accounted for 75% of the european population in 1918 . Monthly allcause deaths of civilians 19061936 were obtained from a book published, in 1954, by the french national institute of demographic study, which compiled official demographic and vital statistics from several countries, including 14 european countries . For each country, allcause death counts were available on a monthly basis, except for england and wales, for which only quarterly data are available . The observed data were standardized to mortality rates per 10 000 inhabitants using the corresponding yearly populationcount estimates . We used a periodic equation to model the monthly baseline mortality rate (b t) of years 19061922: with, respectively, n = 12 or n = 4 when monthly or quarterly data were analyzed . In this model, the baseline mortality rate depends on time t through a constant term 0, a secular trend 1, annual predictors 1 and 1, semiannual predictors 2 and 2 and an error term t . The unknown parameters were estimated at maximum likelihood from the fit of a linear regression to data from a training period, extending from 1906 to 1917 . The estimation was based on the time series truncated from the values observed during the epidemic periods, defined as the 3 consecutive month period with the highest incidence . More precisely, from each annual august 1july 31 window, we removed the trimester with the highest mortality to take into account seasonal influenza, and the associated annual excess mortality . July period was used because seasonal influenza epidemics occur from fall to spring in europe . The baseline model described above we defined excessmortality periods as those starting when observed mortality exceeded the upper limit of the prediction interval [threshold = e (b t) + 196 sd (e t)], for at least 2 consecutive months (one trimester for england and wales) and ending when the observed mortality fell below the upper limit of the prediction interval . Excess mortality was calculated as the difference between observed and expected baseline deaths during the excessmortality period (figure 1). The crosshatched area represents excess mortality, calculated as the difference between observed (black line) and predicted (continuous gray line) mortality, from the beginning of the pandemic period (light gray shading; first month during which observed deaths exceeded the pandemic threshold, dashed gray line) until the end (first month during which observed deaths fell below the pandemic threshold). For finland, we subtracted from excess deaths, the 30 000 deaths attributed to the finnish civil war (january may 1918). Data for the civilian mortality rates in germany presented a step pattern for the period 19141918, probably reflecting mismatched definitions of population counts and deaths as a result of changing geographical borders . Therefore, we first replaced the linear trend of mortality rates during 19141918 by those of the surrounding years (19061913 and 19191936), and obtained excess mortality as described above . We also compared the excess mortality in each of the 14 countries during the 19181919 pandemic with the excess mortality during the rest of the years (19061917; 19201922) to determine how worse the 1918 period was compared with those seasons . Cumulative excess deaths during the 19181919 period were 198 million in 14 countries in europe accounting for 75% of the population, an increase of 86% more deaths from baseline . Extrapolating that figure to the rest of europe, the estimated total mortality in europe during the 1918 pandemic would be 264 (198/075) million deaths, i.e. 11% of the entire population estimated at 250 million in 1918 . The highest cumulative excess / predicted mortality ratio was observed in italy (+ 172%) during the pandemic period (peaked in october 1918), following by bulgaria and portugal (+ 102% each), spain (+ 87%), the netherlands (+ 84%), sweden (+ 74%), germany (+ 73%), switzerland (+ 69%), france (+ 66%), norway (+ 65%), denmark (+ 58%), scotland (+ 57%) and england and wales (+ 55%), with the lowest ratio being seen in finland (+ 33%)(table 1). The highest excessmortality rate (per 10 000 inhabitants) cumulated throughout the entire excessmortality period was observed in portugal (233/10 000 inhabitants), followed by italy, spain, bulgaria, switzerland, finland, france, germany, sweden, netherlands, norway, england and wales, scotland and denmark (figure 2). Excess mortality in 14 european countries represents the excess mortality calculated, based on expected baseline mortality . Existence of a later peak in april 1920 (not taken into account to calculate excess deaths throughout the pandemic period). South gradient in the progressively darker shades of gray, * (/10 000 inhabitants). Overall, the excess mortality found during the pandemic was 35 times higher than the excess mortality found in the rest of the 19061922 period . This ratio ranged between 16 (finland) and 61 (portugal) (table 2). Excess mortality (%) comparison between the 19181919 pandemic periods and the rest of the years (19061917; 19201922) in each of the 14 countries we found a statistically significant negative correlation (spearman s rank correlation = 066; p = 0013) between excess mortality and latitude (figure 2), meaning that northern countries experienced significantly less mortality, while southern europe suffered significantly more excess deaths . Countries were clustered according to their mortality patterns (figure 3): countries with one sharp mortality peak (e.g. Portugal, spain, italy, bulgaria), countries with two major peaks (e.g. Switzerland, scotland, netherlands and france), country with several successive peaks (only finland). Monthly (or trimesterly) mortality rate * (ordinate) in 14 european countries from 1917 to 1921 (abscise). Lines and shading are as defined in the legend to figure 1, * (/10 000 inhabitants). Excess mortality was time dependent: the start and stop months of the excessmortality period varied across the continent and occurred in four waves (figure 4, table 1). Moreover, an early first wave of deaths, occurring between march 1918 and july 1918, was observed in six countries (bulgaria, portugal, germany, finland, switzerland and spain) (figure 4). Importantly, the excessdeath curves could be temporally superposed, albeit not of the same intensity, for 19181919, with mortality peaking in all countries within a 2month window (october november 1918, figure 3), defined as the second wave (figure 4). In five countries (spain, denmark, finland, germany and switzerland), a late peak between january 1920 and april 1920 was recorded . That fourth wave in 1920 was not taken into account in estimating the cumulative excessdeath rates for the entire pandemic period the limitation of any study focused on mortality burden at the epoch of the spanish pandemic depends on the exhaustiveness of the data . During this period, data were often scattered in various places or sources, making their collection for analyses difficult . This is the reason why earlier publications were based on various documents dating from 1918 to 1919 (medical journals, daily newspapers and archives). In this study we took advantage of the existence of vital statistics compiled in european countries by demographers, using a homogeneous method . We used the information available on the monthly variations of death figures to estimate the pandemic mortality burden, based on the values of a 12 years, were for example computed by simply making the difference between the 1918 and 1920 yearly mortality data and the values observed in the preceding and succeeding 3year periods . The comparison of the datasets used in the different mortality assessments, and the results obtained, are shown in figure 5 . The changes in borders and the mass population movements related to world war 1 may be one reason for these discrepancies . For example, in germany, we checked that using the raw data (not corrected for the mismatches of populations and deaths) would have lowered our estimation of 426 574 to 340 000, changing the excess mortality estimate from 73 to 67% . In total, however, the agreement between estimates are more remarkable than the discrepancies . For example, the total european burden was estimated at 2 300 000 in the two first reports, published in 1991 and 2002, based respectively on 18 countries and 21 countries, at 2 005 569 deaths in the paper from murray et al . (total of their 13 european values) versus 1 980 950 deaths in this report . Comparison of published mortality estimates in europe (number of deaths) during the 19181919 influenza pandemic . Circle: johnson and mueller (j); dark circle: ansart et al . (a); square: patterson and pyle (p); triangle: murray et al . As the 14 european countries we have studied in this paper represented 75% of the european population at that time, one can deduce that 26 million excess deaths (11% of the total population) occurred in europe during the period when spanish flu was circulating . American excess deaths during the same period were estimated at 550 000 (corresponding to 065% of the american population) by glezen wp . However, johnsonmueller put the us figure at 675 000 deaths and more recently murray et al . At only 400 000 deaths (047% of the total population). Whatever is taken as estimation of the american pandemic death burden, it is well below the european estimations we provide and others have provided . A possible explanation is that, at the end of ww1, europe was characterised by massive civilian and army movements, a health care system put at its minimum and frail populations . This has very likely heavily impacted the european death toll . Finally, we measure the indirect impact of the influenza virus on the mortality . Herein, we analyzed allcause mortality and not only influenza or pneumonia deaths . Indeed, we were obliged to base our estimation on the available monthly data on concerned total mortality, not influenza and pneumonia (p&i) mortality however, there is a striking linear relationship between the overall annual influenza and pneumonia mortality records, for each of the 11 countries where such data were available, and the total number of excess deaths (figure 6). This is consistent with the hypothesis that p&i mortality is a relatively constant proportion of the total number of deaths, and supports our use of the total number of excess deaths to quantify the death toll of the pandemic . P&ideaths plotted versus allcause excess mortality in 11 european countries. Spearman s rank correlation = 094; p <10 . germany, spain, france, italy, norway, netherlands, portugal, sweden, switzerland, england and wales and scotland . There was a high level of variability between the excess of mortality experienced in the 14 countries we studied, with a minimal value of 33% in finland and a maximal value of 172% in italy . All factors that can be related to the mortality burden such as the existence of coinfectious diseases, especially bacterial pneumonia and tuberculosis, or socioeconomic status were unlikely to have a north south gradient . Possible geographic determinants of the influenza mortality are cold temperature and vitamin d deficiency relative to low sunlight exposure, but they would have implied a higher mortality in northern countries, not the opposite as we observed . We observed a high degree of synchronism in europe, with the highest mortality peak occurring in all countries within a 2month window (oct nov 1918) (3, 4). All but two countries (denmark and italy) experienced at least a twowave pattern . In addition to this common feature, six countries had an earlier first wave of excess mortality before october 1918 (bulgaria, portugal, germany, finland, switzerland and spain) and five countries (spain, denmark, finland, germany and switzerland) had one late excessmortality waves in february and march 1920 . The very peculiar profile of mortality in finland could be explained by their civil war that caused 30 000 deaths in early 1918 (that could not be excluded directly from our analysis because of their unknown spatiotemporal distribution). A fourth wave having occurred in the early spring 1920 similar to the one we found was reported before in specific settings . Including 6 additional months in the calculation of excess mortality (until july 1920), would have increased the burden estimates as follows: 405 800 (+ 153 744) deaths in spain, 60 500 (+ 5768) in finland, 13 600 (+ 3044) in denmark, 510 200 (+ 83 663) in germany and 39 000 (+ 9080) in switzerland . Where the 1918 pandemic first emerged is still being debated . A recent analysis of the 1918 h1n1 genome failed to single out a particular location ., the origin has been successively proposed in asia, in a british army post in france in 1916,, in usa,,,, or in spain . The european origin was not supported by taubenberger et al . In their 1997 report on the sequence of the 1918virus genome extracted from autopsy samples (obtained from the armed forces institute of pathology in washington, dc) of 28 us servicemen who died of the flu during their military service . Our findings can provide clues as to the origin of the pandemic and do not make plausible an european origin: indeed, if the pandemic would have started somewhere in europe, we would have expected to see a spatiotemporal spread outwards from this hypothetical origin, while the data show that all the european countries reached simultaneously their epidemic peaks . All authors contributed to the study and agree with the contents of the manuscript . Conceived and designed the study: sa, ajv, pyb, cp . Collected the data: sa . Contributed materials, analysis tools: fc, af . Wrote the paper: sa, ajv.
They are subjected to continuous operation of stressful factors caused by the exploitation of the roads . These are mainly emissions, dust from the wear of tires and the clutch disc, and the chemical substances used to maintain the roads in the winter (abollino et al . 2002; conde et al . 2009; jiries et al . 2002; petrotou et al . Roadsides receive considerable amounts of these traffic - generated pollutants (garcia and milan 1998). The effect of these factors is the change of physical and chemical conditions of their growth and bioaccumulation of elements deposited into the environment . Among the many chemicals deposited during road use, the greatest impact is brought by heavy metals, including lead and cadmium . Existing in the soil, large quantities of these elements and fresh emission their content in the plants is, however, mainly related with bioaccumulation potential specific for each species . The occurence of high bioaccumulation of heavy metals in plants has been described in many publications (burt et al ., we can observe that a harmful impact of transport on the environment is still modifying (viard et al . . However, large amounts of heavy metals accumulated over the years prove their permanent hazardous effect on plant quality (petrotou et al . 2012). A ban on fuels containing lead has reduced their emissions into the atmosphere and consequently improved the condition of biota adjacent to a road . However, still possible are heavy metal emissions with the dust coming from the wearing parts of working vehicles . It has resulted in the need to control the level of assessment of heavy metal accumulation in organisms inhabiting areas adjacent to the roads . Their content in the plants is, however, mainly connected with bioaccumulation potential specific for each species (aoyama and kuroyanagis 1996; bulinski et al . Accumulation of dust with fine particles of heavy metals in aboveground parts of the plants has a direct impact on contamination of plants growing in their anthropogenic conditions (hendry 1992; jiries 2002; parekh et al . The ability of plants to a diverse accumulation of heavy metals in aboveground part is due to different morphology of plants (deska et al . 2011). A major role in this process play the structure occurring on the surfaces of leaves grooves, hair, and bristles or specific chemicals wax and others (naszradi et al . 2004). They cause a variety of options to keep the dust on the surface of plants . It may also be the differences in accumulation of heavy metals in the different parts of the species (stafilov and jordanovska 1997). It is sometimes caused by genetics, morphology, or the length of biosorption (dudka et al . The most common are the differences between the content of these elements in the leaves and seeds of plants (banuelos and ajwa 1999; dudka et al . They are also often seen as interspecific differences in bioaccumulation of heavy metals (viard et al . Content of heavy metals near the roads can cause both different pollution of grass species and their morphological parts (naszradi et al . 2004; de nicola et al . 2003). The aims of the study were to evaluate the influence of distance from the road on the content of lead and cadmium in aboveground parts of three grass species near a fast road and to assess bioaccumulation of these elements by morphological parts of the grasses . The plant materials in the form of the aboveground parts of three grasses were taken in august 2011 along a 9-km sector of the speed roadway s2 (siedlce bypass) (fig . 1). The track connects cork in ireland with omsk in russia and belongs to the most important tracks of communication in europe, and in poland, it runs along the motorway s2 . The area, where samples were collected, belongs to the mazovian voivodeship, which is located in the middle - east of poland about 80 km on the east from warsaw . Weather conditions of research area were typical for ix - eastern district of agro - climatic of poland . Average annual air temperature ranges from 6.7 to 6.9 c, and in summer, the average daily temperature is 15 c . Annual precipitation is at the level 550650 mm, while they are not frequent, but heavy . In 2011 in may, june, august, and september by selianinov method were poor drought (bac et al . 1993). Average daily movement of motor vehicles (sdr) in the year of study was 9,888 vehicles per day, whereas on the remaining national roads 7,097 engines per day, and in case of international roads 16,667 per day . On the analyzed section of road (ring road for siedlce), the average daily movement of motor vehicles was higher than the average on national roads of poland, and it was 8,136 vehicles per day.fig . 1schematic presentation of the studied sites of s2 road schematic presentation of the studied sites of s2 road chosen species were collected from the grasslands located near the international rout . The following plants were tested: dactylis glomerata, arrhenatherum elatius, and alopecurus pratensis . D. glomerata grows in dense perennial tussocks to 150 cm tall, with leaves of 2050 cm long and up to 1.5 cm broad, and a distinctive tufted triangular flower head of 1015 cm long . Arrhenatherum elatius is a loosely tufted, deciduous, perennial grass with upright to arching, broadly linear, slightly hairy, bright green leaves and erect stems bearing green to purple flower from early summer into autumn . Alopecurus pratensis is a long - lived, tufted perennial grass, with short rhizomes and short ascending stolons, loose or compact tufts, erect culms of 30100 cm tall, and flat, narrow, 26 mm broad, glaucous, glabrous leaf blades . The plant material (a total number of 60 samples) was taken from distances of 1, 5, 10, and 15 m from both the sides of the road at intervals of approximately 100 m. individual samples were collected as morphological parts of plants leaves, shoots, and inflorescences . From the individual samples, five samples for each species of plants and any distance from the edge of the road were taken . The total sampling amounted to 180 (5 samples for each species = 15 4 different distances 3 repetitions). Collected plant samples (not washed) were dried in the temperature of 105 c, weighed and dry mineralized in the temperature of 450 c for 24 h, and then prepared in 10% volume hcl (viard, et al . Content of lead and cadmium has been marked in accordance with atomic absorption spectroscopy (aas) method with the use of aas instrument (thermo elemental, m6 solar type, cambridge uk company). To verify the accuracy of the analytical models, internal quality control patterns were used . It was assumed that the average recovery of tested patterns should range from 85 to 115% of actual value . For research needs, all data in this work were expressed as means standard deviation (sd). Effects of tested factors (grass species, plant part, the distance from the road) on the content of elements in plant were estimated using three - way analysis of variance . Detailed comparisons of the mean values were based on tuckey s test at p 0.05 . In the case of quantitative impact factor (distance), the nature of this impact examined was analyzed using the orthogonal contrasts . To assess the relationship between the content of lead and cadmium in morphological parts of the studied grass species and distance from the road, we used a linear regression method, using polynomial second degree:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${y}_{\mathrm{i}}={a}_1{x}^2+{a}_2x+{a}_0 $$\end{document}yi = a1x2+a2x+a0 where yi is the depended variable (metal content), x is the explanatory variable (the distance from the road), a0 is the intercept, and a2 is the regression coefficient, indicating how much the change of the size of the depended variable (y); when the independent variable increased about 1 unit, the other values were stable . For each equation, the coefficient of determination (r) was estimated which indicated which part of the total variation with y variable explained the regression model (deska et al . 2011). Average lead content in the aboveground parts of tested grass species amounted to 3.56 mg kg dry matter (dm; table 1). In alopecurus pratensis plants, significantly greater amounts of this element (an average of 4.11 mg kg dm) than in the other studied grass species were stated . It has showed a statistical analysis.table 1the content of lead and cadmium in samples of plants: dactylis glomerata, arrhenatherum elatius, and alopecurus pratensis (mg kg)species of grasslead \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{x} $$\end{document}x cadmium \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{x} $$\end{document}x dactylis glomerata 3.27 0.62a0.345 0.02b arrhenatherum elatius 3.30 0.61a0.251 0.01a alopecurus pratensis 4.11 0.52b0.324 0.06ban average for the species3.56 0.270.307 0.02values marked with the same letter are not significantly different in the columns at p 0.05 the content of lead and cadmium in samples of plants: dactylis glomerata, arrhenatherum elatius, and alopecurus pratensis (mg kg) values marked with the same letter are not significantly different in the columns at p 0.05 the lead content found in different grass species from a variety parts of poland reported by klocek et al . The average value reported by these authors is 2.5 mg kg dm . In other countries 2012), it was found that the average size of this element concentration in the grasses was from 0.4 (greece) to 4.6 mg kg dm (slovakia). High uptaking of this element is recorded in nearby communication routes, because the lead uptake by plants is greater in the presence of organic compounds existing in the combustion products (naszradi et al . (2011), the lead content in the soil adjacent to the road on the study section ranged from 91.3 to 101.6 mg kg dm, which is affected on the higher level of their absorption in plants . Having compared the obtained results with a limit of lead content in feed for ruminants (according to klocek et al . (2003), these amount to 10 mg kg dm), we can observe that the tested grasses are classified for use in the animal feeding . It should also be noted that the analyzed species of grasses have a healthy appearance, because their content has not reached the level of lead toxicity (bulinski et al . Plant poisoning with lead, according to klocek and milczarek (2003), may occur when the content is more than 15 mg of this element per kilogram of dry matter . This amount of lead can impair the process of photosynthesis by reducing chlorophyll biosynthesis (parekh et al . The main roadside pollutant concentrations found in these studies are much less than those reported by other studies (mulchi et al . Another significantly important metal in plants growing on the areas adjacent to the speed roadway is cadmium; klocek et al . (2003) claim that critical cadmium content in plants in relation to their usefulness for consumptive aims should not exceed the value of 0.15 mg kg dm, but for fodder aims, it should come to 0.5 mg kg dm . Cadmium content in the studied grass species samples was much lower and ranged from 0.251 to 0.345 mg kg dm (table 1). Statistical analysis showed a significant effect of grass species on the content of this element . The lowest, significantly different from the other species, was the content of this element found in samples of arrhenatherum elatius . (2011) studied an assessment of the cadmium content in the sward of grass species and found species diversity of this element content . In poland, the content of this element in various grass species ranged from 0.1 to 2.6 mg kg dm (klocek et al . 2003). In northeastern poland, cadmium content in plants of d. glomerata ranged from 0.05 to 0.80 g kg, as well as in the southwestern region from 0.10 to 2.60 g kg . While, cadmium content in the grasses found in other studies was in the range of 1.01.6 mg kg dm for belgium (viard et al . 2004) and 0.32.9 mg kg dm for hungary (naszradi et al . Cadmium content in plants depends on the intensity of the dust emission containing this element and on the concentration of cadmium in soil and on physico - chemical parameters of the soil (abollino et al . The cadmium content in soils adjacent to this roadway was in the range from 0.195 to 0.303 mg kg dm (deska et al . 2011). The type of morphological part of tested plant significantly differentiated the lead content in different grass species (table 2). Significantly lower amounts of lead than in the other organs were stated in the leaves of grass species2.82 mg kg dm . In the case of d. glomerata and arrhenatherum elatius, lead content the smallest amount, significantly lower than in the other morphological parts, was determined in the leaves of these plants2.35 and 2.70 mg kg dm). Average lead content in the leaves of d. glomerata was the lowest from the all parts of the studied grass species . In the case of alopecurus pratensis, lead content differed significantly in inflorescences of plants5.04 mg kg dm . It was the highest average from the obtained results . The highest, significantly different from the rest of cadmium values, was found in the leaves of grasses0.387 mg kg dm . In d. glomerata, the content of this element in the inflorescences was significantly lower (0.221 mg kg dm) than in other organs of the studied species . Morphological parts of arrhenatherum elatius did not differ from each other in cadmium content . In the case of alopecurus pratensis, all parts of the tested plants had significantly different concentrations of this element; the least was in the shoots0.162 mg kg dm and the most in the leaves0.485 mg kg dm.table 2the content of lead and cadmium in samples of plants: dactylis glomerata, arrhenatherum elatius, and alopecurus pratensis (mg kg)species of grasssomemorphologicallead \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{x} $$\end{document}x cadmium \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{x} $$\end{document}x dactylis glomerata leaves2.35 0.67a0.466 0.036binflorescence4.02 0.44b0.221 0.074ashoot3.43 0.61b0.368 0.040b arrhenatherum elatius leaves2.70 0.75a0.230 0.039ainflorescence3.54 0.11b0.237 0.089ashoot3.65 0.76b0.285 0.045a alopecurus pratensis leaves3.40 0.52a0.485 0.013cinflorescence5.04 0.35b0.325 0.088bshot3.90 0.25a0.162 0.058aan average for the speciesleaves2.82 0.13a0.387 0.058binflorescence4.20 0.65b0.261 0.049ashoot3.65 0.46b0.272 0.093avalues marked with the same letter do not differ significantly in columns within each species at p 0.05 the content of lead and cadmium in samples of plants: dactylis glomerata, arrhenatherum elatius, and alopecurus pratensis (mg kg) values marked with the same letter do not differ significantly in columns within each species at p 0.05 the distance from the road in different ways influences on the content of the analyzed elements in described grasses (table 3). The largest amounts of lead in plants (4.39 mg kg dm), significantly different from the others, were found in plants growing within 5 m from the edge of the road . This value differed significantly from the lead content in the plant material defined at the other distances from the road . Also, in the study of yusuf et al . (2003), lead exhibited a gradual decrease from 0- to 15-m distance . In the case of cadmium, the largest amount (significantly different from the others) was stated in plants grown in the distances of 1 and 5 m from the road0.380 and 0.377 mg kg dm . Similar correlations of lead content on the distance from the roadway were found for d. glomerata and arrhenatherum elatius . The highest, significantly different, lead content was found in samples taken at a distance of 5 m from the road: an average of 4.72 and 5.08 mg kg dm . The lowest, significantly different from the other trials, was found in samples of plants from distances of 1 and 15 m2.73 and 2.20 mg kg dm (d. glomerata), and 2.52 and 1.64 mg kg dm (arrhenatherum elatius). In the case of alopecurus pratensis, the smallest amounts of lead, significantly different, were found for plants at a distance of 1 m3.13 mg kg dm and the largest, also significantly different, from a distance of 15 m5.18 mg kg dm . Table 3the content of lead and cadmium in plants dactylis glomerata, arrhenatherum elatius, and alopecurus pratensis (mg kg) depending on the distance from the roaddistance from the road(m)plant speciesleadcadmiummean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{x} $$\end{document}x mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{x} $$\end{document}x 1 dactylis glomerata arrhenatherum elatius alopecurus pratensis 2.73 0.35aa2.52 0.41aa3.13 0.35aa0.428 0.050bc0.292 0.018ab0.337 0.018aaaverage for 1 m3.02 0.78a0.380 0.020b5 dactylis glomerata arrhenatherum elatius alopecurus pratensis 4.72 0.60bc5.08 0.10bc4.09 0.27ab0.447 0.020cc0.272 0.061ab0.372 0.014baaverage for 5 m4.39 0.80b0.377 0.017b10 dactylis glomerata arrhenatherum elatius alopecurus pratensis 3.41 0.26ab3.96 0.57ab4.02 0.82ab0.287 0.013ab0.327 0.014ab0.285 0.012aaaverage for 10 m3.52 0.70a0.283 0.013a15 dactylis glomerata arrhenatherum elatius alopecurus pratensis 2.20 0.90aa1.64 0.60aa5.18 0.80bc0.187 0.053ba0.111 0.036aa0.303 0.013caaverage for 15 m3.01 0.97a0.200 0.011avalues marked by the same small letters do not significantly differ in the content of elements in different species for the same distance, at p 0.05 . Values marked with the same capital letters do not significantly differ in the content of elements in plants of one species at different distances, at p 0.05 the content of lead and cadmium in plants dactylis glomerata, arrhenatherum elatius, and alopecurus pratensis (mg kg) depending on the distance from the road values marked by the same small letters do not significantly differ in the content of elements in different species for the same distance, at p 0.05 . Values marked with the same capital letters do not significantly differ in the content of elements in plants of one species at different distances, at p 0.05 the lead content in different samples of the grass species growing at 1 and 10 m from the road did not differ significantly (table 3). In case of other distances, alopecurus pratensis reacted in a different way . At a distance of 5 m, plants of this species contained the highest amounts of this element, and at distance of 15 m the smallest . (2004), a study on the impact of traffic on the content of heavy metals in the grass of festuca arundinacea, phallaris sp ., and d. glomerata gathered beside the road, the lead contents amounted to 1.02.0 mg kg dm, but growing at 5 and 20 m from the road0.82.2 and 0.50.7 mg kg dm . Distance from the road significantly affects the cadmium content in different studied grass species (table 3). In case of d. glomerata, cadmium content was the highest, significantly different from the other species . At distances of 1 and 5 m from the road the lowest amounts of the element, also significantly different, were found at a distance of 15 m0.187 mg kg dm . Arrhenatherum elatius contained significantly different amounts of cadmium, lower than in case of the other species, and they reached values, at 15 m, of 0.111 mg kg dm . Alopecurus pratensis proved no significant influence of distance from the road on bioaccumulation of cadmium by the plant . At 1-m distance from the edge of the road, the greatest amounts of cadmium were found in samples of d. glomerata (0.428 mg kg). Additionally, at 5-m distance, the highest cadmium content was observed in the samples of d. glomerata (0.447 mg kg), and the lowest in arrhenatherum elatius (0.272 mg kg). These amounts differ from the metal content in alopecurus pratensis (0.372 mg kg). At a distance of 10 m, cadmium content in various grass species did not differ . At a distance of 15 m, the smallest amount of this element, significantly different, was found in arrhentherum elatius (0.111 mg kg), and the highest, also significantly different, in alopecurus pratensis (0.303 mg kg). Regression equations describing the effect of the distance from the roadway on the lead content in the tested morphological parts of the grasses indicate high differences of accumulation of this element in the organs of the same species (fig . 2). The most variation in the contents of heavy metals has been demonstrated in plants organs for d. glomerata and alopecurus pratensis.fig . 2course of regression equations describing the effect of distance from the road on the lead content in morphological parts of the studied grass species (p 0.05) course of regression equations describing the effect of distance from the road on the lead content in morphological parts of the studied grass species (p 0.05) in the case of d. glomerata (fig . 2a), the variability of lead content in the inflorescence showed an almost linear course, with the maximum value (6.0 mg kg dm) at a distance of 1 m from the edge of the road . Variability of the lead content in the other organs of plants has been described by characteristic equations (jiries et al . 2011) for this type of phenomena with a maximum at a distance of 78 m from the road . Such a high lead content in inflorescence of d. glomerata may be due to its specific construction, permitting dust keeping (klocek et al . 2c), different relationships between the distance from the road and the lead content for the inflorescences and shoots were stated . Regression equations indicate an increase of the lead content in these plant parts in farther distances from the road from 4.89 to 7.08 mg kg, and from 2.53 to 2.93 mg kg . The same results (calculated by regression coefficient) were obtained in studies of other authors for arrhenatherum elatius in the case of increased traffic (naszradi et al . Variation of the lead content depending on the distance was also found in similar morphological parts of various species (fig . The coefficients of regression equations indicate a high mutual similarity of these functions . In the grasses of d. glomerata and arrhenatherum elatius, similar response to the pollution of roadway area was also related to the shoots of plants . Different courses in all described grass species had regression equations describing the effect of distance from the roadway to lead contamination of plants inflorescences . The regression equations for the averages of all the grasses (fig . 2d) show the different courses of the equation of lead absorption in the inflorescences, in comparison with the equations describing this phenomenon for the shoots and leaves . High values of the determination coefficients (r) show a very good fit of the used polynomial function of the second degree to describe this occurence . It does not concern equation describing the impact of distance on the lead concentration in leaves of d. glomerata . Different values than in case of lead shoved regression equation describing the affect of distance from the road on the amount of cadmium in aboveground plant organs (fig . 3). The calculated ratios of these equations also show a great diversity in accumulation of cadmium in the organs of the same species . As mentioned above, the highest amounts of the element were found in d. glomerata and alopecurus pratensis.fig . 3course of regression equations describing the effect of distance from the road on the cadmium content in morphological parts of the studied grass species (p 0.05) course of regression equations describing the effect of distance from the road on the cadmium content in morphological parts of the studied grass species (p 0.05) in d. glomerata (fig . 3a), the highest content of cadmium in the strip adjacent to the roadway (15 m) was found in the leaves (0.621 and 0.630 mg kg dm) and shoots (0.562 and 0.622 mg kg dm). At the successive distances, the content of this metal in similar parts decreased, and for the shoots, this is evidenced by the low value of the coefficient a1 in the regression equation (fig . Variability of cadmium in the successive distances from the roadway in the inflorescences was described by parabolic equations, with a maximum content at distance of 10 m from the road (0.300 mg kg dm). The increase in the distance from the road induced different effects on the cadmium content in each described plant organs of alopecurus pratensis (fig . The largest amounts of cadmium in its leaves were found near the road lane (0.590 mg kg dm). When the road distance increased, the content of this element almost linearly decreased (fig . Cadmium content in inflorescences increased from 0.290 (1 m) to 0.395 mg kg dm (15 m), and in the shoots of this plant, the most content of this element was found in the middle of the tested strip (0.240 mg kg dm). 3b) was similar as in alopecurus pratensis, but the relationship distance cadmium content is described by a less flattened curve (fig . The highest content in shoots and inflorescences was found in plants taken from the strip of 510 m. calculation for these coefficients of regression equations illustrates the typical response of heavy metals in plants to the occurence caused by the use of vehicles (viard et al . 3d) show the different courses of cadmium absorption equation for tested grass leaves, in comparison to the shoots and leaves . High coefficients of determination (r) show a very good fit of the used polynomial functions to describe this phenomenon for the majority of reported cases . Slightly lower values of coefficient r for just a very few cases indicate a smaller fit of polynomial function to describe this phenomenon, but this description is to be satisfactory (li et al . There are no studies on the impact of traffic pollution on the morphological parts of the grasses . It was found that the lowest amount of heavy metals is accumulated in the seeds of plants, and the highest in their underground parts (garcia and milan 1998). In studies on cadmium and lead content in morphological parts of galega orientalis, the higher lead content was observed in leaves, but in case of cadmium, it was observed in the shoots (deska et al . The content of these elements in the studied plants is the sum of the amounts of the element absorbed by plants through the root system from the soil, taken in the form of fine dusts by stomata and accumulated in the tissue and the amount of dust deposited on the parts of plants (aoyama and kuroyanagis 1996). It should be noted that in the case of plants exposed to dusts containing heavy metals, the great importance is the dust settling on the surface of plant organs . It was proved the influence of surface cleaning of plants metals concentration on the change of their content in plants (rossini oliva and mingorance 2006). Exact wash samples or remove the top layer of tissue resulting in a significant decrease of cadmium and lead content . Large amounts of lead and cadmium accumulate in the leaves of plants growing in areas with high air pollution by dusts containing these elements (aoyama and kuroyanagis 1996). Accumulation of dust can be intensified by various features like surfaces covered with plant hairs, wrinkles, or a spongy or stubbly structure . Grasses having leaf surfaces with varying degrees and covered with bristles, ciliated, or striated can retain the dusts (hendry et al . Even larger traps can be grass inflorescences, which porous, composite structures can hold large quantities of dust . In this study, higher amounts of cadmium and lead in inflorescences of d. glomerata and alopecurus pratensis are probably caused by these processes . The lower content of these elements in arrhenatherum elatius results from the morphological characteristics of this plants species not favorable to the sedimentation of the dust . Additionally, this can affect the species characteristics related with the limited movement of the heavy metals from the soil . The heavy metal content in the shoots is mainly related with the translocation of mobile forms of these elements taken from the soil and with their retention in the structure of the tissues . The obtained results indicate the importance of the species and the harvesting stage of grasses grown in areas with a higher degree of risk of dust sedimentation containing heavy metals regardless of the tested grass species, the most concentration of heavy metals was estimated in the plant materials which were collected at 1-m distance from the road . Average lead content in the aboveground parts of tested grass species amounted to 3.56 mg kg dry matter (dm; table 1). In alopecurus pratensis plants, significantly greater amounts of this element (an average of 4.11 mg kg dm) than in the other studied grass species were stated . It has showed a statistical analysis.table 1the content of lead and cadmium in samples of plants: dactylis glomerata, arrhenatherum elatius, and alopecurus pratensis (mg kg)species of grasslead \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{x} $$\end{document}x cadmium \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{x} $$\end{document}x dactylis glomerata 3.27 0.62a0.345 0.02b arrhenatherum elatius 3.30 0.61a0.251 0.01a alopecurus pratensis 4.11 0.52b0.324 0.06ban average for the species3.56 0.270.307 0.02values marked with the same letter are not significantly different in the columns at p 0.05 the content of lead and cadmium in samples of plants: dactylis glomerata, arrhenatherum elatius, and alopecurus pratensis (mg kg) values marked with the same letter are not significantly different in the columns at p 0.05 the lead content found in different grass species from a variety parts of poland reported by klocek et al . The average value reported by these authors is 2.5 mg kg dm . In other countries 2012), it was found that the average size of this element concentration in the grasses was from 0.4 (greece) to 4.6 mg kg dm (slovakia). High uptaking of this element is recorded in nearby communication routes, because the lead uptake by plants is greater in the presence of organic compounds existing in the combustion products (naszradi et al . (2011), the lead content in the soil adjacent to the road on the study section ranged from 91.3 to 101.6 mg kg dm, which is affected on the higher level of their absorption in plants . Having compared the obtained results with a limit of lead content in feed for ruminants (according to klocek et al . (2003), these amount to 10 mg kg dm), we can observe that the tested grasses are classified for use in the animal feeding . It should also be noted that the analyzed species of grasses have a healthy appearance, because their content has not reached the level of lead toxicity (bulinski et al . Plant poisoning with lead, according to klocek and milczarek (2003), may occur when the content is more than 15 mg of this element per kilogram of dry matter . This amount of lead can impair the process of photosynthesis by reducing chlorophyll biosynthesis (parekh et al . The main roadside pollutant concentrations found in these studies are much less than those reported by other studies (mulchi et al . Another significantly important metal in plants growing on the areas adjacent to the speed roadway is cadmium; klocek et al . (2003) claim that critical cadmium content in plants in relation to their usefulness for consumptive aims should not exceed the value of 0.15 mg kg dm, but for fodder aims, it should come to 0.5 mg kg dm . Cadmium content in the studied grass species samples was much lower and ranged from 0.251 to 0.345 mg kg dm (table 1). Statistical analysis showed a significant effect of grass species on the content of this element . The lowest, significantly different from the other species, was the content of this element found in samples of arrhenatherum elatius . (2011) studied an assessment of the cadmium content in the sward of grass species and found species diversity of this element content . In poland, the content of this element in various grass species ranged from 0.1 to 2.6 mg kg dm (klocek et al . 2003). In northeastern poland, cadmium content in plants of d. glomerata ranged from 0.05 to 0.80 g kg, as well as in the southwestern region from 0.10 to 2.60 g kg . While, cadmium content in the grasses found in other studies was in the range of 1.01.6 mg kg dm for belgium (viard et al . 2004) and 0.32.9 mg kg dm for hungary (naszradi et al . Cadmium content in plants depends on the intensity of the dust emission containing this element and on the concentration of cadmium in soil and on physico - chemical parameters of the soil (abollino et al . The cadmium content in soils adjacent to this roadway was in the range from 0.195 to 0.303 mg kg dm (deska et al . 2011). The type of morphological part of tested plant significantly differentiated the lead content in different grass species (table 2). Significantly lower amounts of lead than in the other organs were stated in the leaves of grass species2.82 mg kg dm . In the case of d. glomerata and arrhenatherum elatius, lead content the smallest amount, significantly lower than in the other morphological parts, was determined in the leaves of these plants2.35 and 2.70 mg kg dm). Average lead content in the leaves of d. glomerata was the lowest from the all parts of the studied grass species . In the case of alopecurus pratensis, lead content differed significantly in inflorescences of plants5.04 mg kg dm . It was the highest average from the obtained results . The highest, significantly different from the rest of cadmium values, was found in the leaves of grasses0.387 mg kg dm . In d. glomerata, the content of this element in the inflorescences was significantly lower (0.221 mg kg dm) than in other organs of the studied species . Morphological parts of arrhenatherum elatius did not differ from each other in cadmium content . In the case of alopecurus pratensis, all parts of the tested plants had significantly different concentrations of this element; the least was in the shoots0.162 mg kg dm and the most in the leaves0.485 mg kg dm.table 2the content of lead and cadmium in samples of plants: dactylis glomerata, arrhenatherum elatius, and alopecurus pratensis (mg kg)species of grasssomemorphologicallead \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{x} $$\end{document}x cadmium \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{x} $$\end{document}x dactylis glomerata leaves2.35 0.67a0.466 0.036binflorescence4.02 0.44b0.221 0.074ashoot3.43 0.61b0.368 0.040b arrhenatherum elatius leaves2.70 0.75a0.230 0.039ainflorescence3.54 0.11b0.237 0.089ashoot3.65 0.76b0.285 0.045a alopecurus pratensis leaves3.40 0.52a0.485 0.013cinflorescence5.04 0.35b0.325 0.088bshot3.90 0.25a0.162 0.058aan average for the speciesleaves2.82 0.13a0.387 0.058binflorescence4.20 0.65b0.261 0.049ashoot3.65 0.46b0.272 0.093avalues marked with the same letter do not differ significantly in columns within each species at p 0.05 the content of lead and cadmium in samples of plants: dactylis glomerata, arrhenatherum elatius, and alopecurus pratensis (mg kg) values marked with the same letter do not differ significantly in columns within each species at p 0.05 the distance from the road in different ways influences on the content of the analyzed elements in described grasses (table 3). The largest amounts of lead in plants (4.39 mg kg dm), significantly different from the others, were found in plants growing within 5 m from the edge of the road . This value differed significantly from the lead content in the plant material defined at the other distances from the road . Also, in the study of yusuf et al . (2003), lead exhibited a gradual decrease from 0- to 15-m distance . In the case of cadmium, the largest amount (significantly different from the others) was stated in plants grown in the distances of 1 and 5 m from the road0.380 and 0.377 mg kg dm . Similar correlations of lead content on the distance from the roadway were found for d. glomerata and arrhenatherum elatius . The highest, significantly different, lead content was found in samples taken at a distance of 5 m from the road: an average of 4.72 and 5.08 mg kg dm . The lowest, significantly different from the other trials, was found in samples of plants from distances of 1 and 15 m2.73 and 2.20 mg kg dm (d. glomerata), and 2.52 and 1.64 mg kg dm (arrhenatherum elatius). In the case of alopecurus pratensis, the smallest amounts of lead, significantly different, were found for plants at a distance of 1 m3.13 mg kg dm and the largest, also significantly different, from a distance of 15 m5.18 mg kg dm . Table 3the content of lead and cadmium in plants dactylis glomerata, arrhenatherum elatius, and alopecurus pratensis (mg kg) depending on the distance from the roaddistance from the road(m)plant speciesleadcadmiummean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{x} $$\end{document}x mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{x} $$\end{document}x 1 dactylis glomerata arrhenatherum elatius alopecurus pratensis 2.73 0.35aa2.52 0.41aa3.13 0.35aa0.428 0.050bc0.292 0.018ab0.337 0.018aaaverage for 1 m3.02 0.78a0.380 0.020b5 dactylis glomerata arrhenatherum elatius alopecurus pratensis 4.72 0.60bc5.08 0.10bc4.09 0.27ab0.447 0.020cc0.272 0.061ab0.372 0.014baaverage for 5 m4.39 0.80b0.377 0.017b10 dactylis glomerata arrhenatherum elatius alopecurus pratensis 3.41 0.26ab3.96 0.57ab4.02 0.82ab0.287 0.013ab0.327 0.014ab0.285 0.012aaaverage for 10 m3.52 0.70a0.283 0.013a15 dactylis glomerata arrhenatherum elatius alopecurus pratensis 2.20 0.90aa1.64 0.60aa5.18 0.80bc0.187 0.053ba0.111 0.036aa0.303 0.013caaverage for 15 m3.01 0.97a0.200 0.011avalues marked by the same small letters do not significantly differ in the content of elements in different species for the same distance, at p 0.05 . Values marked with the same capital letters do not significantly differ in the content of elements in plants of one species at different distances, at p 0.05 the content of lead and cadmium in plants dactylis glomerata, arrhenatherum elatius, and alopecurus pratensis (mg kg) depending on the distance from the road values marked by the same small letters do not significantly differ in the content of elements in different species for the same distance, at p 0.05 . Values marked with the same capital letters do not significantly differ in the content of elements in plants of one species at different distances, at p 0.05 the lead content in different samples of the grass species growing at 1 and 10 m from the road did not differ significantly (table 3). In case of other distances, alopecurus pratensis reacted in a different way . At a distance of 5 m, plants of this species contained the highest amounts of this element, and at distance of 15 m the smallest . In viard et al . (2004), a study on the impact of traffic on the content of heavy metals in the grass of festuca arundinacea, phallaris sp ., and d. glomerata gathered beside the road, the lead contents amounted to 1.02.0 mg kg dm, but growing at 5 and 20 m from the road0.82.2 and 0.50.7 mg kg dm . Distance from the road significantly affects the cadmium content in different studied grass species (table 3). In case of d. glomerata, cadmium content was the highest, significantly different from the other species . At distances of 1 and 5 m from the road the lowest amounts of the element, also significantly different, were found at a distance of 15 m0.187 mg kg dm . Arrhenatherum elatius contained significantly different amounts of cadmium, lower than in case of the other species, and they reached values, at 15 m, of 0.111 mg kg dm . Alopecurus pratensis proved no significant influence of distance from the road on bioaccumulation of cadmium by the plant . At 1-m distance from the edge of the road, the greatest amounts of cadmium were found in samples of d. glomerata (0.428 mg kg). Additionally, at 5-m distance, the highest cadmium content was observed in the samples of d. glomerata (0.447 mg kg), and the lowest in arrhenatherum elatius (0.272 mg kg). These amounts differ from the metal content in alopecurus pratensis (0.372 mg kg). At a distance of 10 m, cadmium content in various grass species did not differ . At a distance of 15 m, the smallest amount of this element, significantly different, was found in arrhentherum elatius (0.111 mg kg), and the highest, also significantly different, in alopecurus pratensis (0.303 mg kg). Regression equations describing the effect of the distance from the roadway on the lead content in the tested morphological parts of the grasses indicate high differences of accumulation of this element in the organs of the same species (fig . 2). The most variation in the contents of heavy metals has been demonstrated in plants organs for d. glomerata and alopecurus pratensis.fig . 2course of regression equations describing the effect of distance from the road on the lead content in morphological parts of the studied grass species (p 0.05) course of regression equations describing the effect of distance from the road on the lead content in morphological parts of the studied grass species (p 0.05) in the case of d. glomerata (fig . 2a), the variability of lead content in the inflorescence showed an almost linear course, with the maximum value (6.0 mg kg dm) at a distance of 1 m from the edge of the road . Variability of the lead content in the other organs of plants has been described by characteristic equations (jiries et al . 2011) for this type of phenomena with a maximum at a distance of 78 m from the road . Such a high lead content in inflorescence of d. glomerata may be due to its specific construction, permitting dust keeping (klocek et al . 2c), different relationships between the distance from the road and the lead content for the inflorescences and shoots were stated . Regression equations indicate an increase of the lead content in these plant parts in farther distances from the road from 4.89 to 7.08 mg kg, and from 2.53 to 2.93 mg kg . The same results (calculated by regression coefficient) were obtained in studies of other authors for arrhenatherum elatius in the case of increased traffic (naszradi et al . Variation of the lead content depending on the distance was also found in similar morphological parts of various species (fig . In the grasses of d. glomerata and arrhenatherum elatius, similar response to the pollution of roadway area was also related to the shoots of plants . Different courses in all described grass species had regression equations describing the effect of distance from the roadway to lead contamination of plants inflorescences . The regression equations for the averages of all the grasses (fig . 2d) show the different courses of the equation of lead absorption in the inflorescences, in comparison with the equations describing this phenomenon for the shoots and leaves . High values of the determination coefficients (r) show a very good fit of the used polynomial function of the second degree to describe this occurence . It does not concern equation describing the impact of distance on the lead concentration in leaves of d. glomerata . Different values than in case of lead shoved regression equation describing the affect of distance from the road on the amount of cadmium in aboveground plant organs (fig . 3). The calculated ratios of these equations also show a great diversity in accumulation of cadmium in the organs of the same species . As mentioned above, the highest amounts of the element were found in d. glomerata and alopecurus pratensis.fig . 3course of regression equations describing the effect of distance from the road on the cadmium content in morphological parts of the studied grass species (p 0.05) course of regression equations describing the effect of distance from the road on the cadmium content in morphological parts of the studied grass species (p 0.05) in d. glomerata (fig . 3a), the highest content of cadmium in the strip adjacent to the roadway (15 m) was found in the leaves (0.621 and 0.630 mg kg dm) and shoots (0.562 and 0.622 mg kg dm). At the successive distances, the content of this metal in similar parts decreased, and for the shoots, this decline is virtually linear . This is evidenced by the low value of the coefficient a1 in the regression equation (fig . Variability of cadmium in the successive distances from the roadway in the inflorescences was described by parabolic equations, with a maximum content at distance of 10 m from the road (0.300 mg kg dm). The increase in the distance from the road induced different effects on the cadmium content in each described plant organs of alopecurus pratensis (fig . The largest amounts of cadmium in its leaves were found near the road lane (0.590 mg kg dm). When the road distance increased, the content of this element almost linearly decreased (fig . Cadmium content in inflorescences increased from 0.290 (1 m) to 0.395 mg kg dm (15 m), and in the shoots of this plant, the most content of this element was found in the middle of the tested strip (0.240 mg kg dm). 3b) was similar as in alopecurus pratensis, but the relationship distance cadmium content is described by a less flattened curve (fig . The highest content in shoots and inflorescences was found in plants taken from the strip of 510 m. calculation for these coefficients of regression equations illustrates the typical response of heavy metals in plants to the occurence caused by the use of vehicles (viard et al . 3d) show the different courses of cadmium absorption equation for tested grass leaves, in comparison to the shoots and leaves . High coefficients of determination (r) show a very good fit of the used polynomial functions to describe this phenomenon for the majority of reported cases . Slightly lower values of coefficient r for just a very few cases indicate a smaller fit of polynomial function to describe this phenomenon, but this description is to be satisfactory (li et al . 2008). There are no studies on the impact of traffic pollution on the morphological parts of the grasses . It was found that the lowest amount of heavy metals is accumulated in the seeds of plants, and the highest in their underground parts (garcia and milan 1998). In studies on cadmium and lead content in morphological parts of galega orientalis, the higher lead content was observed in leaves, but in case of cadmium, it was observed in the shoots (deska et al . The content of these elements in the studied plants is the sum of the amounts of the element absorbed by plants through the root system from the soil, taken in the form of fine dusts by stomata and accumulated in the tissue and the amount of dust deposited on the parts of plants (aoyama and kuroyanagis 1996). It should be noted that in the case of plants exposed to dusts containing heavy metals, the great importance is the dust settling on the surface of plant organs . It was proved the influence of surface cleaning of plants metals concentration on the change of their content in plants (rossini oliva and mingorance 2006). Exact wash samples or remove the top layer of tissue resulting in a significant decrease of cadmium and lead content . Large amounts of lead and cadmium accumulate in the leaves of plants growing in areas with high air pollution by dusts containing these elements (aoyama and kuroyanagis 1996). Accumulation of dust can be intensified by various features like surfaces covered with plant hairs, wrinkles, or a spongy or stubbly structure . Grasses having leaf surfaces with varying degrees and covered with bristles, ciliated, or striated can retain the dusts (hendry et al . Even larger traps can be grass inflorescences, which porous, composite structures can hold large quantities of dust . In this study, higher amounts of cadmium and lead in inflorescences of d. glomerata and alopecurus pratensis are probably caused by these processes . The lower content of these elements in arrhenatherum elatius results from the morphological characteristics of this plants species not favorable to the sedimentation of the dust . Additionally, this can affect the species characteristics related with the limited movement of the heavy metals from the soil . The heavy metal content in the shoots is mainly related with the translocation of mobile forms of these elements taken from the soil and with their retention in the structure of the tissues . The obtained results indicate the importance of the species and the harvesting stage of grasses grown in areas with a higher degree of risk of dust sedimentation containing heavy metals . Proper selection of crop species may limit the risk of animal feeding . Regardless of the tested grass species, the most concentration of heavy metals was estimated in the plant materials which were collected at 1-m distance from the road . The average lead content in the above parts of tested grass species d. glomerata, arrhenatherum elatius, and alopecurus pratensis amounted to 3.56, and for cadmium, it is 0.307 mg kg dm, which indicated even lower values than the standard adopted for the feed using of grasses.there were significant differences in the content of lead and cadmium in the studied species of grasses . Alopecurus pratensis has absorbed significantly higher amounts of lead (average of 4.11 mg kg dm), while in arrhenatherum elatius grass, a lower bioaccumulation of this elementary substance has been noted than in the other species (0.251 mg kg dm).having analyzed the morphological parts of the grass species, we can observe that the lead and cadmium content in d. glomerata, arrhenatherum elatius, and alopecurus pratensis was significantly different . Cadmium concentration in the studied plants was different.distance from the road has significantly affected the contents of the studied metals in morphological parts of the grasses . The occurence is associated with a diversified structure of these plant organs.the obtained results indicate a possibility to select species composition of grasses on areas threatened with heavy metal contamination of dust origin . The grass species recommended for sowing of land contaminated with lead is d. glomerata, and that contaminated with cadmium, alopecurus pratensis the average lead content in the above parts of tested grass species d. glomerata, arrhenatherum elatius, and alopecurus pratensis amounted to 3.56, and for cadmium, it is 0.307 mg kg dm, which indicated even lower values than the standard adopted for the feed using of grasses . There were significant differences in the content of lead and cadmium in the studied species of grasses . Alopecurus pratensis has absorbed significantly higher amounts of lead (average of 4.11 mg kg dm), while in arrhenatherum elatius grass, a lower bioaccumulation of this elementary substance has been noted than in the other species (0.251 mg kg dm). Having analyzed the morphological parts of the grass species, we can observe that the lead and cadmium content in d. glomerata, arrhenatherum elatius, and alopecurus pratensis was significantly different . Distance from the road has significantly affected the contents of the studied metals in morphological parts of the grasses . The obtained results indicate a possibility to select species composition of grasses on areas threatened with heavy metal contamination of dust origin . The grass species recommended for sowing of land contaminated with lead is d. glomerata, and that contaminated with cadmium, alopecurus pratensis
It accounts for <1% of all hernias . Typically, they manifest in multiparous, thin females within their seventh to ninth decades of life . Patients frequently present with symptoms of intestinal obstruction and, occasionally, pain along the distribution of the obturator nerve on the ipsilateral side (howship romberg sign). However, the diagnosis can be difficult to make and delay in diagnosis and surgical intervention contributes directly to high morbidity and mortality rates . The authors report the successful laparoscopic management of an acute presentation of an obstructed obturator hernia . An 85-year - old female with a background of trans - abdominal hysterectomy, hypertension and a previous thyroid lobectomy, presented acutely with a 1-day history of upper abdominal pain radiating down her left leg associated with numerous episodes of emesis . Her bowels had opened three times in the previous 24 h. she was anorexic for the previous 24 h. she reported a similar episode requiring hospitalization 3 weeks before, which was treated non - operatively in another institution . On examination, she was treated presumptively as a small bowel obstruction and non - operative measures were employed and the patient proceeded to have a computed tomography (ct) scan of the abdomen and pelvis, which indicated proximal small bowel obstruction secondary to a left obturator hernia (fig . 1). She proceeded to emergent laparoscopy and subsequent mesh repair of left obturator hernia, containing an obstructed loop of small bowel . Proximal small bowel obstruction secondary to a left obturator hernia within white outline . Under general anaesthetic, the optical technique for access was utilized at the umbilicus with a 5 mm port . Two further working ports, a 5 and 10 mm, were placed in the right lower quadrant and right hypochondrium, under direct vision . Findings included visualization of small bowel dilatation down to small bowel in a left obturator hernia with an incidental left femoral hernia containing omentum (fig . The incarcerated small bowel was reduced using a pair of non - traumatic forceps and deemed viable . Figure 2:intraoperative image showing left sided obturator hernia within white outline and incidental femoral hernia at 11o clock position . Intraoperative image showing left sided obturator hernia within white outline and incidental femoral hernia at 11o clock position . Sommerville, nj, usa) tacked with absorbatack (covidien, mansfield, inc ., this then allowed the mesh to be spread laterally and fixed to the transversalis fascia . The femoral hernia was repaired with 2 2/0 vicryl (ethicon, inc ., the patient did suffer a post - operative pneumonia but was treated appropriately, made a good recovery and was discharged home well . Conditions associated with long - standing increased intra - abdominal pressure, weight loss that leads to a decreased peritoneal fat and multiparity are implicated on the basis of laxity of the parietal peritoneum . All are believed to increase the risk for development of an obturator hernia and women are affected nine times more frequently than men . The obturator membrane covers most of the foramen, except at the anterior superior aspect . The anatomic formation of obturator hernia has been previously described in detail in the literature, but the basic premise of the aetiology and pathogenesis is based on the loss of preperitoneal fat or lymphatic tissue that occupies the obturator canal . Classic features of an obturator hernia: (i) a palpable mass in the groin with the patient supine, and the thigh flexed, adducted and rotated laterally; (ii) intestinal obstruction; (iii) previous attacks of bowel obstruction resolving spontaneously; (iv) the howship romberg sign is medial thigh and hip pain exacerbated by adduction and medial rotation of the thigh and relieved by thigh flexion . The usefulness of ct in diagnosis was first reported by cubillo in 1983 and it has been assisting in the earlier diagnosis of obturator hernia but despite this post - operative mortality has not improved remarkably . However, several studies have shown the feasibility of laparoscopic techniques for the management of incarcerated obturator hernia . The transabdominal preperitoneal repair for obturator hernias is the preferred technique as it allows assessment of the visceral contents . The current trend is to repair the defect using prosthetic material; however, there is no robust evidence in the literature to compare and contrast the superiority of mesh repair over other repairs in obturator hernia . In conclusion, we present this rare case to raise awareness of a rare presentation but with significant morbidity and mortality if a high index of suspicion is not maintained . This case illustrates the usefulness of ct in diagnosing obturator hernia and it also confirms that laparoscopic mesh repair of an incarcerated obturator hernia is safe and technically feasible.
Oral myiasis is a rare condition that refers to the invasion of tissue of the oral cavity by fly larvae . The term myiasis (greek: myia= fly, iasis = disease) was coined by hope in 1840 and it was first described by laurance in 1909 . Zumpt defined myiasis as the infestation of live human and vertebrate animals with dipterous larvae, which feed on living or necrotic tissues, liquid body substances, or ingested food . Human myiasis is reported mainly in developing countries such as asian countries and very rarely from western countries . Myiasis can be classified depending on the condition of the involved tissue as i) accidental myiasis when larvae get ingested along with food, ii) semi - specific myiasis when the larvae are laid on necrotic tissue of the wound and iii). Based on anatomic site, it can be classified as i) cutaneous myiasis, ii) myiasis of external orifices and iii) myiasis of internal organs . Primary myiasis is caused by biophagous larvae (feed on living tissues) and also called as obligatory myiasis . Secondary myiasis is caused by the necrobiophagous larvae (feed on dead tissues) and also called as facultative myiasis . The most common anatomical sites for myiasis are the nose, eyes, skin wounds, sinuses, ears, lungs, gut, gall bladder, vagina, nasal cavities, and rarely, the mouth . Whereas cutaneous myiasis involves invasion of the skin through the wounds . The common predisposing factors are incompetent lips, poor oral hygiene, severe halitosis, anterior open bite, mouth breathing during sleep, facial trauma, extraction wounds, ulcerative lesions and carcinoma . Most of the patients are senile, alcoholics, mentally handicapped, cerebral palsied,[121416] and also those living in poor conditions, with no age limitation . Droma etal . Reported that incidence of myiasis is more in anterior maxillary region and men are more affected than women . Traumatic wounds in orofacial region, when neglected by patients themselves as well as care takers, can lead to development of myiasis . The present report describes a case of oral myiasis in a 14 year old boy with a history of trauma to the maxillary anterior region and it was neglected by him and parents . A 14 year old boy with learning disability presented to department of pedodontics and preventive dentistry, sjm dental collegeand hospital, chitradurga, with a chief complaint of upper lip swelling and itching sensation on the right side of the face since more than 2 weeks [figure 1]. The patient gave a history of trauma to maxillary anterior dentoalveolar region and it was neglected for many days . On examination, there was swelling of the face on the right side, which extended from infraorbital region to upper lip [figure 1]. Intraoral examination revealed a deep lacerated wound which was infected and maggots were noticed on the upper vestibule and the nasal floor . The patient had an incompetent lip, swollen upper gingiva, and poor oral hygiene, and general symptoms like pain, fever and malaise were present . A cotton swab impregnated with turpentine was placed near the upper vestibule (wound) area for 510 minutes . Many of the maggots came out from the nose, which were then removed one by one with the help of curved artery forceps [figures 2, 3]. During the 3 days interval, the length of the each maggot ranged from 6 to17 mm [figure 6]. Then, the wound was irrigated with hydrogen peroxide, antiseptic betadine solution and broad - spectrum antibiotics, and analgesics were prescribed . The patient was recalled for regular follow - up for wound debridement and the wound healed uneventfully . Swelling of the right side of the face and the upper lip arrow shows larvae coming out from the nose number of maggots removed from this area collected larvae from the lesion each maggot was measured myiasis is an uncommon disease in humans and common in rural areas compared to urban areas . The most common sites are the nose, eye, ear, anus, vagina, and rarely, the oral cavity . Predisposing factors are extraction wounds, poor oral hygiene, psychiatric patients, mouth breathing during sleep, facial trauma, open neglected wounds, necrotic tissues, suppurative lesions, severe halitosis, senility, cerebral palsy, mental retardation, hemiplegia and factors that favor persistent non - closure of the mouth. [121416] primary oral myiasis commonly affects the anterior part of the mouth . The patient reported here was residing in a rural area, with low socioeconomic background . The life cycle of a house fly begins with the egg stage followed by the larva, the pupa, and finally the adult fly . The conditions required for egg laying and survival of the larvae are moisture, necrotic tissue and suitable temperature . The larval stage lasts from 6 to 8 days, in which period they are parasitic to human beings . They are photophobic, and therefore tend to hide deep into the tissues, which also helps them to secure a suitable niche to develop into pupa . In the present case also, the larvae were present deep into the tissue and the nasal floor . Proteolytic enzymes released by the surrounding bacteria decompose the tissue and the larvae feed on this rotten issue . Oral myiasis can also be classified as- 1) larvae living outside the body, 2) larvae burrow into unbroken skin and develop under it . 4) eggs or young larvae are deposited in the wounds or natural cavities in the body . When there are multiple larvae and in advanced stages of development and there is tissue destruction, local application of various agents like turpentine oil, larvicidal drug like negasunt, ethyl chloride, ether, mercuric chloride, creosote, saline, iodoform, chloroform, clove oil, calomel, phenol mixture, olive oil, gentian violet, alcoholic solution in association with tobacco, camphor, sodium hypochlorite can be used for complete removal of all larvae . These agents asphyxiate the aerobic larvae and force them to a superficial position and these larvae are removed with less damage to tissues and larvae as well . The rupture of the larvae might cause allergic or foreign body reaction and secondary infection, so care must be taken not to rupture the maggots . In the present case, maggots were removed completely in 3 days interval by using turpentine oil . Myiasis of orofacial region can be prevented by educating the people from rural areas and low socioeconomic groups about personal hygiene, taking care of any wound, control of fly population and maintenance of sanitation of the surroundings . We, the dentists, must educate parents / guardians to make them aware of the conditions, and the parents should bring children as early as possible for dental intervention to prevent further complications.
Dexamethasone, see scheme 1 for molecular structure, is a synthetic member of the glucocorticoid class of steroid drugs that has anti - inflammatory and immunosuppressant effects . It is more potent than cortisol in its glucocorticoid effect, while having minimal mineralocorticoid effect . Based on the who's list of essential medicines, dexamethasone is of the most important medications needed in a basic health system . It is used to treat many inflammatory and autoimmune conditions, such as rheumatoid arthritis and bronchospasm [2, 3]. In addition, it is given in small amounts before and/or after some forms of dental surgery and useful to counteract allergic anaphylactic shock, if given in high doses . It is present in certain eye drops and as a nasal spray and certain ear drops [4, 5]. With respect to the widespread use of the drug, developing the rapid, low cost, and reliable procedure for quantitation of it in real samples with different matrices is necessary . Various reports have been found for quantitative determination of dexamethasone in real samples with different matrices . They are including spectrophotometry, liquid chromatography [7, 8], liquid chromatography - mass spectrometry [911], and electrochemical methods . The methods have limitations such as high cost and hard operation [711] and low repeatability . Kinetic spectrophotometric method that has advantages such as high sensitivity, sufficient accuracy, simplicity, speed, and the necessity of less expensive apparatus makes it as an attractive method for the determination of trace elements in samples with different matrices such as foods and biological and pharmaceutical [14, 15] samples . In continuing of our research interest for the determination of drugs, a simple the method is based on the catalytic effect of dexamethasone on the orange g - bromate reaction system . The developed method has been successfully applied for the determination of dexamethasone in pharmaceutical and biological samples . To the best of our knowledge, we do not have any report for the determination of dexamethasone using kinetic spectrophotometric method . A single beam agilent uv - vis spectrophotometer (8453, usa) with 1 cm glass cell was used to measure the absorbance . A thermostated water bath (heidolph, germany) was used to keep the temperature of all solutions at the working temperature (25.0 0.1c). Dexamethasone as dexamethasone phosphate (sigma) stock solution 100.0 mg l was prepared just before use by dissolving appropriate amount of dexamethasone phosphate in water and diluted to the mark in a 100 ml calibrated flask . A solution of orange g (6.6 10 mol l) was prepared by dissolving 0.2985 g of orange g (merck) in water and diluting to 1.0 l with water . Sulfuric acid solution (4.0 mol l) was prepared by appropriate dilution of conc . Sulfuric acid (merck). A 0.05 mol l of potassium bromate solution was prepared by dissolving 8.3504 g of kbro3 (merck) in water and diluting to 1.0 ml in a calibrated flask . The catalyzed reaction was studied spectrophotometrically by monitoring the change in absorbance of the reaction mixture at 478.5 nm (max). For this purpose, to a series of 10 ml volumetric flasks, 0.8 ml of 6.6 10 mol l of orange g solution, 1.5 ml of 4.0 mol l sulfuric acid solution, and the standard solutions containing 0.020.54 mg of dexamethasone were added . Then, 1.0 ml of 0.05 mol l of bromate solution was added and diluted to the mark . A time measurement was started just after adding the last drop of the bromate solution . After thorough mixing, a portion of the solution was transferred to a glass cell . The absorbance of catalysed reaction (a s) was measured against water at max and 20c for time interval 30420 s. the measurement in the absence of dexamethasone was repeated to obtain the values for the uncatalysed reaction (a b). Finally, the difference in the absorbance change was considered as the response (a = a s a b). Ear - eye drop (0.1%) and injection solution (8.0 mg) were used as pharmaceutical samples . Each sample was diluted properly and appropriate amount of the solution was used in each analysis . They were spiked with dexamethasone and solid phase extraction technique with c18 cartridge (supelco inc ., 10 ml) was used for purification and preconcentration of dexamethasone from the samples . The purification and preconcentration procedure was performed as discussed in . According to the procedure, orange g, a yellowish synthetic azo - based dye, widely used as color marker for following the electrophoresis process, ph indicator, dyeing of textiles, paper, and leather, and preparing coloring inks . The capability of the reaction system (orange g, sulfuric acid, and bromate) for the determination of dexamethasone was evaluated by following the change in absorbance in absence (figure 1) and presence (inset of figure 1) of it . Comparison of the two spectra indicated that trace amounts of dexamethasone can increase the reaction rate seriously . Therefore, the proposed reaction system can be used for the determination of dexamethasone . The suggested reaction mechanism for proposed reaction system the uncatalysed reactions that resulted in blank signal (a b) was carry out in a cyclic way by these reactions: (1)orange gred+bro3+6h+orange gox + br+3h2o (2)6bro3+10h++12br6br2 + 6h2o (3)orange g(red)+br2+h+orange gox+2br in the presence of dexamethasone (4)dexamethasonered+br2+h+2br+dexamethasone(ox) where red and ox are the reduced and oxidized forms of the reactant, respectively . Since dexamethasone along with the orange g participates on br generation, the possibility of br2 generation was increased (reaction (2)) and resulted in increasing the possibility of decolorization of orange g (reaction (3)). Therefore, the change in absorbance in presence of dexamethasone was increased dramatically . In order to obtain the maximum sensitivity as employing the proposed procedure, the effect of orange g concentration on the rate of reaction was studied over the range 52.879.2 mol l. as it can be seen in figure 2, the sensitivity was increased up to 66.0 mol l of orange g. at higher concentrations, the reaction rate was decreased which may be attributed to the dye aggregation . Thus, 66.0 mol l of orange g as optimum concentration was selected for further study . The effect of sulfuric acid concentration on the catalyzed and uncatalyzed reactions was studied over the range of 0.6 to 0.8 mmol l (figure 3). The maximum sensitivity was obtained at 0.76 mmol l, whereas at higher concentrations the sensitivity was decreased . Protonation of orange g at higher acid concentrations that makes its oxidaion to be quite difficult resulted to the disorder . Therefore, 0.76 mol l of sulfuric acid was used for further study . The effect of bromate concentration on the reaction rate was studied in concentration range 4.55.5 mmol l. as shown in figure 4, the net reaction rate was increased up to 5.0 mmol l which was selected as being the optimum concentration of oxidant . Under optimum experimental conditions, the effect of the temperature on the reaction rate was studied in the range of 15 to 45c . Increasing the temperature up to 20c caused an increase in the sensitivity, whereas at higher temperatures it decreased . The optimum time was found by measuring the change in the absorbance during 30540 s. the reaction rate increased up to 420 s, and in longer times the reaction rate was almost constant . Calibration graph was constructed by plotting the response against dexamethasone concentration . Using the recommended procedure and under optimized conditions that are outlined above, calibration graph was linear over the range 0.254.0 mg l of dexamethasone including two segments of 0.212.0 and 12.054.0 mg l. the regression equation of the two segments gives (5) and (6), respectively . (5)a=0.0154dexamethasone+0.0071 r2=0.9987, (6)a=0.0032dexamethasone+0.1574 r2=0.9984, where a is the difference in the absorbance between the blank and the sample and [dexamethasone] is the dexamethasone concentration in mg l and r is the correlation coefficient . The limit of detection (3s b / m; s b is the standard deviation of the blank signal and m is the slope of calibration curve) was 0.14 mg the tolerance limit was defined as the concentration of the added species causing an error more than 5% on analytical signal . The obtained results show that chloride and nitrite have seriously interfering effect, whereas they do not exist in real sample matrix . Also, the interfering effect of cortisol that has the same effect on dexamethasone and can be coexisting with the analyte in real samples matrices was investigated . The tolerance limit of 8.3 fold than 2.0 mg l of dexamethasone which was not more than the upper limit of cortisol in blood confirms the proposed method is free from the cortisol interfering effect . Evaluating the reliability and analytical applicability of the developed method makes it potentially useful for the quantitative determination of dexamethasone in real samples with different matrices . The experimental t values for eye - ear drop (1.72 and 1.73) and injection solution (1.74 and 0.88) are different from the critical value (4.30, 95% confidence level, and two degrees of freedom). Regarding the difference between the experimental and critical t values, the systematic error for the determination of dexamethasone in pharmaceutical samples using the developed method is negligible . Also, the procedure was used for the determination of dexamethasone in urine and serum samples . After sample preparation (section 2.4.2) they were spiked with different amounts (2.0, 8.0, and 20.0 mg the recoveries of the spiked urine and serum samples vary over the range 99.4102.0% and 99.0100.7%, respectively . The recovery values are near 100% and confirm that the systematic error during the quantitative determination of dexamethasone in biological samples is slight . Successive applications of developed method for drug determination in pharmaceutical preparations and urine samples were performed . Therefore, the developed method is free from interfering effect of matrix effect and suitable for analysis of dexamethasone in different samples . This study reports a sensitive and relatively selective spectrophotometric method for the determination of dexamethasone using a new reaction system . The developed method possesses distinct advantages over other existing methods in cost, simplicity, ease of operation, and applicability to real samples analysis . Also, the reliability of the method permits the analysis of pharmaceutical and biological samples satisfactorily.
Diabetes mellitus type 2 is strictly related to cad (coronary artery disease); in fact 7080% of diabetic patients die of cardiovascular complications; moreover, these patients have a risk of myocardial infarction about four times higher than that found in the general population [13]. Several noninvasive techniques are available for this purpose, including stress ecg and spect (single photon emission computed tomography) [4, 5] and most recently coronary ct . In fact the latest ct generation with 64 slices has emerged as a truthful alternative to conventional ca (coronary angiography), with excellent diagnostic accuracy allowing us to identify and quantify the degree and extent of coronary artery disease, including the study of wall arteries [69]. Previously published results have proved the high values of sensitivity, specificity, and negative predictive value (almost 100%) of ct for the assessment of coronary disease, even in patients treated with stent and bypass [10, 11]. However, in case of important coronary calcifications, the ct examination presents several limitations in residual vessel lumen evaluation . Otherwise, mdct (multidetector computed tomography) sometimes shows some limitations in the grading of coronary stenosis due to motion artefacts or severe vessel calcifications . It is well known that diabetes causes a large amount of vessel calcification, resulting in lower diagnostic accuracy of ct in the detection and evaluation of coronary stenosis in diabetic patients . In this case, it could be useful to work with hybrid imaging, merging the anatomical images of ct to the functional images of spect, overcoming the limits of the two techniques . Our purpose was to combine the results of the mdct morphological data and the spect data using hybrid imaging to overcome the limits of the mdct in the evaluation of coronary stenosis in diabetic patients with large amount of calcium in the coronary arteries . Between january 2009 and december 2011, we enrolled 120 consecutive diabetic patients (84 males and 36 females), mean age 67 years old (range 5078), and performed a coronary ct . All the patients had one or more cardiac symptoms such as stable angina, atypical chest pain, and dyspnea . We evaluated the cardiovascular risk factors for each patient, looking for hyperglycaemia (glycated haemoglobin> 7.5%), hypertension (bp> 130/85 mmhg), hypercholesterolemia (cholesterol> 190 mg / dl), obesity (bmi 30 kg / cm), smoking, and family history of cad (table 1). Twenty patients with a history of known cad (1 with previous bypass graft surgery, 19 with previous successful angioplasty) were included, while patients with a serious arrhythmia, known allergy to iodinated contrast agents and kidney failure, were excluded . Invasive coronary angiography was performed in patients in which the ct examination results were doubtful . Afterwards the imaging fusion results were compared to the coronary angiography evaluation . All patients, provided informed consent to the examinations and the study was approved by the local ethics committee . We used a 64-slice ct scanner (lightspeed vct, general electric medical systems, milwaukee, wi, usa) and a retrospective synchronization technique . Patients with a heart rate higher than 65 bpm were previously (almost 5 days before) treated with oral beta - blocker therapy . A preliminary unenhanced scan was done to determine the scan extent and to calculate the calcium score (smartscore protocol). The acquisition stack extended from the ascending aorta superiorly (approximately 1 cm above the tracheal bifurcation) and the heart apex inferiorly (lower portion of the lowest hemidiaphragm), therefore enabling the evaluation of the entire cardiac volume . Scan parameters for the unenhanced baseline scan were beam collimation 8 2.5 mm, slice thickness 2.5 mm, table feed 1 cm/4 slices, tube rotation speed 0.35 s, tube voltage 120 kv, intensity 300 ma, fov 25 cm, craniocaudal scan direction . A second image stack was then acquired after intravenous administration of iodinated contrast material using a dual - head automated injector (stellant, medrad, pittsburgh, pa, usa). A dose of 80 ml of nonionic iodinated contrast material (iomeron 400, bracco, milan, italy) was administered through an 18-gauge needle cannula placed in an antecubital vein, followed by 40 ml of saline solution, both at a rate of 5 ml / s . To synchronize the beginning of the scan with the arrival of the contrast agent in the coronary arteries, the bolus - tracking technique was used . Parameters for the contrast - enhanced scan were beam collimation 64 0.625 mm, slice thickness 0.625 mm, reconstruction increment 0.625 mm, table feed 2.9 mm / rotation, tube rotation 0.35 s, tube voltage 120 kv, intensity 400800 ma, fov 25 cm, craniocaudal scan direction . Image reconstruction was carried out using three temporal windows at 70%, 75%, and 80% of the cardiac cycle, corresponding to the r - r interval or mid to end diastole . In the event of motion artefacts due to sudden changes in heart rate, other reconstruction windows were used (from 40% to 65% of the r - r cycle). The ct datasets were analysed by the agreement of two independent, experienced readers, who used axial source images, multiplanar reformations, volume rendering, and thin - slab maximum - intensity projections on a remote workstation (advantage workstation 4.4; ge healthcare). Each coronary was judged as negative for coronary disease in the presence of one stenosis <50% and positive in the presence of one stenosis 50% [1416]. Gated - spect was performed on a double - headed camera system (spect - tc vg millennium, ge healthcare, usa). We adopted the single day protocol: stress - rest protocol with 300 mbq tc - mibi or tetrofosmin at peak exercise during bicycle ergometry or dipyridamole infusion and 900 mbq at rest (n 203, 77.2%) after 3 hours . Spect images were acquired 1 hour after the radiopharmaceutical injection both for the rest and the stress phases . Thirty - two images of 25 sec per frame (matrix 64 64, zoom 1.33) were acquired using the step and shoot technique (90 g / head). Energy discrimination was provided by a 20% window centred over the 140 kev photon peak of tc . Transverse images were reconstructed by the filtered back projection method, with a butterworth filter (order 10; cutoff 4.0) for processing and a ramp filter for back projection using a xeleris console . The left ventricular myocardium was divided up into 17 segments; each of the 17 segments was scored according to the guideline for semiquantitative analysis (semiquantitative scoring system: the five point model: 0 = normal; 1 = mildly reduced - not definitely abnormal; 2 = moderate reduced - definitely abnormal; 3 = severe reduced; 4 = absent radiotracer distribution). The fusion between stress - spect images and ct axial images was done using the cardiac iq fusion protocol on a remote workstation (advantage workstation 4.4; ge healthcare). This software allows a perfect matching between the images using as reference points the ct myocardium outlines and the spect epicardial surface of the left ventricle; furthermore this software cuts the aorta and the veins . Several 3d protocols permit a better visualization of the coronaries, the wall septum, and the right ventricle . The result of the fusion is an image with a high spatial resolution and a better definition of the coronary stenosis . These hybrid images have been evaluated separately by a radiologist and a nuclear medicine doctor with eventual agreement . Cca was performed using a philips flat - panel system with multiple projections (medical philips system, the netherlands). Stenoses were evaluated as a percentage of the reference diameter determined in two orthogonal projections, taking the mean of the two samples as the final value . Data were expressed as means plus one standard deviation (sd) or as percentages . Diagnostic accuracy for evaluating significant stenoses was calculated by comparing spect / tc with cca images . The comparison between groups was obtained by analysis of variance or the test, as appropriate . Mean heart rate of the patients during the examination was 65 bpm (range 5677 bpm). After the ct scans, 12 (10%) patients were excluded because of motion artefacts due to an increase in heart rate during the acquisition . The quality of the images of the other 108 (90%) patients was excellent in 76 patients (70.2%), good in 16 patients (15%), sufficient in 8 patients (7.4%), and low in 8 patients (7.4%). Considering the three main coronary branches in the overall count, left anterior descending (lad) artery, right coronary artery (rca), and left circumflex artery (lcx), 324 coronaries were evaluated . A total of 52 vessels (32.8%) had stenoses> 50%, in particular 28 (8.6%) were in the lad, 16 (4.9%) in the rca, and 8 (2.4%) in lcx; 228 coronaries (70.4%) were found to have nonsignificant stenoses (<50%) (table 2). We observed that 44 coronaries (13.6%) were not evaluable or doubtful due to the presence of a large amount of vessel calcification (table 2). The mean calcium score value in not evaluable or doubtful coronaries was 823 36 . Considering the 324 areas corresponding to the ct coronary arteries in particular, 44 (13.6%) perfusion defects were located in the anterior and septal wall, 28 (8.6%) in the inferior wall, and 8 (2.5%) in the lateral wall . Furthermore, gated - spect showed 232 (71.6%) areas without perfusion defects and 12 (3.7%) doubtful cases because of artefacts due to mammary gland attenuation in the female patients (table 2). The 52 (16%) coronary arteries with significant stenosis at ct were confirmed as perfusion defects at spect . Of 228 coronaries (70.4%) found to have nonsignificant stenosis at ct, 212 (65.4%) were confirmed as normal perfusion areas while 16 (4.9%) were described as perfusion defects at spect . Of 44 (13.6%) nonevaluable coronaries at ct, 12 (3.7%) were described as perfusion defects at spect, 20 (6.2%) as normal perfusion areas, and 12 (3.7%) as doubtful cases because of artefacts due to mammary gland attenuation in the female patients (table 3). Of 324 coronary arteries and corresponding areas, the hybrid spect / ct evaluation showed 92 (28.4%) of areas with hypoperfusion and 232 (71.6%) with normal perfusion (table 2). In the first group of 52 (16%) coronaries with significant stenosis at ct and perfusion defect at spect, hybrid images demonstrated a hypoperfusion (figure 1). In the second group of 228 (70.4%) with nonsignificant stenosis at ct, hybrid images demonstrated a normal perfusion in 212 (65.4%) cases and a low hypoperfusion in 16 (4.9%) cases (figure 2); of these 16 cases, 12 (3.7%) cases were described at ct as stenosis of 4050%, while the other 4 cases were seen at ct as nontransmural myocardial necrosis (with known history of non - st infarction). In the third group of 44 (13.6%) doubtful coronaries at ct, hybrid images demonstrated 20 (6.2%) normal perfusion areas and 24 (7.4%) hypoperfusion areas (seen at spect as 12 perfusion defects and 12 doubtful cases), described as nonquantifiable calcified stenosis at ct and were finally evaluated as significant stenosis (table 3). The third group of 44 coronaries was also studied with a conventional ca, confirming the results of hybrid images (24 significant stenoses and 20 nonsignificant stenoses). The patients with 24 significant stenoses also underwent a ptca (percutaneous transluminal coronary angioplasty) with stenting . Therefore the evaluation of hybrid images permitted the identification of all the stenoses and solved the doubts related to the 44 coronaries not well evaluated at ct and spect separately, with a diagnostic accuracy of 100% in comparison versus ca (table 3). Myocardial perfusion scintigraphy represents the most widely used technique validated in the prognostic stratification of diabetic patients with known or suspected ischemic heart disease in order to predict the short and medium terms (generally 1 - 2 years) for cardiac events such death and myocardial infarction [18, 19]. However, the perfusion images obtained with spect show a good sensitivity and a low specificity in the detection of patients with cad . The diagnostic accuracy of myocardial scintigraphy is affected by different variables . Unlike positron emission tomography (pet), single - photon emission computed tomography (spect) provides no correction for attenuation . This causes the presence of false positives in some areas (false hypoperfusion obtained from attenuation of photons through the tissues of the body), along the passage from the heart to the gamma camera . Mostly the artefacts are related to the hepatic uptake, due to the interposition of the diaphragm and, in female patients, in the anterior - lateral wall due to the mammary gland; because of this reason, in our study, spect was unable to assess the myocardial perfusion in 12 female patients . Furthermore spect reported some false positives when a patient cannot reach 85% of the theoretical maximum heart rate during the exercise testing; this event occurs in obese patients, in older patients, in patients with arterial disease of the lower limbs, and in subjects treated with beta - blockers . In these cases because of these reasons, there is a high variability of values of sensitivity and specificity reported in previously published meta - analysis [1821]. Specificity and sensitivity have been improved by the introduction of gated technique: especially in women, the number of false positives can be reduced, observing at the same time the perfusion and the functionality of the myocardial region . However, sometimes the myocardial region that seems to have a normal perfusion after a stress test could hide a perfusion defect and would not be able to determine a myocardial stunning [21, 22]. Coronary ct, when used in selected populations of patients with low - intermediate cardiovascular risk, has been shown to provide essential morphological information about the coronary tree . The diagnostic accuracy of cardiac ct is widely demonstrated by high values of sensitivity, specificity, and negative predictive value [23, 24]. Actually, the most important limit in the evaluation of the vessel lumen is the presence of large coronary artery calcification, causing a blooming type artefact, due to the hardening of the x - ray beam during the passage through the calcified plaques . This artefact could cause a significant invalidation of lumen visualization determining false positive in coronary ct and consequently sending a certain number of patients with stenosis <50% to undergo a ca . In our study the patients, being diabetic, had large calcifications of the coronary arteries, creating doubts in the interpretation of quantitative coronary stenosis in 44 coronary arteries . During the postprocessing phase, the use of some convolution filters (kernel) can decrease the blooming artefact; in particular applying the high - resolution filters (bone and detail), the relative density of calcium is reduced, the margins of calcification plaque are better delineated, and the width of the vascular lumen increases . However, in a large number of cases, the quantification of stenosis caused by calcified plaque remains still difficult and it is often necessary to send the patient to ca examination or a spect . Part of medical literature has tried to define the agatston values above which performing ct would be inappropriate . However, although sensitivity and specificity are reduced, the overall diagnostic accuracy is not reduced and, in our study, it has been implemented with the fusion imaging ct / spect . The skillful use of hybrid images, obtained by merging ct and spect images, is to have both morphological and functional data with a high spatial resolution, allowing a better stratification of diabetic patients, identifying the patient with a significant stenosis and hypoperfusion who needs revascularization and avoiding a ca in patients with unquantifiable calcification plaques at ct and spect [26, 27]. The hybrid images can overcome the limits of coronary ct, permitting the evaluation of large vessel calcification, metal stents, and small size vessels . However, this problem usually does not occur in the evaluation of noncalcified native coronary arteries; in fact ong et al . (2006) demonstrated that in patients with calcium score <142 agatston score the interpretability of the coronary tree was 93.6% against 86.9% of those patients with values> 142 agatston score . In our experience, hybrid spect / ct images provide help in the evaluation and grading of calcified stenoses in ct and overcome the problem of the abnormal uptake of the radiopharmaceutical by the breast tissue in spect . In particular in the group of 44 (13.6%) doubtful coronaries at ct, hybrid spect / ct images demonstrated 20 (6.2%) normal perfusion areas and 24 (7.4%) hypoperfusion areas (seen at spect as 12 perfusion defects and 12 doubtful cases because of breast tissue), described as unquantifiable calcified stenosis at ct and finally evaluated as significant stenosis . Therefore the evaluation of hybrid images permitted the identification of all the stenoses and solved the doubts related to the 44 coronaries not well evaluated at ct and spect separately, with a diagnostic accuracy of 100% in comparison to ca . These results are extremely interesting when applied to a group of patients such as diabetics, in which, until now, the limit of the coronary ct examination in the evaluation of calcified coronary was high . On the other hand, the correct identification of an area of myocardial hypoperfusion at spect examination is enhanced by the morphological information of the coronary arteries with a significant decrease in the number of false positive and an increase of the prognostic value of scintigraphy . In fact, spect provides a high prognostic accuracy in the prediction of cardiac event (especially in the short - term followup), although it gives only functional information . The perfusion defects can be localized and assigned to a specific coronary artery only if the spect exam is complemented by a morphological assessment . According to medical literature, hybrid imaging adds diagnostic clinical value, improving patient risk stratification and showing a higher sensitivity and specificity in the detection of the haemodynamically relevant coronary artery stenoses compared to the side - by - side analysis [2931]. Furthermore the improvement of diagnostic accuracy was obtained with the latest ct scanners, as high - pitch dual - source ct coronary angiography and 320-row cardiac ct, which allow to improve the imaging of coronary plaque and obtain the evaluation of myocardial viability with a high diagnostic accuracy, a reduced radiation exposure and amount of contrast agent, even in patients with heart rates of> 80 bpm or atrial fibrillation [3235]. Our study has the limit of a high exposure of radiation because the patients underwent two different examinations (ct and spect) and because we used a retrospective gating during the ct scan; nevertheless, the actual use of the prospective gating and the application of new advanced reconstruction techniques that reduce image noise and improve low contrast detectability and image quality can give higher diagnostic performance of ct scan at very significant lower dose [36, 37]. Another limitation of the present study was the only anatomical and not functional evaluation of coronary stenoses . Therefore, the usefulness of the fusion between ct and spect images is to overcome the limits of ct and the limits of spect with a high diagnostic accuracy, especially in patients with high cardiovascular risk and high presence of physical limitations, like the diabetic patients.
It is estimated that over 70% of adults have at least one episode of low back pain during their lifetimes.2 prevalence is higher in young, economically active adults in south american populations;3 indeed, low back pain is the second most common reason for absenteeism from work, and one of the most common reasons for medical consultation.4 one important risk factor for low back pain is weakness of superficial trunk and abdominal muscles,5 - 9 and strengthening of these muscles is often associated with significant improvements of clbp, as well as with decreased functional disability.10 - 15 another independent risk factor for clbp is the weakness and lack of motor control of deep trunk muscles, such as the lumbar multifidus (lm) and transversus abdominis (tra) muscles.16,17 ferreira et al.18 and hodges et al.19 demonstrated that the tra had insufficient control and speed of muscle contraction delayed in individuals with clbp . Kinesiotherapeutic protocols addressing both the superficial and the deep muscles seem to be effective in the treatment of clbp.20 - 22 most clinical protocols combine different exercises and techniques, making it difficult to isolate the efficacy of specific strategies.10,11 this is of great clinical importance and needs to be further clarified through research . Therefore, in this study, we compared the efficacy of ss exercises with strengthening of abdominal and trunk muscles (st) on pain, functional capacity, and tra activation capacity in individuals with clbp . Our hypothesis was that the lumbar stabilization would be more efficient than the muscle strength in the improvement of chronic low back pain . Our sample was selected from a list of patients being seen at the department of orthopedics, university hospital, sao paulo university . Therefore, the sample consisted of 30 patients (four men and 11 women in each group) with nonspecific clbp . They were randomized by means of opaque envelopes to one of two treatment groups: ss and st . Inclusion criteria were: low back pain for more than 3 months (pain felt between t12 and the gluteal fold), patients willing and able to participate in an exercise program safely and without cognitive impairments that would limit their participation . Exclusion criteria were history of back surgery, rheumatologic disorders, spine infections and spine exercise training in the 3 months before the onset of the study . This study was approved by the ethics committee of the university hospital (protocol 700/06) and of the school of medicine (protocol 1249/06), university of so paulo . Participants were assessed at baseline and at the end of the treatment by an investigator (physiotherapist) who was blinded to the randomization, the severity of pain, functional disability, and tra activation capacity . Pain was assessed using a visual analogical scale (vas) and the mcgill pain questionnaire.23 the vas consists of a 10cm line, with the left extremity indicating no pain and the right extremity indicating unbearable pain . Participants were asked to use the scale to indicate their current level of pain . The mcgill pain questionnaire consists of a list of 78 pain descriptors organized into 4 major classes (sensory, affective, evaluative, and miscellaneous) and 20 subclasses, each made up of at least 2 and at most 6 words, to which are assigned intensity values . Functional disability was estimated by the oswestry disability questionnaire,24 a functional scale assessing the impact of low back pain on daily activities . Other questionnaires are available for the measurement of the evaluation of lowback pain, but mcgill and oswestry were considered the most appropriate in the context of this project.26,27 the score is calculated by the addition of the values assigned for each of the 10 individual questions and is used to categorize disability as: mild or no disability (020%); moderate disability (21%40%); severe disability (41% to 60%); incapacity (61% to 80%); restricted to bed (81% to 100%). Tra activation capacity was assessed by using the stabilizer pressure biofeedback unit (pbu, chattanooga group, australia). The pbu consists of a combined gauge / inflation bulb connected to a pressure cell . It is a simple device that registers changing pressure in an airfilled pressure cell allowing body movement, especially spinal movement, to be detected during exercise . The pressure cell measures from 0200 mmhg, with a precision of 2 mmhg . Changes in body position modify the pressure, and they are registered by the sphygmomanometer.25 the device was placed on the tra (above the anterior superior iliac spines) while participants were in ventral decubitus over a rigid surface . The depression of the abdominal muscles over the spinal cord typically decreases the pressure by 410 mmhg.20 before individuals were asked to contract the muscle, the device was inflated to a pressure of 70 mmhg . The participants were instructed to draw the lower stomach gently off the pressure sensor without moving the back or the hips and to sustain it for 10 seconds, measured by a stop watch . Interventions were conducted over 6 weeks, twice per week, each session lasting 30 minutes . Sessions were supervised by the investigator, and participants were instructed to report any adverse event, whether it was related to the exercises or not . Groups were instructed not to participate in any other physical program during the study and not to exercise while at home . In the segmental stabilization (ss) group, exercises focused on the tra and lm muscles according to the protocol proposed by richardson et al.17,28 in the superficial strengthening (st) group, exercises focused on the rectus abdominis (ra), abdominus obliquus internus (oi), abdominus obliquus externus (oe), and erector spinae (es).29 three series of 15 repetitions were done for each exercise (table 1). Sample size was calculated assuming a power of 80% to detect a 30% improvement in pain (vas), with a standard deviation of 2 points and a significance level of 5% . The relative gain with treatment was calculated with the following equation: anova one way was used for intergroup and intragroup comparisons . For tra activation, the binomial our sample was selected from a list of patients being seen at the department of orthopedics, university hospital, sao paulo university . Therefore, the sample consisted of 30 patients (four men and 11 women in each group) with nonspecific clbp . They were randomized by means of opaque envelopes to one of two treatment groups: ss and st . Inclusion criteria were: low back pain for more than 3 months (pain felt between t12 and the gluteal fold), patients willing and able to participate in an exercise program safely and without cognitive impairments that would limit their participation . Exclusion criteria were history of back surgery, rheumatologic disorders, spine infections and spine exercise training in the 3 months before the onset of the study . This study was approved by the ethics committee of the university hospital (protocol 700/06) and of the school of medicine (protocol 1249/06), university of so paulo . Participants were assessed at baseline and at the end of the treatment by an investigator (physiotherapist) who was blinded to the randomization, the severity of pain, functional disability, and tra activation capacity . Pain was assessed using a visual analogical scale (vas) and the mcgill pain questionnaire.23 the vas consists of a 10cm line, with the left extremity indicating no pain and the right extremity indicating unbearable pain . The mcgill pain questionnaire consists of a list of 78 pain descriptors organized into 4 major classes (sensory, affective, evaluative, and miscellaneous) and 20 subclasses, each made up of at least 2 and at most 6 words, to which are assigned intensity values . Functional disability was estimated by the oswestry disability questionnaire,24 a functional scale assessing the impact of low back pain on daily activities . Other questionnaires are available for the measurement of the evaluation of lowback pain, but mcgill and oswestry were considered the most appropriate in the context of this project.26,27 the score is calculated by the addition of the values assigned for each of the 10 individual questions and is used to categorize disability as: mild or no disability (020%); moderate disability (21%40%); severe disability (41% to 60%); incapacity (61% to 80%); restricted to bed (81% to 100%). Tra activation capacity was assessed by using the stabilizer pressure biofeedback unit (pbu, chattanooga group, australia). The pbu consists of a combined gauge / inflation bulb connected to a pressure cell . It is a simple device that registers changing pressure in an airfilled pressure cell allowing body movement, especially spinal movement, to be detected during exercise . The pressure cell measures from 0200 mmhg, with a precision of 2 mmhg . Changes in body position modify the pressure, and they are registered by the sphygmomanometer.25 the device was placed on the tra (above the anterior superior iliac spines) while participants were in ventral decubitus over a rigid surface . The depression of the abdominal muscles over the spinal cord typically decreases the pressure by 410 mmhg.20 before individuals were asked to contract the muscle, the device was inflated to a pressure of 70 mmhg . The participants were instructed to draw the lower stomach gently off the pressure sensor without moving the back or the hips and to sustain it for 10 seconds, measured by a stop watch . Interventions were conducted over 6 weeks, twice per week, each session lasting 30 minutes . Sessions were supervised by the investigator, and participants were instructed to report any adverse event, whether it was related to the exercises or not . Groups were instructed not to participate in any other physical program during the study and not to exercise while at home . In the segmental stabilization (ss) group, exercises focused on the tra and lm muscles according to the protocol proposed by richardson et al.17,28 in the superficial strengthening (st) group, exercises focused on the rectus abdominis (ra), abdominus obliquus internus (oi), abdominus obliquus externus (oe), and erector spinae (es).29 three series of 15 repetitions were done for each exercise (table 1). Sample size was calculated assuming a power of 80% to detect a 30% improvement in pain (vas), with a standard deviation of 2 points and a significance level of 5% . The relative gain with treatment was calculated with the following equation: anova one way was used for intergroup and intragroup comparisons . For tra activation, no significant differences were seen for age, weight, height, and body mass index . All variables significantly improved with treatment (p<0.001), with the exception of tra contraction (p = 0.99). The ss group yielded significantly higher gains in all variables when compared to the st group (p<0.001). No significant differences were seen for age, weight, height, and body mass index . All variables significantly improved with treatment (p<0.001), with the exception of tra contraction (p = 0.99). The ss group yielded significantly higher gains in all variables when compared to the st group (p<0.001). The aim of this study was to compare the efficacy of ss and st exercises in the relief of clbp symptoms . Both treatments were effective in relieving pain and in decreasing functional impairment, but only the ss treatment improved tra muscle activation . The pbu test has been validated by imaging19 and electromyography, 17 tests that are considered to be the goldstandard measurements of tra performance . These tests demonstrated that individuals with low back pain have an impaired ability to depress the abdominal wall . Hides et al.30 suggested that the tra is important in sustaining the spinal cord and that its conditioning is accompanied by functional improvement . The ss group exercised the tra and lm muscles . On average, participants had optimal depression of the abdominal wall, as measured by the pbu, with a gain of 48.3% with the program, findings that are similar to those obtained by cairms et al.31 ferreira et al.18 and teyhen et al.32 also suggested that tra exercising improves muscle activation in individuals with low back pain . According to jull and richardson 30 and richardson et al.,31 normal pbu responses range from 4 to 10 mmhg; for hodges et al.,19 mean normal values were around 5,82 mmhg . For pain, the ss technique yielded impressive improvements (99% when measured by the vas and 90% by the mcgill questionnaire); functional disability improved by 90% . Our findings are supported by other studies,21,22 where ss translated into pain and functional capacity improvements . The better results of the ss group may be explained by the fact that this technique addressed two muscles primarily affected by low back pain . Hides16 identified selective atrophy of the lm after the first episode of back pain; the atrophy was unlikely to revert without specific training, and the lower muscular stability predisposed an individual to further episodes of low back pain . In individuals with low back pain, the tra has decreased anticipatory capacity, meaning that it has reduced segmental protective function.19 richardson et al.17 suggested that both muscles are primary stabilizers of the lumbar segment, minimizing compressive forces on spinal structures . The st group performed exercises that aimed to strengthen the superficial muscles of the abdomen and trunk . The regimen yielded significant pain and functional capacity improvements, which were also demonstrated by other studies.10,11 rodacki et al.33 suggested that abdominal exercises are associated with low back pain improvement, since during abdominal contraction the pressure on the intervertebral disks was decreased as a consequence of the increased intraabdominal pressure . Cairms et al.31 found that individuals with a history of but no current low back pain had impairments in tra contraction . The better improvement in all variables yielded by the ss relative to st may be explained by the hierarchical structure of the muscular control system . According to bergmark,34 two systems the local system is formed by the deep muscles directly involved with the joints, and their primary function is stabilizing the segments, avoiding articular micromovements . These muscles do not normally perform joint movements, which makes contracting them more difficult, and this is exacerbated by pain . The second system is formed by the superficial muscles, which secondarily stabilize the spinal cord, further minimizing compressive forces . The main function of this system is to generate and control axial movements, and it makes little contribution to segmental stability . Hides et al.16 found that even after pain remission in patients with low back pain, proper deep muscle reestablishment often did not happen and that specific physical therapy focusing on those muscles was necessary . Snijders et al.35 postulated that the cocontraction of the tra and lm muscles is the basis of the lumbosacral biomechanic stability and that these muscles act by reducing the compressive overloads, attenuating or eradicating pain perception . According to norris,36 the rectus abdominis is the most important trunk flexor, while the obliquus muscles support flexion but also rotation, and lateral inclination as well as providing secondary stability during exercise.37 our findings suggest that both protocols are of clinical utility in the improvement of chronic low back pain . Segmental stabilization and strengthening exercises effectively reduce pain and functional disability in individuals with chronic low back pain . Improvement in all variables was superior in the segmental stabilization group opposed to the strengthening group . Limitations of the study were that there were no intermediate and longterm follow up examinations.
African oil bean (pentaclethra macrophylla benth) is a member of the family leguminosae . It is popular in nigeria where it is known by several names such as apara in yoruba, ukana in efik, and, the most prominent, ugba / ukpaka in igbo . Ugba is a popular igbo condiment and delicacy made from traditional household solid state fermentation of african oil bean seeds . The fermentation is a mixed culture alkaline process involving a variety of microorganisms [1, 2]. Although a variety of microorganisms are involved in the process, only the bacillus spp . The methods of production and also the length of fermentation vary from one producer to another and with the final intended use resulting in nonuniform products . However, the basic method involves boiling the seeds for up to 12 hours, removing and slicing the cotyledons, soaking / washing the sliced cotyledons in several changes of water, and wrapping the sliced cotyledons for the fermentation to take place . Beans that have been fermented for 2 - 3 days are taken as a snack delicacy directly or following seasoning and some further processing . Well fermented beans (up to 5 or more days) are added to soup as flavouring . Prepared in different ways, ugba is an essential food item for various traditional ceremonies, and in instances it may be used as meat substitute in certain soups / gravies particularly for the rural poor . Flavour is considered the quality index of ugba and plays an important role in consumer acceptability . Although fermented african oil bean seeds have typical flavour and appealing organoleptic quality, differences in flavour range and intensities exist . These vary perhaps as attributes of producer organisms and are due to the various volatile compounds produced by the fermenting population . A great deal of work has been implemented in the microbiological characterisation of the fermentation process . However, unlike asian and pacific fermented seasoning, no work has yet been reported, to our knowledge, on the flavour components of ugba and their evolution during the fermentation process . In fact, this knowledge gap is not restricted to ugba but appears to cut across the entire gamut of african fermented foods, particularly of the genre of seasoning agents . Knowledge of the volatile and flavour characteristics of the fermented oil bean seeds and their evolution will aid in the quality control of the process, modification of the flavour, and eventual synthesis of the ugba flavour products for possible use in the seasoning of various factory processed and even home foods . This is important in view of declining production of seeds, due to deforestation, and the need to extend use of the appealing aroma of ugba to other food types . The aim of this study was to identify the volatile flavour components of african oil bean seeds during natural and pure culture fermentation and to relate these to the fermentation progress . African oil bean seeds were purchased from dealers in a local market in nsukka, enugu state . The seeds were first boiled for 68 hrs to soften the coat and enhance peeling . The cotyledons were then sliced longitudinally into 4.5 cm 0.1 - 0.2 cm slices, washed and boiled in water for 1 - 2 hrs, and drained and soaked in water for 1012 hrs . After soaking, the slices were washed in several changes of water to reduce bitterness and allowed to drain in a basket . The slices were then wrapped in clean dry banana leaves (musa sapientum linn .) In 4050 g wraps . The wraps were allowed to ferment for 3 days at room temperature (28 2c) to produce ugba . One kilogram (1 kg) of african oil bean seeds was boiled dehulled, peeled, washed, and sliced for the traditional process . The slices were further washed and 20 g portions were placed into conical flasks and capped tightly with cotton wool and aluminium foil . The flasks containing the slices were sterilized by autoclaving at 110c for 10 mins . Pure cultures of b. subtilis and b. megaterium isolated from the traditional process and identified by standard microbiological procedures were inoculated independently in 20 ml sterile 0.1% peptone water and incubated for 24 hrs . After incubation, the cultures were centrifuged and the culture pellets were washed repeatedly with sterile saline and distilled water . 0.1 ml of the cells were emulsified in 9.9 ml distilled water and pour plate method was used to ascertain the population of the cells and to check for culture purity . Each flask was separately, aseptically inoculated with 1 ml suspension (5.0 10 ml) of b. subtilis or b. megaterium individually using a sterile pipette and mixed with the sample by simple manual mixing . Fermentation was allowed to proceed for 72 hrs at room temperature with occasional manual mixing by shaking the flask . To check for purity of the process during the period of fermentation, 1 g of the fermenting slices was removed at 24-hour interval and at the end of fermentation and agitated in 9 ml 0.1% sterile peptone water . Smear of the sample was streaked on nutrient agar plate for isolation of the particular organism acting as a single pure culture . Triplicate pure culture flasks were homogenised and sampled for analysis of volatile compounds . Only pure culture processes were analysed for volatile compounds . Samples for analysis were collected from naturally fermented ugba and also from ugba fermented with pure cultures of b. subtilis and b. megaterium . One gram (1 g) of the fermenting sample was ground and homogenized in 9 ml diethyl ether to extract volatile flavour compounds . 1 filter paper was used to separate the mixture; then millipore filtration using membrane filter of 0.45 m pore size was used to remove particulate matter from the resulting filtrate . The resulting filtrate was placed in small ampoules, sealed, and stored at 30c until being required for analysis . Analyses were done on gc - ms (qp 2010 plus; shimadzu corp, japan) operated in electron - impact (ei) mode with an ionization voltage of 70 ev . Sample aliquot of 1 l was injected and analyses were done with split injection mode . The gc column oven temperature was 60c and injector temperature was 250c with 53.2 ml / min flow and interface temperature of 250c . Oven temperature was held at 60c for 2 mins, ramped to 180c and held for 3 mins, and finally programmed at 280c and held for 5 mins . The ion source temperature was 200c and the solvent cut time was 2.50 mins . Gc chromatograms of the fermenting african oil bean seeds were examined and each peak identified was confirmed by direct comparison of their chromatographic retention times, retention indices, and mass spectra with those of the authentic compounds and/or data in the nist05 mass spectral library . At the end of the traditional fermentation, the produced ugba had a very light brown colour and exhibited classical aroma of the product, with a light, but sharp, ammoniacal smell . There was no visible sign of any slime on the slices which remained firm but slightly softer than the control / starting material . A total of 36 aroma compounds made up of 12 hydrocarbons, 10 esters, 5 alcohols, 2 phenols, 2 ketones, and one of each of furan, amine, acid, thiophene, and lactone were identified at different fermentation periods during natural fermentation (table 1). At the end of the pure culture process, the products of fermentation with two organisms were generally similar in colour, both being very light brown with no visible slime on the slices . The product of the b. subtilis fermentation, however, had only a slightly deeper ammoniacal smell than that of b. megaterium . Both products tasted similar to the product of natural process but were slightly bitterer than the natural process . Similarly, the product of b. megaterium was bitterer than that made with b. subtilis . The extract of oil seed fermented with b. subtilis yielded 30 aroma compounds comprising 10 hydrocarbons, 8 esters, 3 alcohols, 2 sulfur compounds, 2 amines, and one of each of acid, aldehyde, phenol, ketone, and furan (table 2). On the other hand, sample fermented with b. megaterium produced 29 aroma compounds consisting of 9 hydrocarbons, 10 esters, 2 nitrogenous compounds, 2 ketones, 3 alcohols, and one of each of lactone, aldehyde, furan, and amine (table 3). Volatile compounds have been shown to be partly responsible for the aroma of several fermented foods [711]. They are probably important players in the flavour of ugba, as they have been shown to be important in a variety of bacillus spp . Fermented foods . Ugba flavours are formed during fermentation as a result of breakdown of plant compounds as well as formation of microbial metabolites by the active populations responsible for the fermentation process . The ammoniacal flavour observed in ugba is believed to result from the alkaline degradation of protein component of the seed by the fermenting bacillus spp . . Many of the aroma compounds identified in this study derived from the breakdown of lipids (fatty acid), proteins (amino acid), and other bioavailable compounds in ugba through the activities of the microbial enzymes . Hydrocarbons, esters, alcohols, phenols, ketones, furan, and amines were detected in all the samples examined and the consistency of these various volatiles would suggest roles for them in the final flavour of the product, although minor . However, neither the flavour thresholds of these compounds in this product nor their precise individual contributions are yet known . This may explain their abundance as volatile compounds in the fermented african oil bean seeds, since the seeds are rich in oil . Nakamura et al . Suggested that hydrocarbons do not play a significant role as flavour compounds in roasted sesame since they possess a relatively weak aroma . If this be the case in ugba, then the principal flavour compounds may be other compounds coproduced in the fermentation process . It is to be expected, as with all complex products, that the final flavour of ugba will derive from the interaction of a variety of compounds . Methyl esters of fatty acids are one of the largest group of volatile aroma compounds produced during the fermentation in both mixed culture natural fermentation and pure culture processes . These were largely produced during 3648 hr of fermentation and persisted in various forms to the end of the process . They are produced enzymatically during fermentation by the methanolysis of acyl - coa that is formed during fatty acid synthesis or degradation . These compounds have been reported to be responsible for quality sensory properties of various fermented foods . 9-octadecenoic acid, methyl ester; pentanoic acid, methyl ester; 11,14-eicosadienoic acid, methyl ester; and methyl 12-methyl tetradecanoate are methyl esters of fatty acids which occurred commonly and in significant amounts (from the peak areas) in all the samples . Histamine was identified at 0 hr fermentation and cyclopentanamine was identified in the sample fermented with b. subtilis at 36 hr . The presence of these compounds may be attributed to the enzymatic decarboxylation of amino acid in the seeds during fermentation . The presence of nitrogenous compounds, butyl isocyanide and acetonitrile, in the sample fermented with b. megaterium at 12 hr and 48 hr, is indicative of deaminase activity during the fermentation . Unfermented ugba has been reported to contain cyanide and other toxic compounds / antimetabolites as has been reported in a variety of oil seeds . Some volatile sulfur compounds in food are obtained by a variety of enzymatic reactions and/or as products of secondary metabolism of leucine and cysteine . Methane sulfonic anhydride and t - butyl sulfinate, o - methyl, were present at 12 hr and 60 hr, respectively, in the sample fermented with b. subtilis . Considerable amounts of various alcohols were identified during the fermentation in all samples . Of these, many alcohols have been described as possessing undesirable odor which partially contributes to raw or beany a small number of aldehydes, ketones, and acids were identified in this study . It is conceivable that these compounds contribute to the final flavour of fermented african oil bean seeds . Aldehyde can also be obtained as metabolic by - products of the enzymatic transamination or oxidative deamination of amino acid . 2,4-hexadienal and 14-heptadecenal are aldehydes identified in the samples fermented with b. subtilis and b. megaterium at 60 hr of fermentation . Although b. subtilis and b. megaterium are present in natural fermentation process the profile of volatiles differed between pure culture and traditional one . This is understandable, as the mixture of other microorganisms present in the natural process may modulate the variety, quantity, and persistence of different volatile compounds . Phenol, 2,4-bis(dimethylbenzyl) phenol, and 2,4-bis(dimethylbenzyl)-6-t - butylphenol identified during natural fermentation were not detected in pure culture fermentation and may be attributed to the smoked banana leaves used in wrapping the slices prior to fermentation . Although relatively small amounts of aldehyde, ketone, furans, amines, lactones acids, and thiophenes were identified in this study, they are expected to play important roles in the aroma of ugba because of their low detection / aroma thresholds as has been reported in roasted chicory, in the dry scented thai flowers used in tea production and in roasted malaysian almond nuts . A vast array of volatile compounds has been identified in fermented pentaclethra macrophylla seeds (ugba). The variety and quantity of these volatiles change with the time of fermentation and the participating populations . The dynamic nature of evolution of volatile compounds is interesting for modulating the flavour of the final product . It remains to be demonstrated, the relative amounts, flavour thresholds, and specific contribution of the various compounds to the final flavour of the ready to eat fermented seeds . Clearly, a proper understanding of the roles of the various flavour compounds will assist in the modulation of ugba flavour for the various possible uses of this important fermented seasoning and for a possible use in food processing industries and homes as a seasoning.
Recent results from the national institutes of health (nih) frequent hemodialysis network (fhn) daily trial and the following rehabilitation, economics and everyday - dialysis outcome measurements (freedom) study have provided confirmation of the clinical and quality of life (qol) benefits of short daily hemodialysis (sdhd) when compared to conventional thrice weekly in - center hemodialysis (chd). The fhn study was conducted in - center; however, sdhd in the usa is more commonly performed as a home therapy and is not offered as a standard treatment modality in most dialysis clinics . Although it has been nearly 50 years since the early pioneers such as belding scribner and stanley shaldon began dialyzing patients in their homes, peaking at 40% of all us dialysis patients in 1973, the popularity of home hemodialysis (hd) has been limited in the usa for a number of reasons, including lack of suitable equipment and increased cost . The last few years, however, have seen a revival in home hd, resulting in renewed interest in the various forms of more frequent therapy, including sdhd . The revival of home dialysis was led in the mid 1990s by robert uldall, andreas pierratos and others in canada, gradually spreading throughout the usa . This re - emergence has been made possible by recent advances in technology, including portable and more user - friendly machines designed specifically for use in the home environment . In 2005, the fda cleared the first hd machine for home use in the usa, the nxstage system one. Fda clearance was based on an open - label, prospective, two - treatment two - period crossover study on 32 patients to compare sdhd performed in - center (prescribed six times / week) versus sdhd performed at home . The study was conducted at six centers in the usa under an investigational device exemption . This study showed sdhd was delivered as efficiently in the home environment as in - center, with 98.5% of treatments performed successfully in - center versus 97.3% at home . Notably, the composite rate of intra - dialytic and inter - dialytic adverse events was significantly higher during the in - center phase when compared with the home phase (5.3 versus 2.1 adverse events/100 treatments; p = 0.007), suggesting hd therapy is at least as safe at home as in - center . When comparing clinical parameters from the period immediately preceding the study when patients were treated with conventional thrice weekly center hd, home sdhd was associated with reductions in blood pressure, antihypertensive medications and interdialytic weight gain . Since the clearance of the nxstage system one in the usa, the number of patients now performing home dialysis is estimated to be 5000, with the majority of these patients performing sdhd . Although this represents a considerable increase over the past 5 years, it is still only 1% of the total end - stage renal disease (esrd) population . Sdhd offers the promise of improved clinical outcomes in patients with kidney failure, and given the positive results recently released from both the fhn and freedom studies outlined below, sdhd has the potential to become a more viable and popular choice amongst many esrd patients . Results recently published from the fhn daily trial, funded by the nih, showed that in - center sdhd (prescribed six times / week), provided significant benefits in both composite co - primary outcomes of death or 12-month change in left ventricular mass (lvm) and death or 12-month change in the rand-36 physical health composite (phc) score . Specifically, the fhn daily trial showed patients randomized to sdhd reduced their lvm by 16.3 35.3 g (p <0.001) and improved their phc score by 3.3 8.9 points (p = 0.004), after 12 months . However, patients on sdhd were more likely to undergo interventions related to vascular access, a result which will require further elucidation with the ongoing analysis of the trial . The fhn trial will be a tremendous source of data to further dissect the advantages seen with daily therapy in the coming years . The freedom study is an ongoing prospective cohort study investigating the clinical and economic benefits of sdhd . The objectives of the freedom study are to compare a cohort of patients starting sdhd using the nxstage system one to a matched cohort of patients receiving chd for all - cause hospitalizations and non - treatment - related medical expenditures . Using a 10-to-1 ratio, totaling 5000 patients, the matched thrice weekly in - center hd cohort will be obtained from the us renal data system (usrds) database . In addition, changes in qol measures, urea kinetics, management of anemia, bone and mineral metabolism, nutrition, vascular access interventions and use of medications will be examined . The freedom study will involve up to 70 clinical sites and 500 participants, with a minimum 1-year follow - up . Study participants complete qol surveys at the time of study enrollment, at 4 and 12 months and every 6 months thereafter . Interim data recently released from the freedom study has shown that sdhd, performed in the home environment, is associated with significant improvements in several important clinical and qol measures when compared to conventional thrice weekly in - center hd . Interim results from the freedom study investigating the effect of sdhd on depressive symptoms were recently published . Depressive symptoms were examined in 248 participants at enrollment and at 4 and 12 months by administering the validated beck depression inventory (bdi)-ii survey . The study protocol requires that the site investigator be notified immediately of a bdi score> 10, with mild and moderate to severe depressive symptoms defined as bdi scores of 1115 and> 15, respectively . In summary, sdhd was associated with a significant improvement in mean bdi score over 12 months [11.2, 95% confidence interval (ci) 9.612.9, versus 7.8 (95% ci 6.59.1); p <0.001], in the per - protocol (pp) cohort . Similar results were found in the intent - to - treat (itt) cohort . For participants with moderate to severe depressive symptoms, the bdi scores almost halved over 12 months [24.4 (95% ci 19.429.4) versus 12.6 (95% ci 8.017.2); p <0.001]. It should be noted that a bdi score> 15 previously has been shown to be highly predictive of a diagnosis of clinical depression . The percentage of participants with depressive symptoms (bdi score> 10) significantly decreased during 12 months (41 versus 27%; p <0.03). Similarly in the fhn study, patients participating in the daily arm did show a trend toward improvement after 12 months when compared to baseline (12.6 8.7 versus 10.4 8.5, p = 0.1). Although this trend was not statistically significant, one might speculate that undergoing daily dialysis therapy at home may contribute to this improvement . Considering the practical feasibility of daily dialysis both from a logistical and a patient comfort perspective, one might hypothesize that the home setting may further enhance the benefits of sdhd experienced in - center . The long - term effect of sdhd on post - dialysis recovery time was also assessed in the freedom study by administering the following previously validated question: how long does it take you to recover from a dialysis session and resume your normal, usual activities? Interim results were recently published and confirmed that sdhd is associated with a significant decrease in post - dialysis recovery time [476 min (95% ci 359594) versus 63 min (95% ci 3295); p <0.001]. The percentage of participants experiencing prolonged post - dialysis recovery time (> 60 min) also significantly decreased over the 12-month period (81 versus 35%; p <0.001). The fhn daily trial showed significant improvements in control of hyperphosphatemia for patients on sdhd . A significant reduction in mean serum phosphate was also reported in an interim report from the freedom study, along with significant reductions in the calcium phosphate product, serum creatinine, serum potassium and a trend toward a reduction in blood urea nitrogen . Both the fhn daily trial and an interim analysis from the freedom study showed no change in serum albumin levels . Well - known uremic symptoms, including restless legs symptoms (rls) and poor sleep quality are common in the hd population . Poor sleep quality and rls have both been linked to increased risk of death for these patients [9, 10]. A recent interim report from the freedom study demonstrated initiation of sdhd at home is associated with significant improvement in rls and sleep disturbances . Results from 235 patients found 40% suffered from rls at baseline, which was associated with poorer sleep quality and respiratory disturbances . Among patients with rls, the mean irls (international restless legs severity scale) score improved significantly at month 12, after adjustment for use of rls - related medications (18 versus 11, p <0.0001). Among patients with moderate - to - severe rls (irls score 15), there was an even greater improvement in the irls score (23 versus 13, p <0.001). Over the 12-month period, there was decline in the percentage of patients reporting rls (35 versus 26%, p = 0.05) and those reporting moderate - to - severe restless legs symptoms (59 versus 43%, p = 0.06). There was a similar and sustained improvement in several scales of the medical outcomes study sleep survey over 12 months, after adjustment for presence of rls and use of anxiolytics and hypnotics . Recent results from both the fhn daily trial and freedom study have shown that sdhd, performed either in - center or at home, is associated with several important clinical and qol benefits when compared to conventional thrice weekly hd . The improvements in left ventricular mass, control of hypertension and improvement in hyperphosphatemia demonstrated in the fhn trial, in conjunction with the positive interim findings from the freedom study, including improvements in depressive symptoms, post - dialysis fatigue, various laboratory parameters, restless leg syndrome and sleep disorders, confirm the anecdotal benefits of sdhd seen and reported by many practicing nephrologists and their patients . Until now, a complex variety of reasons has hindered the more widespread use of sdhd in the usa and globally . However, the results of these studies may provide a new impetus for esrd patients and nephrologists to reconsider the paradigm of thrice weekly center hd as the default therapy. Such a paradigm shift will likely increase the possibility for improved clinical outcomes and overall patient care for those affected by esrd.
Along with the introduction of effective therapeutic agents such as somatostatin analogue (ssa), multityrosine kinase inhibitor and mammalian target of rapamycin inhibitor, much improvement has been made in the treatment of neuroendocrine tumors (net). Especially, the promid and radiant studies proved the effectiveness of these new therapeutic agents in the treatment of mid gut and pancreatic net, respectively . However, in the case of metastatic gastric net, there is neither an established treatment strategy nor a prospective study because gastric nets are not only rare but also mostly diagnosed in the early stage, which can be controlled by surgical resection . Therefore, gastric net has been considered a part of the gastroenteropancreatic net category and there have been no studies exclusively aimed at gastric net . In the treatment of metastatic net, a treatment approach according to the presence of hormonal symptom, embryological origin of primary site and pathologic grade is usually accepted as a principle . However, in the case of gastric net, a separate treatment approach depending on a specific classification, which can be recognized based on clinical and histologic characteristics, is considered . In the classification of gastric net, type i (74% of the gastric nets) is associated with chronic atrophic gastritis and hypergastrinemia, and type ii (6% of the gastric nets) is associated with multiple endocrine neoplasia type i, zollinger - ellison syndrome and hypergastrinemia . Lastly, sporadic type iii gastric neuroendocrine carcinoma (nec; 13% of the gastric nets) is gastrin - independent and carries the worst prognosis . In this paper, we describe the case of a female gastric net patient with multiple liver metastases who was unresponsive to biologic therapy and cytotoxic chemotherapy . In addition, we look at the therapeutic strategy for metastatic gastric net together with a literature review . Upper endoscopic examination revealed a localized, fixed and firm subepithelial tumor (diameter, 2.2 cm) in the peripyloric area, but there was no evidence of gastric ulcer in the entire stomach (fig . A stomach computed tomography (ct) was performed and revealed two non - enhancing, low - attenuated liver masses in s4 (2.2 cm) and s5 (1.6 cm) suggesting metastasis (fig . Laparoscopic antrectomy with gastrojejunostomy and liver biopsy were performed, but hepatic metastatectomy was not tried due to multiplicity . The biopsy result of both gastric mass and metastatic hepatic nodule revealed a well - differentiated nec (net g2 by 2010 who classification). Pathologically, the tumor was 37 21 mm in size with a negative margin (distance from resected margin: 0.5 cm) and infiltrated the muscular propria layer . Microscopically, the tumor cells were medium - sized and uniform in shape and showed moderate cellularity with mild mitosis (<1/50 per high - power field) (fig . On immunohistochemical staining, the tumor cells were positive for chromogranin a and cd56a (fig . 3d), and the serum gastrin level was increased to 1,832 pg / ml (normal range 0108). The vitamin b 12 level was 350 pg / ml (normal range 211911), chromogranin a level was 356.29 ng / ml (normal range 2794), 5-hydroxy indole acetic acid level was 3.04 mg / day (normal range <10) and serotonin (5-hydroxytryptamin) was 48 ng / ml (normal range <200). However, ct and mri did not localize any tumor assumed of gastrinoma . Considering the gastrin level and histologic grade moreover, she complained of abdominal pain with diarrhea and was therefore treated with long - acting repeatable (lar) sandostatin at a dose of 20 mg intramuscularly every 28 days . However, abdominal ct, which was performed 3 months later, revealed progression of the hepatic metastases without tumor recurrence in the operated bed (fig . The patient received an etoposide plus cisplatin (ep) combination chemotherapy (etoposide 100 mg / m on days 13, cisplatin 75 mg / m on day 1). Despite 3 cycles of ep chemotherapy, response evaluation identified a stable disease status, and eventually, her disease progressed after 6 cycles of ep chemotherapy (fig . 2c). Because of the patient's economic situation and lack of grounds, targeted agents such as sunitinib and everolimus were infeasible . Therefore, we chose a combination therapy with interferon- (ifn) and sandostatin lar . On response evaluation after 3 months of treatment, she showed favorable response with regression of liver metastasis (fig . Since oberndorfer first denominated the carcinoid tumor in 1907, there has been much improvement in the classification and treatment of net . Especially the introduction of the new who pathologic classification system, which is based on the ki-67 labeling index and a few landmark studies such as promid and radiant, provided a useful guide to the oncologists dealing with gastroenteropancreatic net . Based on these studies, ssa is known to be effective in the treatment of midgut nets, and cytotoxic chemotherapy such as the ep regimen is known to be effective in the treatment of foregut nets, including pancreatic nets . However, net can arise in most organs of the body and can show a variety of clinical features according to its origin and the degree of differentiation . Moreover, in the case of nets that originate from rare primary sites, standard treatment strategies have not been defined yet . Although a division according to the embryological origin, which divides nets into foregut, midgut and hindgut nets, has been used in practice, it cannot guarantee the homogeneity between the different organs inside the same embryological category . That is to say, although gastric net embryologically originates from the foregut, we do not know exactly whether it shows an aspect similar to net of the pancreas . Moreover, gastric net has a few distinctive features in its pathogenesis and natural course . Gastric net is known to be derived from the enterochromaffin - like (ecl) cells, which produce histamine, express the gastrin receptor and regulate gastric acid secretion . Gastrin, which is released by g cells in the antrum of the stomach, duodenum and the pancreas, stimulate the release of histamine in ecl cells . Therefore, neoplastic changes in ecl cells are often associated with elevated serum gastrin levels . Based on this unique pathogenetic background, a separate classification system for gastric net was first defined by rindi et al . In 1993 . According to this classification, gastric net can be subdivided into types i, ii and iii, depending on the gastrin level and source of hypergastrinemia . A treatment strategy according to that system because of the relatively benign clinical course, low metastatic potential and low histologic grade of type i and ii gastric nets, the treatment strategy for these types of nets was focused on local control, and the principal treatment was conservative, including endoscopic follow - up or endoscopic mucosal resection . Even in the case of multicentric tumor with liver metastasis, antrectomy and the consequent normalization of gastrin levels was considered sufficient for the control of the remnant lesion . As for medical treatment, biologic therapy such as ssa and ifn a few studies also demonstrated a therapeutic value of octreotide in nets of gastroduodenal origin [6, 7]. Besides that, khuroo et al . Showed that long - acting ssa injections reduced serum gastrin and serum chromogranin a levels with a 50% reduction in the visible number of tumors in 3 patients with type i metastatic gastric carcinoids . Ifn- and - have also been used in the treatment of gastrointestinal nets, regardless of functionality . Immune - mediated cytotoxicity, inhibition of progression of the cell cycle from the s to the g2 phase and inhibition of tumor angiogenesis were proposed as main mechanisms of ifn . However, systemic side effects such as flu - like symptoms and hypothyroidism hindered widespread usage as a first - line treatment . According to the european neuroendocrine tumor society (enets) guidelines, ssa is known to be valuable for the subgroups of patients with slowly progressive and low proliferative nets (g1) of gastroduodenal origin, and in cases of metastatic g3 necs or well - differentiated but rapidly progressive metastatic disease, combinations of etoposide and cisplatin are recommended . However, it is not certain whether the histologic grade depending on the rate of cellular proliferation correlates with the classification based on gastrin levels and how to integrate the gastric net and who classifications into the same treatment strategy for gastric nets, especially in cases of metastatic disease . Moreover, there is the opinion that dysplastic cells are less dependent on gastrin stimulation and do not regress in response to reduced circulating gastrin levels . Indeed, a few metastatic cases with a bad prognosis have been reported in type i or ii gastric nets [11, 12]. In our case, the biopsy results showed a well - differentiated, g2 nec with 4% of ki-67 mitosis and an elevated gastrin level of 1,832 pg / ml . However, there was no evidence of endocrine neoplasia at the other site . Based on the histologic grade and gastrin level, type therefore, we expected good response to ssa, but somatostatin did not achieve response and disease progression was faster than expected . However, after 6 cycles of ep chemotherapy, the tumor eventually progressed within only 2 months . Because the general condition of our patient was infeasible for another cytotoxic chemotherapy, we again used ssa for the relief of functional symptoms and stabilization of the tumor . In addition, we added ifn- and obtained favorable response . According to previous studies comparing ssa or ifn alone with a combination of the two drugs, response was not superior in any of these groups, whereas the side effects leading to an interruption of the therapy were more frequent in the combination group . However, according to kolby et al ., the combination of ifn- and ssa reduced tumor progression better and for a longer time than either agent alone and was well tolerated because ssa can reduce side effects of ifn-, although the study was aimed at midgut net . One review article about the combination of ifn- and ssa presented data showing an advantage of additional ifn- after progression following ssa alone . However, there is not enough statistical evidence for an upfront use of the combination of ifn- and ssa . In summary, for the treatment of metastatic gastric net, both the clinical classification according to the gastrin level and the pathologic grade should be considered . Ssa can be considered a first - line medical treatment for type i and ii metastatic gastric net . However, the combination of ssa and inf- can be an effective option in cases showing aggressive tumor behavior or after failure of ssa alone.
Oral leukoplakia (olep) is defined as a white plaque of questionable risk that is diagnosed after excluding other known diseases or disorders that carry no additional risk for cancer . Olep is a clinical term that requires the application of strict diagnostic criteria for its classification . Olep may present a wide spectrum of histopathological patterns, including hyperkeratosis, epithelial dysplasia, carcinoma in situ, and squamous cell carcinoma (scc). The ki67 protein is localized to the nucleus in all actively dividing cells, and its expression level is proportional to the severity of dysplasia in olep . The upregulation of ki67 in both dysplastic and malignant tissues qualify ki67 as a reliable marker of cell proliferation with prognostic significance . Cyclooxygenase (cox)-2 is an enzyme that catalyzes the synthesis of prostaglandins (pg) whose expression is associated with carcinogenesis owing to its roles in apoptosis, angiogenesis, inflammation and immunosuppression, invasion, and metastasis . Cox-2 expression was investigated for its relation with specific markers of these multiple mechanisms and correlations between high levels of cox-2 expression and carcinogenic progression were reported . Additionally, cox-2 was reported to activate several classes of chemical carcinogens and its peroxidase function was contributed to the activation of procarcinogens . The mode of action of cox-2 in carcinogenesis was linked to pgs, especially of the e series . Due to their effects on proliferation, angiogenesis, immune surveillance and apoptosis, increased synthesis of pgs are believed to be important in the pathogenesis of cancer [8 - 10]. Cox-2 was found to be overexpressed in various carcinomas as well and high levels have been detected in scc compared to normal epithelium [11 - 14]. The aim of this clinicopathological study was to assess the prognostic value of cox-2 and ki67 expression for oral leukoplakia by determining their association with histomorphological data . To model the process of malignant transformation, samples of normal oral mucosa and squamous cell carcinoma were also investigated . Tissue samples were collected from olep patients who were referred to the department of oral diagnosis and radiology in the faculty of dentistry at marmara university, istanbul, turkey . Informed consent was obtained from the patients and the study was approved by the ethics committee of marmara university (mar - yc-2006 - 0123). Patients using non - steroidal anti - inflammatory drugs at the time of diagnosis and in the month prior to their clinical examination were excluded from the study because of the potential effects of these agents on cox-2 levels . Smoking and alcohol habits were recorded . With respect to the former, patients were divided into two groups . Smokers were individuals who were smoking regularly at the time of diagnosis and for at least 1 year prior . Non - smokers comprised patients who reported never having smoked as well as former smokers who had previously smoked but were not doing so at time of diagnosis . Individuals who had one or more drinks three or more times per week were defined as habitual drinkers and placed in the alcohol consumption group, with one unit of an alcoholic drink corresponding to 12 g of ethanol . Clinically, leukoplakia was subdivided into homogeneous (c1) and non - homogeneous (c2) types . C1 was defined as flat, thin, and uniformly white; c2 referred to predominantly white, or white and red lesions that were irregular in shape, flat, nodular, or exophytic . A conservative excisional biopsy was performed for any lesion smaller than 2 cm in diameter; larger lesions were treated according to histopathological results by applying previously described management protocols . Biopsied specimens were evaluated in the department of tumour pathology at the institute of oncology of istanbul university . The final diagnosis was confirmed by histopathological examinations and any other definable lesions were excluded . Olep samples were divided into four subgroups according to histopathological features: hyperkeratosis (hk) and - when epithelial dysplasia was present - three oral intraepithelial neoplasia (oin) subgroups classified according to severity as mild dysplasia (oin1), moderate dysplasia (oin2), and severe dysplasia and carcinoma in situ (oin3). A total of 20 samples, presenting 10 samples of intact normal oral mucosa (n) and 10 samples of scc were retrieved from the archives of the department of tumour pathology and used as negative and positive control groups, respectively . The control tissue samples were used only in the immunohistochemical evaluation in order to form a gradually increasing model for the process of malignant transformation (figure 1) and not evaluated in terms of habits of smoking and alcohol consumption . Olep = oral leukoplakia; nsaid = non - steroidal anti - inflammatory drugs; hk = hyperkeratosis; oin = oral intraepithelial neoplasia; n = normal mucosa; scc = squamous cell carcinoma . The 50 tissue samples were embedded in paraffin and serial sections were cut at a thickness of 5 m that were collected on charged slides, then permeabilized and dried overnight in an autoclave at 56 c . After deparaffinization, sections were subjected to antigen retrieval by heating in a microwave four times for 5 min in citrate buffer (ph 6.0), then cooling to room temperature and washing in phosphate buffered saline (pbs) for 5 min . Endogenous peroxidase activity was quenched by incubating the sections in 3% h2o2 followed by washes in distilled water . Slides were incubated for 2 h with rabbit anti - cox-2 (thermo scientific, lab vision corporation, fremont, ca 94538 - 6406, usa) and rabbit monoclonal anti - ki-67 (thermo scientific, thermo fisher scientific, anatomical pathology, fremont, ca 94538, usa) antibodies, while negative control sections were treated with pbs . All slides were incubated with biotinylated goat anti - rabbit secondary antibody (tr-015-bn; lab vision corp .) For 25 min, followed by a streptavidin peroxidase reagent for 25 min; the chromogenic substrate 3-amino-9-ethylcarbazole was used to visualize immunoreactivity . Sections were counterstained with mayer s hematoxylin, covered with a coverslip, and evaluated under a light microscope (olympus, tokyo, japan). Quantification of cox-2 and ki67 staining cox-2 and ki67 staining was evaluated by a pathologist who was blinded to the experimental protocol . Slides were scanned at low magnification under a light microscope and five areas with the highest degree of staining were selected for cell counts . Only nuclear staining was accepted as positive for ki67 . For cox-2, cytoplasmic and membranous staining the number of cells in the epithelium and in the tumour island of scc specimens was counted . The immunoreactivity score was calculated as the mean percentage of positively stained cells to the total number of cells . Note the strong reaction in the cytoplasms of oral intraepithelial neoplasia cells marked by arrows . Positive staining was observed in the nucleus of oral intraepithelial neoplasia cells marked by arrows . Kruskal - wallis test was used to compare variables among multiple groups with mann - whitney u test (bonferroni corrected) as a post - hoc test . Fisher s exact and fisher - freeman - halton exact tests were used for qualitative comparisons of data . The means (m) and standard deviations (sd) of ages, expressions of cox-2 and ki67 were expressed as (m [sd]). Clinicopathological findings of olep patients clinicopathological characteristics of the study group are shown in table 1 . Patients ranged in age from 30 to 75 years, (54.33 [11.03]), with 19 presenting hk (13 (68.4%) males, 6 (31.6%) females) and 11 presenting oin (7 (63.6%) males, 4 (36.4%) females). The mean ages of the hk and oin patients were similar (55.05 years vs. 53.09 [12.93] years) (p = 0.966). The occurrence of oin was associated with a non - homogeneous clinical aspect (p = 0.004), but was also observed in lesions with a homogeneous appearance (three oin1 and two oin2 patients). The homogeneous - type olep was more frequently observed in the hk group (p = 0.004). Clinicopathological characteristics of the study group statistically no significant, mann whitney u test . Sd = standard deviation; hk = hyperkeratosis; oin = oral intraepithelial neoplasia; n = number of samples; m = male; f = female; s = smoker; ns = non - smoker; u = using; nu = non - user; c1 = homogenous type; c2 = non - homogenous type . There was no difference between groups in terms of smoking and alcohol consumption (p = 0.156; p = 0.327; respectively). Lesions were more often localized on the tongue for oin while buccal mucosa localization was higher for hk (p = 0.002) (table 2). Localization of oral leukoplakia lesions by histopathological subgroups n = number of samples; hk = hyperkeratosis; oin = oral intraepithelial neoplasia . Immunohistochemical findings cox-2 and ki-67 expression increased relative to the degree of lesion severity in the histopathological subgroups, with the scc group having the highest numbers of cox-2 - and ki67-positive cells (table 3). The mean number of cox-2 expressing cells was lowest in n tissue samples, higher in the scc and oin than in the hk group, and higher in the scc than in the oin group (p = 0.0001). Cox-2 and ki67 expressions in olep samples and controls statistically significant at the level p <0.05, kruskal wallis test . Sd = standard deviation; olep = oral leukoplakia; n = normal mucosa; hk = hyperkeratosis; oin = oral intraepithelial neoplasia; scc = squamous cell carcinoma . Among oin subgroups ki-67 expression was detected in the nuclei of cells and in ascending order; these were n (5.33%), hk (36.01%), oin (39.49%), and scc (74.66%). There was no significant difference between the hk and oin groups, but a difference was found between scc and oin groups (p = 0.0001) (table 3). The prevalence of malignant transformations of olep is between 0.13% to 17.5% for observation periods ranging from 1 to 30 years . Several factors are associated with an increased risk of malignant transformation, and the clinical appearance of olep is considered a major benchmark . The occurrence of oin is relatively low for the c1 clinical type, and higher rates of malignant transformation have been reported for the c2 type . In the present study, 5/23 (21.7%) c1 and 6/7 (85.7%) c2 olep cases presented oin . However, given that oin was detected in c1 cases, a biopsy is recommended as a mandatory procedure for all olep types . Tobacco usage in different forms is the most common antecedent for olep lesions, with suggestions that some lesions may regress upon cessation of tobacco use . According to one report, the risk for malignant transformation among individuals with scc that had ceased using tobacco for more than 10 years was similar to that of non - smokers . The patients in the present study were predominantly smokers (80%), and two patients in the non - smokers group had ceased smoking for more than 10 years and both presented with hk . Nonetheless, due to dynamic nature of olep, regression is not expected in all olep cases . Paradoxically, some olep lesions in non - smokers were reported to have worse prognosis than in smokers, and women without smoking habits had a higher risk of malignant transformation among the groups examined in one study . Consistent with these findings, all the oin 3 patients in the present study were non - smokers . It is suggested that the high - risk nature of lesions in non - smokers is associated with intrinsic factors such as inherited or acquired predisposition, for which a more aggressive treatment strategy is recommended . Alcohol consumption is another risk factor for olep, but its most potent effects are exerted through synergy with tobacco usage, with geographical variations reportedly playing a role . In this study, a slightly higher rate of alcohol consumption was detected in oin than in hk (27.3% vs. 10.5%), although this difference was not statistically significant . The increased proliferative capacity in olep has been confirmed by a higher expression level of ki67, which was proposed as a marker for the occurrence and severity of oin in oral mucosa . Its association with lesion severity underscores ki67 expression as a prognostic factor for olep, which was suggested in the present study; the highest ki67 expression was observed in scc, followed by oin and hk, indicating that this marker of proliferation may be a predictor of progressive malignancy . On the other hand, although there was a gradual increase in ki67 expression, no significant difference was detected between the hk and oin groups . Cox-2 is an inducible enzyme whose expression is low or negative in most tissues, but a few hours after a single stimulation the enzymatic activity of cox-2 was reported to increase more than 10-fold and then return promptly back to the basal level . This expression is influenced by growth- and tumour - promoting factors, inflammatory stimuli, and oncogenes under a variety of pathophysiological conditions . Cox-2 expression has been implicated in the malignancy of scc of the tongue as well as oin and cox-2 overexpression was found to be predictive of patient survival in scc . Elevated levels of cox-2 were also observed in the majority of dysplastic squamous epithelia and scc, in contrast to the weak expression detected in normal esophageal tissue . Another study reported progressively higher cox-2 levels in normal mucosa, oin, and scc . Consistent with these results, here it was found that cox-2 expression was lowest in normal mucosa, higher in hk followed by oin, and highest in scc . Nevertheless, as a limitation of the present study, the number of the study group is low (30 samples of olep) and the subgroups are not equal (2 samples of oin3). Therefore, further studies are required with larger populations in order to indicate cox-2 and ki67 as prognostic markers of olep . Our results suggest that cyclooxygenase-2 and ki67 expression may have a prognostic value for evaluating the malignant transformation of oral leukoplakia, and that the etiopathogenesis of oral leukoplakia should be investigated in terms of risk factors other than smoking and alcohol consumption . Semih ozbayrak has worked in the field of oral medicine and dentomaxillofacial radiology in department of oral diagnosis and radiology, faculty of dentistry, marmara university, istanbul, turkey (1986 - 2013) and provided indispensable advice and guidance throughout this investigation.
Osteosarcoma is a malignancy of mesenchymal cells that have the ability to produce osteoid or immature bone . Osteosarcoma of the head and neck is relatively rare and constitutes only around 8.4% of all osteosarcomas, while in the jaw there is an estimated incidence of 0.7 per million population . Gnathic osteosarcomas are broadly categorized into central (intramedullary) and peripheral (surface) subtypes . Surface tumors are further divided into parosteal well - differentiated (low - grade), periosteallow- to intermediate - grade and high - grade surface osteosarcomas . In 1949, geschickter and copeland were the first to describe parosteal osteosarcomas under the heading of parosteal osteoma a group of parosteal bone tumors of which the seemingly benign clinical and histologic aspect was belied by frequent relapse and metastasis . Unni et al ., defined the juxtacortical osteosarcoma as a distinctive type of malignant bone tumor that originates on external surface of the bone specifically in relation to periosteum and/or immediate connective tissue . The first report of parosteal osteosarcoma in craniofacial site was reported in 1961 by som and peimer . It has predilection to occur in the 2and 4decade mainly affecting distal femur . In the long bones females have a greater risk than men (3:2), whereas in jaws men are more affected as (1.75:1) compared to women . In the jaws it can be mistaken for benign lesions as the clinical behavior and radiographic appearances are usually benign . Histologically, parosteal osteosarcoma is well - differentiated and is characterized by spindle cell stroma with minimal atypia and rare mitotic figures separating irregular trabeculae of woven bone . Parosteal osteosarcoma of craniofacial area behave similar to long bone counterparts with slow growth and low grade malignancy that do not tend to metastasize . This paper describes a case of parosteal osteosarcoma and emphasizes the need for differential diagnosis of this rare entity with benign reactive and neoplastic osseous lesions . A 22-year - old female patient presented with the complaint of swelling in the right side of the face since 9 months, which was not associated with pain, discharge or neurological symptoms . On extraoral examination, diffuse swelling of about 4 4 cm in midface region was noted on the right side of the face . Intraorally, swelling involved right maxillary buccal aspect of the ridge from second premolar to third molar measuring around 3.5 2.5 cm in dimensions, extending anterioposteriorly from the mesial aspect of maxillary second premolar to distal aspect of third molar and superioinferiorly from the marginal gingival level to the vestibule causing obliteration, without any palatal expansion . Exophytic bony swelling on right maxillary alveolar ridge occlusal radiograph revealed dome - shaped swelling with radiopaque shadow and patchy radiopacity on buccal aspect of maxilla [figure 2]. Axial and coronal computed tomography (ct) scans revealed well - defined, hyperdense bony mass with patchy radiopacity emanating from the buccal cortical plate with broad base . Three - dimensional ct (3d - ct) showed lobulated exophytic bony mass attached with broad base to the buccal cortical plate formed by the network of multiple inter coalescing striae of trabeculae [figure 3c]. The h and e stained sections showed parallel array of bony trabeculae in a background of spindle cells and angular cells which showed minimal atpia . Areas of osteoid admist the angular and spindle cells were also detected [figures 4 - 6]. The patient was treated with partial maxillectomy and is under follow - up since the past 1.5 years . Occlusal view showing well - defined radiopaque lesion with patchy radiopacity (a) axial ct showing hyperdense radiopaque lesion, slightly dense at base than at periphery . (c) 3d - ct showing exophytic bony mass showing network of multiple intercoalescing radiating striae . 3d - ct = three - dimensional computed tomography irregular osteoid matrix separated by spindle and angular cells . (h&e stain, 100) photomicrograph showing thick often parallel (streamers) array of bony trabeculae in a spindle stroma . Parosteal osteogenic sarcoma is an uncommon malignant tumor . In a large case series of 998 osteosarcomas of entire skeleton, dahlin et al . Bras et al ., reviewed seven cases of gnathic parosteal osteosarcomas of which five were reported in mandible and two in maxilla with an average age of 35.4 years . Simon et al ., in there view of 12 parosteal osteosarcomas, reported five in maxillary and seven in mandibular region with an average age of 34 years . A peak incidence between the ages of 20 and 30 years with 2:3 male to female ratio however, we report parosteal osteosarcoma in a 22-year - old female patient, in the maxillary region . To date only 27 cases of juxtacortical osteosarcomas have been reported of which 12 were of parosteal variant, nine were of periosteal variant and six were of periosteal / parosteal type . The present case brings the total number of parosteal osteosarcomas of jaws to 13, a rarity in itself in the literature . Concurrent with the literature presenting features in our case were a painless, slowly growing, exophytic osseous swelling with nonlobular outer surface . Pain was mentioned as dull aching in half of the reported cases and it appeared as exophytic hard nodule on the attached gingiva appearing as soft tissue epulides mimicking benign osseous proliferative growth . Occasionally surface ulceration was noted . Radiographically, parosteal osteosarcomas are radiodense, lobulated, cauliflower - like or oval masses with a broad stalk attached to the external cortex of the underlying bone . Parosteal variant is considered to arise from the outer layer of cortex, therefore does not elevate the periosteum like periosteal variant . Between underlying bone and the tumor, a thin radiolucent cleavage plane called string sign, a characteristic but not a constant finding, is seen in only 30% of the cases . String sign is a radiolucent line histopathologically corresponding to unmineralized thickened periosteum interposed between the cortex and the tumor mass . It is more difficult to demonstrate in the jaws because their anatomical form does not allow the necessary radiographic projections to be obtained . Various other radiographic patterns like cotton wool image with distinct margin, patchy radiopacity and tumor mass with diffuse hazy opacification with few fine bony spicules radiating from the lower border of the lesion also have been reported . In contrast, periosteal osteosarcoma presents with a radiographically intact cortex with no involvement of the underlying marrow cavity . The tumor matrix is not as radiographically dense or homogenous as parosteal osteosarcoma and has a poorly defined periphery . Since treatment and prognosis varies with both the variants of surface osteosarcomas, radiologic differentiation is important . Computerized tomography scan helps to demonstrate the tumor confinement, attachment to the cortex and involvement of marrow . Ct and magnetic resonance imaging (mri) images are extremely useful for staging, detecting the presence of cortical erosion, regions of dedifferentiation, intramedullary extension or satellite lesions in the surrounding soft tissues . Scintigraphy and chest ct are useful in confirming the solitary nature of the tumor and absence of metastatic disease . In the present case, many interlacing coalescing trabeculae of varying thickness forming a meshwork from the lower border of the intact cortex as seen in 3dct was an interesting finding not reported in previous cases . Radiographic differential diagnosis includes tori, exostosis and peripheral osteomas more commonly; and other rare entities like nora's lesion (bizarre periosteal ossifying periostitis), osteochondroma, myositis ossificans and hyperostosis . Tori and exostosis occurring at the same site are painless, slow growing masses with limited growth potential and can attach to cortex of jaw via pedicle or wide base . Often the correct diagnosis is established on histologic basis . Peripheral osteomas tend to be more radiodense because of absence of cartilagenous areas and the rare trabeular straie tend to be fine and closer together, like the edge of the feather . However, the aggressive behavior with rapid expansion and medullary involvement should help in ruling out their presence . Osteochondroma, a rare entity in jaws, shows continuity of cortex and underlying medullary bone with the base of the lesion . Hyperostosis, an uncommon lesion encountered under the pontics in the posterior region can be confused with parosteal variant . Histologically, it is well - differentiated and characterized by spindle cell stroma with minimal atypia and rare mitotic figures separating irregular trabeculae of woven bone . With time, the trabeculae often coalesce and form a large mass of solid bone . About 4050% of parosteal osteosarcomas exhibit foci of cartilage . In the present case fine lace - like osteoid tissues were present among hyperchromatic tumor cells with foci of cartilaginous tissue . Microscopic evaluation of the specimen helps in screening the dedifferentiation areas and areas that represent high grade behavior . Approximately 10% of parosteal osteosarcomas dedifferentiate to high - grade osteosarcoma with a corresponding worsening of prognosis . The histologic differential diagnosis of parosteal osteogenic sarcoma of the jaws includes nora's lesion, benign fibro - osseous lesions like fibrous dysplasia and ossifying fibroma, low - grade intraosseous osteosarcomas and osteomas . Nora's lesion histopathologically shows characteristic cartilage capping and distinct zones which distinguishes it from parosteal osteosarcoma . Fibrous dysplasia and ossifying fibroma apart from their histopathology, the intramedullary location negates the possibility of them in differential diagnosis . Osteoma in conjunction with mature bone lacks the spindle cell proliferation seen in parosteal osteogenic sarcoma and do not show cellular polymorphism, anaplasia and mitotic activity . Low - grade intraosseous osteosarcoma shows overlapping histologic features with parosteal osteosarcoma . In most of the cases, minimal local reccurence occurs with wide excision of adjacent bone and periosteum by removing satellite lesions and preventing local spread along the bone surface . Now, chemotherapy following wide excision is the recommended treatment protocol for recurrent cases and those exhibiting highly malignant features on histological examination . Low - grade parosteal osteosarcoma has an overall good prognosis and has low metastatic potential . Local control is more limited in the maxilla than the mandible and thus mandibular parosteal osteosarcomas overall have better prognosis than those of the maxilla . Therefore maxilla has high recurrent rate . With the number of recurrences, the incidence of intramedullary involvement and dedifferentiation to high - grade sarcoma increases . To conclude, this case illustrates the importance of correlation of clinical, radiological and histopathological features in confirming the diagnosis of parosteal osteosarcoma . Because of the less aggressive biological behavior
Labor is induced for a variety of reasons, such as preventing prolongation of pregnancy (1), rupture of membranes in the absence of labor, and diseases that threaten the mother or fetus (2). The readiness of the cervix is important for successfully inducing labor and reducing the need for assisted birth and cesarean section (1, 3, 4). Hence, significant attention has been focused on finding appropriate methods to prepare the cervix before inducing labor (3, 5). Recently, prostaglandins or mechanical techniques have been used for ripening the cervix, but these methods have some limitations . For instance, prostaglandins should be used in the labor room or a place where it is possible to monitor the uterus activity and the fetal heart rate; in case of uterine tachysystolic activity, they should be immediately removed . Furthermore, intracervical gels, vaginal inserts, and mechanical techniques cannot be used if membranes are ruptured; if oxytocin is required, it should be provided at least 6 - 12 hours after administering prostaglandins (1, 3, 5). Experiences of the past few decades have shown that chemical medications have adverse effects despite their efficacy, so herbal medications have become more attractive (6, 7). Saffron, scientifically known as crocus sativus l, is a stemless herb found in compounds such as essential oil (containing trepenes), picrocrocin, and crocin (8, 9). It is traditionally used to accelerate labor with no prescription or sometimes as prescribed by traditional healthcare workers . A review of the literature did not yield any human studies on the effects of saffron on labor induction . Meanwhile, studies on rats showed that oral consumption of saffron can induce premature labor and abortion (10 - 12). Descriptive studies suggest that it may have an effect on human abortion (13 - 15). Furthermore, clinical trials reported the effects of saffron on reducing symptoms of premenstrual syndrome (16), primary dysmenorrhea (17), mild - to - moderate (18) and major depression (19), with no significant side effects in human subjects . Some traditional medical textbooks mention that saffron has inducing effects on the smooth muscles of the uterus, and chinese medicine recommends it be used for menorrhagia, difficult labor, and postpartum hemorrhage (20). However, it is believed that saffron can harm the fetus in the first trimester during organogenesis . Still, moderate consumption (0.5 - 2 g per day) after the first trimester can promote the elasticity of uterine tissue and facilitate labor (9, 21, 22). The present study aimed at investigating the effects of the oral consumption of saffron on cervical ripening (primary outcome), and certain delivery and fetal outcomes (secondary outcomes) in full - term pregnant women (at gestational weeks 39 - 41) without indications of requiring cesarean section . The study was conducted as a randomized, double - blind, placebo - controlled trial . Fifty singleton pregnant women with zero to two parity and a gestational age of 39 to 41 weeks were recruited among outpatients presenting to two private offices and a clinic in shohada hospital for prenatal care . Each of the women had intact amniotic sac, no active uterine contractions, a bishop s score of less than 4, a reactive non - stress test, a normal fetus with estimated weight of 2500 to 4000 in cephalic presentation, and plans to have a vaginal delivery at shohada hospital of bonab, iran . Women with the following criteria were excluded: (1) no access to a phone line; (2) illiterate or only primary education; (3) a history of cesarean section or any possible indication that cesarean section would be required for the current pregnancy; (4) a history of cryotherapy or cauterization on the cervix; (5) a known chronic and systematic disease; or (6) smokers, alcohol drinkers, or drug abusers . Shohada hospital is the only center for delivery in bonab (a city with a population of 129,795) (23). It is a public center affiliated with tabriz university of medical sciences which has maternity and pediatric wards and is responsible for the care of people living in bonab and some of the small cities and villages surrounding the city . Almost all vaginal deliveries (about 150 per month), which comprise about half of all deliveries at the hospital, are facilitated by 20 midwives under the indirect supervision of 4 obstetricians . The research protocol was approved by the ethics committee of tabriz university of medical sciences (code 91219) and registered at the iranian registry of clinical trials (iranian registry of clinical trials (irct) 201212233706n19) on 17 march 2013 before starting participant recruitment . The allocation sequence was determined using a computerized program using block randomization with block sizes of 4 and 6 and an allocation ratio of 1:1 and stratified according to a history of previous delivery (yes / no). Sequentially numbered sealed opaque envelopes containing three saffron or three placebo pills were used for the allocation concealment . The allocation sequence and the packages were prepared by a person not involved in the recruitment, data collection, or analysis . The participants and other people involved in the recruitment, data collection, or analysis were not aware of the type of intervention for each participant . Participants in the intervention group received saffron in the form of 3 pills (250 mg in each pill) and participants in the control group received a placebo 3 times in 24 hours (one every eight hours). They took the first pill immediately after enrolling in the study under the supervision of the investigator . They were instructed orally and in writing on how to take the two other pills at home . The saffron pills and the placebos were produced in the industrial pharmacy laboratory of the tabriz university of medical sciences and under direct supervision of the pharmacist from our research team . Before preparation of the pills, the purchased active ingredient was identified and controlled for quality and safety by the herbarium of the faculty of pharmacy at the tabriz university of medical sciences . The company purchases saffron from farms in khorasan province and performs tests on the humidity percentage, acid insoluble ash, crocin (responsible for the color), picrocin (responsible for bitterness and taste), and saffranel (responsible for the aroma) using a spectrophotometer at wavelengths of 257, 330, and 440 nm, and microbial tests to monitor contamination and to safety of the product before packaging (24). Each saffron pill had dried saffron stigma (250 mg), microcrystalline cellulose (filler), sodium stearate glycolate of 3% (disintegrant), and magnesium stearate 1% (lubricant). The placebo pills had the same materials except for the saffron stigma and were identical to the saffron pills in terms of color and size . The primary outcome was readiness of cervix assessed by the bishop s score at 10 - 12 and 20 - 24 hours after initiation of intervention, and just after onset of uterine active contractions . The bishop s score is a quantifiable method used to predict outcomes of labor induction involving 5 components, including dilatation, effacement, fetal station, cervical consistency, and position, each of which has been described by bishop (1964). The possible score ranges from 0 to 13; the higher score, the better the readiness of the cervix (2). Secondary outcomes included delivery - related variables such as the interval between starting the intervention and the onset of active uterine contractions, the duration of the first and second stages of delivery, the frequency of vaginal delivery, and hemorrhage assessed by measuring hemoglobin and hematocrit at the admission to the hospital and 12 - 24 hours after delivery . Fetal and neonatal outcomes such as the apgar score measured at the first and fifth minutes, meconium staining, and the need for admission into the neonatal ward were also considered . To recruit participants, potentially eligible women were examined using abdominal palpation in 10 minutes to rule out active uterine contractions, and a non - stress test conducted in 20 minutes to rule out any fetal distress . The eligibility criteria was assessed by a midwife and confirmed by an obstetrician . After obtaining informed written consent from eligible women and assessing their baseline characteristics, the women were given pre - prepared packages containing the pills to the participants in their recruitment order into the study . They were also provided with a diary to record the times when the pills were taken and to report any side events . It was emphasized that they needed to immediately report any complications including hemorrhage, decrease in fetal movement, perforation of the amniotic sac, or any other serious adverse events by calling the investigator or referring to the hospital . The women were also instructed on how to count fetal movements and to notice the start of active contractions (tightening of abdominal muscles three or more times in ten minutes). It was also emphasized that the participants were to come back for follow - up assessment 10 - 12 and 20 - 24 hours after taking the first pill, and also whenever active uterine contractions had started . Furthermore, the participants were called every 3 - 4 hours during the day, and before and after a 6 hour interval during the night within the first 24 hours to remind them about the instructions . Any other interventions, including using misoprostol or oxytocin for augmentation or induction, were recorded . After delivery, the first and fifth apgar scores and weight of the infants were checked by the investigators . Hospital records of the women and their infants were checked for any intervention that may have been administered . At time of admission to the hospital and 12 - 24 hours after delivery the initial sample size included 20 people in each group based on the guidelines of pirdadeh et al.s study (25), where m1 = 2.50 (bishop s mean score), sd1 = 1.29, m2 = 3.75 (a 50% increase in bishop s mean score), sd1 = sd2 = 1.29, = 0.05 and = 0.1 . Considering the possible dropout rate, some imputation was done before data analysis . For participants who did not attend the 10 - 12 hours follow - up, if the bishop s scores at the baseline and at the 20 - 24 hours assessment were the same, the same score was also used for the 10 - 12 hours assessment score . If the two values were not the same, the participant was omitted from the analysis at the time - point with no recorded information . For any participants who delivered before 20 hours after intervention, the maximum bishop s score was used for the 20 - 24 hour score after intervention . Any person who had a cesarean section before the follow - up score assessments prior to starting active uterine contractions was omitted from the bishop score analysis . The normality of the distribution of the quantitative variables according to study group was assessed using the kolmogorov bishop s scores, along with their log10 and ln, had no normal distribution . Therefore, a mann - whitney u test was used to compare the groups in terms of the primary outcome and also for in terms of the bishop s test components . An independent t - test was used for the other quantitative outcomes which had normal distribution . Pearson chi - square, linear - by - linear, or fisher s exact tests were used for comparison of the groups in terms of the qualitative outcomes . The study was conducted as a randomized, double - blind, placebo - controlled trial . Fifty singleton pregnant women with zero to two parity and a gestational age of 39 to 41 weeks were recruited among outpatients presenting to two private offices and a clinic in shohada hospital for prenatal care . Each of the women had intact amniotic sac, no active uterine contractions, a bishop s score of less than 4, a reactive non - stress test, a normal fetus with estimated weight of 2500 to 4000 in cephalic presentation, and plans to have a vaginal delivery at shohada hospital of bonab, iran . Women with the following criteria were excluded: (1) no access to a phone line; (2) illiterate or only primary education; (3) a history of cesarean section or any possible indication that cesarean section would be required for the current pregnancy; (4) a history of cryotherapy or cauterization on the cervix; (5) a known chronic and systematic disease; or (6) smokers, alcohol drinkers, or drug abusers . Shohada hospital is the only center for delivery in bonab (a city with a population of 129,795) (23). It is a public center affiliated with tabriz university of medical sciences which has maternity and pediatric wards and is responsible for the care of people living in bonab and some of the small cities and villages surrounding the city . Almost all vaginal deliveries (about 150 per month), which comprise about half of all deliveries at the hospital, are facilitated by 20 midwives under the indirect supervision of 4 obstetricians . The research protocol was approved by the ethics committee of tabriz university of medical sciences (code 91219) and registered at the iranian registry of clinical trials (iranian registry of clinical trials (irct) 201212233706n19) on 17 march 2013 before starting participant recruitment . The allocation sequence was determined using a computerized program using block randomization with block sizes of 4 and 6 and an allocation ratio of 1:1 and stratified according to a history of previous delivery (yes / no). Sequentially numbered sealed opaque envelopes containing three saffron or three placebo pills were used for the allocation concealment . The allocation sequence and the packages were prepared by a person not involved in the recruitment, data collection, or analysis . The participants and other people involved in the recruitment, data collection, or analysis were not aware of the type of intervention for each participant . Participants in the intervention group received saffron in the form of 3 pills (250 mg in each pill) and participants in the control group received a placebo 3 times in 24 hours (one every eight hours). They took the first pill immediately after enrolling in the study under the supervision of the investigator . They were instructed orally and in writing on how to take the two other pills at home . The saffron pills and the placebos were produced in the industrial pharmacy laboratory of the tabriz university of medical sciences and under direct supervision of the pharmacist from our research team . Before preparation of the pills, the purchased active ingredient was identified and controlled for quality and safety by the herbarium of the faculty of pharmacy at the tabriz university of medical sciences . The company purchases saffron from farms in khorasan province and performs tests on the humidity percentage, acid insoluble ash, crocin (responsible for the color), picrocin (responsible for bitterness and taste), and saffranel (responsible for the aroma) using a spectrophotometer at wavelengths of 257, 330, and 440 nm, and microbial tests to monitor contamination and to safety of the product before packaging (24). Each saffron pill had dried saffron stigma (250 mg), microcrystalline cellulose (filler), sodium stearate glycolate of 3% (disintegrant), and magnesium stearate 1% (lubricant). The placebo pills had the same materials except for the saffron stigma and were identical to the saffron pills in terms of color and size . The primary outcome was readiness of cervix assessed by the bishop s score at 10 - 12 and 20 - 24 hours after initiation of intervention, and just after onset of uterine active contractions . The bishop s score is a quantifiable method used to predict outcomes of labor induction involving 5 components, including dilatation, effacement, fetal station, cervical consistency, and position, each of which has been described by bishop (1964). The possible score ranges from 0 to 13; the higher score, the better the readiness of the cervix (2). Secondary outcomes included delivery - related variables such as the interval between starting the intervention and the onset of active uterine contractions, the duration of the first and second stages of delivery, the frequency of vaginal delivery, and hemorrhage assessed by measuring hemoglobin and hematocrit at the admission to the hospital and 12 - 24 hours after delivery . Fetal and neonatal outcomes such as the apgar score measured at the first and fifth minutes, meconium staining, and the need for admission into the neonatal ward were also considered . To recruit participants, potentially eligible women were examined using abdominal palpation in 10 minutes to rule out active uterine contractions, and a non - stress test conducted in 20 minutes to rule out any fetal distress . The eligibility criteria was assessed by a midwife and confirmed by an obstetrician . After obtaining informed written consent from eligible women and assessing their baseline characteristics, the women were given pre - prepared packages containing the pills to the participants in their recruitment order into the study . They were also provided with a diary to record the times when the pills were taken and to report any side events . It was emphasized that they needed to immediately report any complications including hemorrhage, decrease in fetal movement, perforation of the amniotic sac, or any other serious adverse events by calling the investigator or referring to the hospital . The women were also instructed on how to count fetal movements and to notice the start of active contractions (tightening of abdominal muscles three or more times in ten minutes). It was also emphasized that the participants were to come back for follow - up assessment 10 - 12 and 20 - 24 hours after taking the first pill, and also whenever active uterine contractions had started . Furthermore, the participants were called every 3 - 4 hours during the day, and before and after a 6 hour interval during the night within the first 24 hours to remind them about the instructions . Any other interventions, including using misoprostol or oxytocin for augmentation or induction, were recorded . After delivery, the first and fifth apgar scores and weight of the infants were checked by the investigators . Hospital records of the women and their infants were checked for any intervention that may have been administered . At time of admission to the hospital and 12 - 24 hours after delivery the initial sample size included 20 people in each group based on the guidelines of pirdadeh et al.s study (25), where m1 = 2.50 (bishop s mean score), sd1 = 1.29, m2 = 3.75 (a 50% increase in bishop s mean score), sd1 = sd2 = 1.29, = 0.05 and = 0.1 . Considering the possible dropout rate, 25 people were ultimately selected for each group . Some imputation was done before data analysis . For participants who did not attend the 10 - 12 hours follow - up, if the bishop s scores at the baseline and at the 20 - 24 hours assessment were the same, the same score was also used for the 10 - 12 hours assessment score . If the two values were not the same, the participant was omitted from the analysis at the time - point with no recorded information . For any participants who delivered before 20 hours after intervention, the maximum bishop s score was used for the 20 - 24 hour score after intervention . Any person who had a cesarean section before the follow - up score assessments prior to starting the normality of the distribution of the quantitative variables according to study group was assessed using the kolmogorov bishop s scores, along with their log10 and ln, had no normal distribution . Therefore, a mann - whitney u test was used to compare the groups in terms of the primary outcome and also for in terms of the bishop s test components . An independent t - test was used for the other quantitative outcomes which had normal distribution . Pearson chi - square, linear - by - linear, or fisher s exact tests were used for comparison of the groups in terms of the qualitative outcomes . Participant recruitment was carried out from may 23 until july 17, and follow - up ended on july 29, 2013 . All women randomized into the groups (25 women in each group) two participants from the placebo group did not take the second and third pills due to opposition from their husbands . Two participants from each group did not attend the 10 - 12 hour follow - ups . Only one participant from the placebo group did not report for the 20 - 24 hour follow - up . One person from the placebo group had a cesarean section at 13 hours after intervention before starting active uterine contractions due to meconium staining of the fetus . The hemoglobin and hematocrit of 1 person could not be measured from the saffron group, and for 3 persons from the placebo group 12 - 24 hours after delivery due to their early discharge (figure 1). A, two participants did not take the second and third pills due to husband opposition; b, two participants from each groups did not report for the 10 - 12 hour follow-up.from the placebo group, for 1 person, the baseline and 20 - 24 hour bishop s score assessments were the same, and the same score was considered for the 10 - 12 hour assessment score . One person in this group did not refer for both the 10 - 12 and 20 - 24 hour follow - ups and was omitted from the analysis at those points in time . For 2 participants from the intervention group who did not report for the 10 - 12 hour follow - up, and due to differences between baseline and 20 - 24 hours bishop s score assessment, their results were omitted from the analysis; c, one person did not refer for the 20 - 24 hour follow - up and another had a cesarean section before onset of active uterine contractions 13 hours after intervention due to meconium staining of the fetus; d, hemoglobin and hematocrit were not assessed for 1 person in the saffron group and for 3 in the placebo group due to their early discharge the groups were similar in terms of demographic and fertility characteristics . About half (52%) of the women the gestational age of about three fourths (76%) was 273 - 280 days (table 1). Results of independent t - test . Results of linear - by - linear chi - square test . Results of fisher s exact test . Results of pearson chi - square test . The mean (sd) of the bishop s score for the saffron group increased from 2.6 (0.6) at baseline to 3.0 (1.1) at 10 - 12 hours, to 5.2 (2.6) at 20 - 24 hours and to 7.2 (0.9) at just after starting active uterine contractions . The corresponding figures for the placebo group were 2.6 (0.6), 2.6 (0.6), 4.0 (2.4), and 6.2 (1.1), respectively . The difference between the groups was not statistically significant at the baseline (p = 0.792) and at 10 - 12 hours (p = 0.159). The score was significantly higher in the saffron group at 20 - 24 hours (p = 0.029) and at just after starting active uterine contractions (p = 0.003) (table 2). Sd, standard deviation; med (p25-p75): median (percentile 25-percentile 75). The higher the score, the more readiness . Results of the independent t - test . Results of fisher s exact test . Comparing the groups in terms of the bishop s components showed statistically significant differences only in terms of effacement at 10 - 12 hours (p = 0.019) and 20 - 24 hours (p = 0.023), and station (p = 0.003) and consistency (p = 0.006) at just after starting active uterine contractions (table 3). Data indicates number (percent). In 2 participants from the intervention group and 1 person from the control group who did not report for the 10 - 12 hour follow - up, the dilatation and station at the baseline and at 20 - 24 hours were the same, so the same score was considered for the 10 - 12 hour assessment score . In the control group, 1 person who had a cesarean section at 13 hours after intervention due to meconium staining of the fetus, and 1 person who did not report for the 10 - 12 and 20 - 24 hour follow - ups were omitted from the analysis . For 2 participants from the intervention group and 1 from the control group who delivered before 20 hours after intervention, the maximum bishop s score was considered for the 20 - 24 hour score after intervention . However, the differences were not statistically significant (p = 0.117 and p = 0.609, respectively). Also, there was no statistically significant difference between the groups in terms of the timing of starting spontaneous active uterine contractions (p = 0.372), the duration of the first (p = 0.173) and second (p = 0.615) stages of labor, and hemoglobin (p = 0.854) and hematocrit (p = 0.878) levels at 12 hours after delivery (table 2). Mean neonatal weight was 3266 g (sd 430) in the saffron group and 3272 g (sd 278) in the placebo group (p = 0.954). There was a lower, but not statistically significant, frequency of meconium staining of the fetus (one vs. four, p = 0.110) and neonatal admission in the neonatal ward (1 vs. 6, p = 0.098) in the saffron group compared with the placebo group . Also, there was no statistically significant difference between the groups in terms of the first (p = 0.235) and fifth (p = 1.00) minute neonatal apgar scores, with no apgar scores of less than 7 among either of the groups . This study is the first trial on full - term pregnant women that attempts to determine the effect of oral saffron tablets on cervical ripening in outpatients . The saffron pill recipients had a significant increase in the bishop s mean score 20 - 24 hours after the intervention and onset of uterine contractions as compared with the placebo group, but the time of the onset of contractions was not significantly different between the two groups . The length of the first and second phases of labor was shorter, and fewer women required cesarean section or labor induction, but the differences were not statistically significant . Studies in rats indicate that saffron can stimulate the uterus, cause abortion, and preterm labor (10 - 12). Additional observations have shown an increased probability of abortion in pregnant farmers who had been working in saffron farms during the first trimester of pregnancy (14, 15). Examination of the bishop s score components showed that saffron has the highest effect on effacement and cervical ripening . Considering that one of the causes of abortion in the first trimester is a reduced level of progesterone, and progesterone antagonists such as mifepristone play a role in cervical ripening at the end of pregnancy (2), saffron might therefore have an effect on abortion or premature labor as a progesterone antagonist . Furthermore, another study has revealed the effect of saffron on treating primary dysmenorrhea (17). The main cause of primary dysmenorrhea is prostaglandins and their effect on uterine contractions and narrowing of the cervical canal (24). Therefore, it is possible that saffron may improve primary dysmenorrhea and readiness of the cervix in term - pregnant women with similar mechanisms, i.e., cervical ripening, but further lab investigations and clinical trials are needed for additional support . A review study by gulmezoglu et al . (1) showed that cervical ripening before labor induction leads to shorter latent and active phases . Although the results of the present study showed shortening of the first and second phases of labor in the saffron group, the difference was not significant . This may be related to the small sample size in this study . A major concern about using misoprostol and oxytocin for labor induction is extreme uterine contractions, and increased risk of the need for cesarean section as well as other side effects for the mother and the fetus (26 - 28). No abnormal uterine contractions occurred in the present study and the need for cesarean section and meconium staining were reduced, although the reductions were not statistically significant . Furthermore, another major concern about medicinally intervening is the possibility of adverse side effects . (26) used isosorbide mononitrate for labor induction in outpatients, of whom 66% complained of headache with different intensities, while there were no reports of similar such side effects in the present study . Another study that utilized saffron tablets for therapeutic and research purposes at higher doses than were used in the present study did not report a significant effect in clinical parameters or the occurrence of any side effects (17, 29). Performing outpatient interventions and having patients take the tablets themselves is a strength of this intervention . In their review study, kelly et al . Concluded that there is little available data on the efficacy or potential risks of labor induction in outpatients, so it is not possible to determine the applicability and safety of such labor induction at this particular time (30). On the contrary, dowswell et al . (31) revealed in their review study that some medications and forms of acupuncture were safe to use for cervical ripening in low - risk pregnant women . Because there were no interventional studies on the effect of saffron in pregnant women and considering its possible maternal and fetal side effects, the intervention was performed for a short time and at low doses in low - risk full - term women . Therefore, the results cannot be generalized for labor induction in other situations, particularly in post - term and high - risk pregnancies . Moreover, further studies are required to determine the optimal dose, the most effective route of administration, and the optimal duration of performing such an intervention for cervical ripening and labor induction . Moreover, it will be useful to investigate the effect of this method along with other conventional methods of labor induction . Given the results of the present study, it seems that oral saffron is effective for the cervical ripening of full - term pregnant women . Although the number of cesarean sections and the amount of meconium staining were lower in the saffron recipients, the difference was not significant . However, the study limitations, including the sample size, do not allow for any definite conclusion for the use of saffron in clinical practice, and more research is needed to assess its effect on delivery and neonatal outcomes and its potential side effects . Given the results of the present study, it seems that oral saffron is effective for the cervical ripening of full - term pregnant women . Although the number of cesarean sections and the amount of meconium staining were lower in the saffron recipients, the difference was not significant . However, the study limitations, including the sample size, do not allow for any definite conclusion for the use of saffron in clinical practice, and more research is needed to assess its effect on delivery and neonatal outcomes and its potential side effects.
The online version of this article (doi:10.1007/s40121 - 014 - 0043 - 9) contains supplementary material, which is available to authorized users . A member of the togaviridae family, infection with the chikungunya virus (chikv) has emerged as a major public health concern, resulting in outbreaks of fever and debilitating arthralgia in the caribbean and globally . The first autochthonous (native) transmission of chikv in the caribbean was confirmed in december 2013 on the french island of saint martin . Rapid spread and local transmission of chikv occurred in the caribbean and the americas within 9 months (fig . 1). This has resulted in 651,344 suspected and 9,182 laboratory - confirmed chikungunya cases in the caribbean and the americas as reported by the centers for disease control and prevention . The effective management of the rapid spread of chikungunya is integrally related to the geographic distribution and re - emergence of competent mosquito vectors, evolving chikv genotypes, susceptible human populations, environmental influences, and social mobilization . Understanding the contribution of these factors is important to improving the control of the global transmission of this disease.fig . 1introduction of chikungunya to the caribbean by epidemiological week first reported december 2013 to august 2014 . Data from paho / who statistics introduction of chikungunya to the caribbean by epidemiological week first reported december 2013 to august 2014 . There are three distinct chikv genotypes: west african, east central south african [ecsa; or indian ocean lineage (iol)], and asian [1, 5, 6]. These genotypes represent the evolution of the virus in keeping with the geographic distribution over the years . In africa, this virus is transmitted through enzootic (sylvatic) and urban (human mosquito cycle) cycles among non - human and human primates by aedes mosquitoes species . Previous authors report the probable existence of the chikv in africa for hundreds of years, with the first suspected epidemic documented in 1779 . Subsequent reports included outbreaks in nigeria (1964), the ivory coast (1981), and senegal (1983) associated with the west african strain . The resurgence of chikungunya in the twentieth century was first documented in 1953, in the newala district of tangaiynika (tanzania). The chikv continued to spread through the suspected vectors aedesfurcifer / aedescordellieri, to other south african countries, inclusive of rhodesia (zimbabwe; 1961, 1962, and 1971). Epidemics associated with the ecsa lineage subsequently occurred in cameroon (2006) and gabon (2007) with aedesafricanus and aedesalbopictus reported as the principal vectors, respectively . In gabon in 2007, concurrent chikv and dengue fever epidemics were also experienced, with chikv transmission linked to the a.albopictus mosquito . The iol strain originated in coastal kenya in 2004 and 2005 (affecting 75% of the lamu island s population, via the vector aedesaegypti) and then spread to the reunion island (affecting one - third of the population) and was subsequently introduced to other islands of the indian ocean and continental india, via the vector a.albopictus [1, 57, 12]. It is believed that the 2007 outbreak in italy was initiated through the importation of the chikv by a traveler from reunion island . Other countries in which international travel facilitated the spread of chikv included france (from travelers from reunion island; 77%), comoros (14%), and mauritius (9%). Autochthonous transmission in france, however, was not detected until the year 2010, in association with a. albopictus . Studies have reported a high efficiency of transmission of chikv in the temperate climates by the a. albopictus species . There are several aedes species which are competent in the transmission of the chikv, with a.aegypti and emerging populations of a.albopictus identified as responsible vectors in asia . The asian strain identified in thailand in 1958 was transmitted by the suspected vector, a. aegypti . This asian strain was also reported in outbreaks in india (19621965) and southeast asia (19581960 and 19621964), also in association with the a. aegypti mosquito . Epidemics of the 1980s and 1990s in southeast asian countries (philippines, thailand, myanmar, and indonesia) were associated with the asian endemic / epidemic chikv lineage . Since then, the asian endemic / epidemic chikv lineage has continued to circulate sporadically, transmitted primarily among humans by a.aegypti and was again documented in the 2006 outbreak in malaysia [1, 7]. Emergence of an iol strain with resulting replacement of the endemic asian strain occurred during the southeast asia epidemics of 20062009 [1, 7]. It is thought that the e1 envelope glycoprotein mutation of the endemic asian genotype resulted in the decreased adaptability of the chikv to a.albopictus, with decreased transmission, and the facilitation of chikv evolution and strain replacement . The caribbean region continues to encounter challenges with the control of mosquito populations including a.aegypti and other aedes species . The aedes mosquito was introduced to the caribbean through the african slave trade . Following its introduction, a significant increase in the a. aegypti population density was associated with the intercontinental exchange activities of the second world war . Public health interventions of national health ministries and the pan american health organization in the 1950s and 1960s successfully reduced a.aegypti vector indexes by 1972 . This success, however, was short lived and the re - infestations of the latin american and caribbean regions by a.aegypti in the late 1970s was accompanied by the introduction of a.albopictus in the early 1980s to some caribbean countries . Although both a.aegypti and a.albopictus may be found in the caribbean, a.aegypti remains the principal vector responsible for the transmission of chikv and dengue in this region . To date, the chikv lineage identified in the 2014 caribbean outbreak belongs to the old asian lineage . The presence of the asian lineage chikv, compounded by a nave caribbean population and high prevalence of a.aegypti, makes the probability of establishment of endemicity of chikungunya in the caribbean high . This risk of establishment of endemicity extends to most tropical and subtropical regions of latin america areas and the southern usa, which is presently experiencing challenges with a.aegypti re - infestation . In the months preceding the caribbean s first case, china and the philippines accounted for 27.5% of travelers into the caribbean . There were ten cases reported in spain from travelers from haiti and dominica republic during april to june 2014 . The expansion of the chikv to north and south america from the caribbean is of great concern . In their review of air travel from the caribbean, noted that the final destination of 52.1% of international travelers from the chikungunya - infected caribbean territories was the usa . The most likely cities to be travelled to from the caribbean were new york (13.8%), paris (11.7%), miami (7.8%) and san juan, puerto rico (3.9%). It is expected that the change of climatic conditions, compounded by the increase of the warmer weather in temperate zones, will facilitate the further spread of this disease . The months june through to november are designated as the hurricane season in the caribbean, or the wet season . The wet season has been shown to be associated with the increased breeding of the a.aegypti mosquitos . Florida reported its first two cases of autochthonous chikv transmission in july 2014 and, as of september 9, 2014, remains the only us state with autochthonous transmission . Florida now has eight cases of local transmission and 184 travel - associated laboratory - confirmed chikungunya cases as reported to arbornet . The impact of the introduction of chikv into the caribbean region, a known tourist destination exchange of an estimated 20 million persons annually of which 9 million are us residents, remains to be seen . The acquisition of chikv by travelers to the caribbean enhances the spread to neighboring caribbean countries and globally . As of epidemiological week 35, among the caribbean countries affected, those with the highest incidence rates (incidence/100,000 population) include: guadeloupe (16,465), martinique (15,087), saint martin (french part 13,401), saint barthelemy (11,707), dominica (5,068), dominican republic (4,128), and haiti (627) (fig . 1). The emergence of chikungunya in the caribbean unfolds a story of the dynamic evolution and interaction of microbes and risk factors underlying the determinants of health . The interaction of economic trade and its role in the transmission of vectors and infectious agents needs to be reviewed and policies reinforced and implemented . . Continued education to increase the awareness of this disease is needed to curtail the population growth of mosquitoes and to mitigate the spread of chikungunya by travelers to the usa and other regions . Improved awareness of the epidemiology and clinical manifestations preventative measures and epidemiological monitoring of the evolving chikv epidemic in the caribbean must be improved and sustained . This article does not contain any new studies with human or animal subjects performed by any of the authors . Lizette mowatt and sandra t. jackson have no conflict of interest to declare with respect to this article . This article does not contain any new studies with human or animal subjects performed by any of the authors . This article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
There has been a striking upsurge in tuberculosis (tb) vaccine research in china in recent years . More than 80 papers reporting such research have been published since 2004 (figure 1), many in english language journals . This has been driven and funded largely by chinese government agencies, since the scale of the tb problem and the difficulties in implementing fully effective control measures were recognized ., we can note that there are at least two additional restricting factors limiting the progress of the field in china: few research groups have access to facilities for safely working with animals infected with a dangerous pathogenic organism such as the tubercle bacillus; infrastructure for undertaking vaccine clinical trials to international standards is sparse . In consequence, the universal problem of how to select among the promising approaches and candidate vaccines is particularly acute in china . Nevertheless, the basic research problems are being tackled with some enthusiasm and creativity, and the new 5-year program to improve tb control that was announced by the chinese ministry of health on 1 april 2009 will have an impact . The antigens investigated to date and the locations of research groups are listed in tables 1 and 2, respectively . A substantial body of work has gone into characterizing the immune responses to be found in tb infection and following vaccination . The twin purposes of identifying candidate antigens for use in vaccines and diagnostic tests and of defining immunological markers for latent infection and disease states drive these studies . These studies have not always been followed up with tests of protective or therapeutic efficacy in animal infection models . The increased immunogenicity of mouse dendritic cells transfected with antigen heat shock protein 65 (hsp65) has been tested in wuhan and in hong kong, and differences have been found in the polarizing effect of intracellular mycobacterium bovis bacille calmette guerin (bcg) vaccine bacteria on antigen presentation by human adult and cord blood dendritic cells . Various forms of recombinant bcg expressing early secreted antigen 6 of mycobacterium tuberculosis (esat6), or antigen 85b (ag85b) and antigen rv3425, or ag85b and esat6 with tumor - necrosis factor-, or esat6 and human granulocyte macrophage - colony stimulating factor (gm - csf) have shown enhanced immunogenicity . Recombinant m. smegmatis expressing m. tuberculosis culture filtrate protein 10 (cfp10)/esat6 fusion protein was found stimulatory for macrophage inducible nitric oxide synthetase . Recombinant m. smegmatis and bcg strains have been made that can express cloned antigens at a range of different levels under the control of modified fura gene promoters . Other bcg recombinants expressing esat6, esat6/interleukin 2 (il-2) or ag85b / esat6 fusions have been made . A range of known antigens has been tested for immunogenicity as mixtures, fusion proteins and peptides . These include chimeric ag85b / esat6 with adjuvants monophosphoryl lipid a (mpl) and trehalose 6,6'-dimycolate, hsp16.3 with dimethyl - dioctadecyl - ammonium bromide / mpl (dda / mpl) adjuvant, fusion protein m. tuberculosis protein 64 (mpt64)esat6, resuscitation - promoting factor b (rv1009), or cfp10, esat6 or rpfe (rv2450) with nitrocellulose, or rv3772 and rv3425 with incomplete freund's adjuvant; or ag85b / mpt64190198/mtb8.4 plus a novel adjuvant of dda with bcg extract . In - silico analysis of putative mhc class 1-restricted epitopes present in antigens encoded within the region of difference 1 (rd-1) to rd-16 regions of m. bovis genome has revealed potential high - affinity hla binders and profiles of human humoral responses to 38-kda, mtb48, cfp10/esat6 antigens have been defined . Screening of human immune sera against an expression library of m. tuberculosis open reading frames revealed three novel antigens among the top 20 most strongly recognized: rv1987, rv3807c and rv3887c . More than a dozen studies have used dna vaccination as a means of delivering antigens in immunogenicity tests . Many have shown th1-biased immunogenicity without additional adjuvanting, for example with ag85b, ag85b / esat6 fusion, esat6/cfp10 fusion, mtb8.4/38-kda / ag85b fusion and epitopes from esat6, ag85a, cfp10 and ag85b inserted within hsp65 . Targeting the expressed product for degradation via the ubiquitin pathway has been used to enhance mhc class 1 presentation of epitopes from mpt64 and 38-kda, mpt64, and esat6 . Enhanced responses to encoded antigens have been obtained by additionally encoding cytokines, such as interleukin 21 (il-21), with ag85a or ag85a / esat6, and gm - csf with ag85a . Inclusion of dna expressing il-12 enhanced prime / boost responses to bcg and to plasmids expressing ag85a and esat6 . Esat6 dna priming and protein boosting has also been shown to give enhanced th1 responses . Many antigen preparations have been tested for their capacity to protect against challenge infection with virulent m. tuberculosis . Subcutaneous esat6/cfp10, or esat6/mpt64 fusion proteins on nitrocellulose protected mice against h37rv challenge, but not as effectively as bcg . Hsp65/il-2 fusion protein was found to elicit better protection than hsp65 given with dda / mpl adjuvant and protection was equivalent to that produced by bcg . Similarly, either hspx, a dormancy associated antigen, or synthetic epitope 91104 gave protection equivalent to bcg when given with dda / mpl . / mpt64190198/mtb8.4 fusion protein with dda adjuvant boosted protection against challenge in bcg primed mice . A similar fusion protein in which mtb8.4 was replaced by hspx gave a similar boost, but boosting with a mixture of the two fusion proteins was even better . A fusion protein of esat6 and ag85a also significantly boosted protection against h37rv in mice . Recombinant bcg expressing the ag85b / mpt64190198/mtb8.4 fusion protein gave slightly better protection to mice against h37rv challenge than parent bcg or bcg expressing rag85b alone . Recombinant bcg expressing a fusion protein of human interleukin (hil)-12p70 and esat6 showed increased immunogenicity but less protective effect . Recombinant salmonella typhimurium that both expressed esat6/ag85b fusion protein and delivered it as a dna vaccine when given orogastrically gave protection similar to subcutaneous bcg and the combination was superior to either vaccine alone . The earliest reports indicated protection in mice superior to bcg when a divalent construct expressing both ag85b and mpt64, or a mixture of plasmids expressing ag85b, mpt64, mpt63 and esat6 was used . The protection given by a mixture of three plasmids expressing mpt83, ag85b and esat6 was enhanced by including dda adjuvant and an encoded fusion protein of ag85b and mpt64 was superior to the separately encoded antigens . Encapsulation in poly(lactide - co - glycolide) microspheres with dda enhanced the protective efficacy in mice of dna - encoding ag85b / mpt64/mpt83 fusion antigen, and strikingly the dna mixed with dda was superior to bcg in protecting cattle against challenge . Inclusion of a plasmid expressing il-2 improved protection by this plasmid or by plasmid expressing mtb8.4 . A mixture of plasmids encoding ag85b, mpt64, mpt70 and tb10.4 boosted protection by bcg, as did a plasmid expressing a cfp21/mpt64 fusion protein . Ag85b or ag85a were superior to esat6 when compared separately for protection as dna vaccines . Dna expressing a fusion of mpb64/ag85b / esat6 was superior to a mixture of plasmids expressing the separate antigens and gave protection equivalent to bcg . The protective effect of dna expressing hsp65 against bcg challenge was enhanced by incorporating epitopes of esat6, ag85a / b and cfp10 within the hsp65 backbone . The protective effect of hsp65 dna against h37rv challenge was increased by expression as a fusion with hil-2, but did not surpass that of bcg . Expression of ag85b fused to bovine herpes virus 1 vp22 protein, which facilitates dissemination of antigen to adjacent cells, resulted in protection against h37rv challenge in mice that was better than protection by bcg . Few studies have been conducted with animals other than mice: a mixture of ag85b, hspx and cfp10/esat6 fusion together with cpg and aloh as adjuvant was immunogenic in mice but gave little protection against challenge with h37rv in guinea pigs; in contrast, combined dna vaccines encoding antigens ag85b, mpt64 and mpt83 given with dda appeared to be better than bcg in protecting cattle . Interest in therapeutic vaccination has been sustained by clinical studies of a commercial chinese product, m. vaccae extract . Recent meta - analyses of published data concluded that this product gave significant benefit in preventing tb in people at high risk (13 studies), but there was only a minor benefit from treating new tb cases (54 studies). In a unique and contrasting approach, a recombinant m. smegmatis delivering dna expressing human granulysin and murine il-12 was recently found to be therapeutic against h37rv infection . Most research into therapeutic vaccines for tb has focused on the use of naked dna vaccination . Dna had a significant therapeutic effect against h37rv in mice and the fusion hsp65/il-2 was significantly better . Treatment of infected mice with a dna vaccine expressing a fusion protein of mycobacterial hsp70 and leukocyte cluster of differentiation antigen 80 substantially reduced acid - fast bacteria and pathology in liver and spleen, whereas bcg had no effect . Although treatment with dna expressing ag85b was therapeutic, treatment with dna expressing mpt64 was not, and the mixture was less effective than the ag85b vaccine on its own, and inclusion of dna expressing il-12 gave a slight additional benefit . Mice infected with a clinical isolate resistant to isoniazid and rifampicin responded well to treatment with the drugs plus dna expressing chimeric ag85a / esat6, and appeared to respond better to treatment with the drugs plus dna expressing ag85a than to the dna vaccine alone; ag85a dna alone was at least as effective as ag85a plus rifampicin in treating mice infected with a strain resistant to rifampicin and isoniazid, ag85a an immunogenic mixture of dna vaccines expressing ag85b, mpt64 and mpt83 has been found to work as a potent adjunct to isoniazid plus pyrazinamide therapy in mice and to be therapeutic in cattle infected with m. bovis, reducing both pathology and bacterial numbers; a mixture expressing ag85b, mpt64 and hsp65 was similarly effective . Inclusion of immunostimulatory nucleotide motifs in the gene transcript enhanced the therapeutic efficacy of a plasmid expressing hsp65 when tested against h37rv in mice . It is evident in considering this body of recent tb vaccine research in china that much of it has been tactical in nature, establishing credentials both nationally and internationally, and building new research bases . Additionally, there are creative and insightful studies of particular relevance to the needs of china . Researchers are now able to exploit cutting edge technologies in designing new tb vaccines and are increasingly able to test the vaccines in relevant animal models of infection . The evidence that dna vaccines can provide effective therapy for tb in cattle may be a significant pointer to the future . The potential benefits of adding immunotherapies / therapeutic vaccines to tb chemotherapy have been recognized in china, but both preventive and therapeutic approaches against human tb await development of clinical trial capacity for their proper assessment.

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