MRSA contamination was shown in 72 buses (36.2%). The majority of the isolates belonged to three major clones: Clone A was identified as EMRSA-15 defined by pattern PFGE A, spa types t2357/t747/t025/t379/t910, ST22, and SCCmec IVh . Clone B was the New York/Japan clone characterized by PFGE B-t002/t10682-ST5-II . Clone C included isolates with characteristics of the international community-acquired USA300 or related clones, PFGE C-t008-ST8-IVa/IVc/IVg/IVnt/VI . The first two clones are currently the two major lineages circulating in Portuguese hospitals. The hands of 15 individuals were contaminated with MRSA belonging to the nosocomial clones A or B. Eleven of these individuals were not nasal carriers of MRSA and all but one had travelled by public transportation, namely by bus, prior to sampling. In conclusion, public buses in two major cities in Portugal are often contaminated with MRSA representing clones dominant in hospitals in the particular geographic area. MRSA contamination of public transport and the transfer of the bacteria to the hands of passengers may represent a route through which hospital-acquired MRSA clones may spread to the community.In a previous study we have shown that public buses in Oporto, the second largest city in Portugal, were highly contaminated with MRSA. Here we describe the results of a similar study performed in another urban area of Portugal–Lisbon, the capital. Between May 2011 and May 2012, hand touched surfaces of 199 public buses in Lisbon were screened for MRSA contamination. Subsequently, the hands of 575 passengers who frequently use these bus lines were also screened. All hand carriers of MRSA were further screened for nasal carriage. The isolates were characterized by PFGE, staphylococcal cassette chromosome (SCC)  Staphylococcus aureus (MRSA) is a major healthcare associated (HA-MRSA) human pathogen responsible for mild to severe life threatening infections worldwide Methicillin-resistant Although, in 2011 the EARSS-Net reported stabilizing or even decreasing MRSA rates in several European countries, Portugal continues to show an increasing trend, reaching a nosocomial prevalence of 54.3%, which represents the highest rate in Europe In contrast, until very recently, the MRSA prevalence in the community has been extremely low. Studies including isolates from nasal swabs of young healthy individuals, nasopharyngeal swabs of children attending day care centers and isolates from soft tissues infections (SSTI) in children attending a pediatric emergency department, reported a prevalence of MRSA lower than 0.9% However, in 2010, the MRSA rate reached 25.9% among SSTI in adults attending heath care centers S. aureus transmission occurs mainly by skin contact, we also screened the hands of individuals who frequently travelled by bus and who may represent a stage in the spread of MRSA clones within the community.The purpose of the present study was to extend these tests to public buses circulating in another urban area in the country - Lisbon, the capital of Portugal. Since The study and the protocol have been previously presented to all students of Escola Superior de Saúde da Cruz Vermelha Portuguesa (ESSCVP) and a verbal informed consent was obtained for each participant  at the time of hand and nasal screenings, which was registered in the respective record sheet.The study and the consent procedure were approved by the Research Department and Ethics Committee of ESSCVP (Gabinete de Investigação and Centro de Bioética da ESSCVP).The transportation agency to which the buses belong is a major public transportation network in Lisbon, with 78 different routes served by 735 vehicles. Daily cleaning (at the end of the day) of the interior of the vehicles is limited to trash removal and floor vacuuming, while in depth cleaning, which occurs every three months only, includes cleaning of the hand touched surfaces with an alkaline detergent (VQ 300 Desengordurante Multi Usos).In sampling for MRSA contamination, we selected surfaces with high levels of hand contact. These included handrails, seat rails, handgrips, stop buttons and the surface of the validation tickets machine. Sampling was always conducted in both sides of a bus, and an attempt was made to sample the same number of surfaces per bus. Sampling was performed every two or three weeks between May 2011 and May 2012 (with the exception of August), at the end of the day and before any cleaning procedure took place.An attempt was made to collect samples from a variety of routes. In each sampling day, six to 12 buses were arbitrarily chosen, some serving major hospitals and others not. Since vehicles were usually assigned to a different route each day, the vehicle identifier number and the route of service were registered at the time of sampling.Samples were collected using two sterile cotton gauzes per vehicle (one for each side), moistened with Tryptic Soy Broth (TSB) . After swabbing the hand touched surfaces, the gauzes were introduced into 100 ml TSB bottles and taken to the laboratory for incubation.Samples were recovered in two periods: in May 2012 for 2 days and in January 2013 for 3 consecutive days. The hands of students and workers who travelled by bus or by other means of public transportation to ESSCVP in Lisbon were swabbed. Individuals were screened in the morning, between 8.00 am and 10.00 am, immediately after entering the school building and before they could wash their hands or touch other surfaces. Samples were collected by swabbing both hands of each individual with a sterile cotton gauze moistened with sterile water and then submerged into TSB for further transport to the laboratory.Individuals showing MRSA contamination on their hands were further swabbed for nasal carriage within a week in order to identify MRSA nasal colonization. Whenever possible, a second hands screening was performed at the same time to reevaluate the persistence or re-contamination of the hands. Nasal screenings were performed on both nares of each individual with a sterile dry cotton swab, which was stored in Stuart transport medium and transferred to the laboratory.All gauzes, from buses and hand screenings, were incubated in TSB at 37°C overnight with orbital agitation (135 rpm) for enrichment and then plated with a sterile cotton swab, onto Tryptic Soy Agar (TSA)  and onto a chromogenic selective media for MRSA, CHROMagar MRSA . Nasal swabs were directly plated onto TSA and CHROMagar MRSA. After 24 to 48 h of incubation at 37°C, all presumptive MRSA colonies (mauve-colored colonies) were tested for coagulase by latex agglutination test Staphaurex Plus  or by agglutination of rabbit plasma in tubes  in case of prior ambiguous results.nuc and mecA genes, respectively MRSA isolates were confirmed at the species level and for methicillin resistance by amplification of the PFGE was performed for all isolates after SmaI digestion as described by Chung et al. spa typing was performed on at least one representative of each PFGE subtype, as previously described spa types were assigned through the Ridom web server (http://spaserver.ridom.de). Representatives of the different PFGE types were further characterized by MLST http://www.mlst.net).mec (SCCmec) was performed using specific primers for each SCCmec type through a multiplex PCR strategy mec gene complex class and ccrAB/ccrC allotypes were determined as previously described mec type IV isolates were subtyped by multiplex PCR Characterization of the staphylococcal cassette chromosome arcA and opp3 loci The presence of PVL was tested in all isolates, as previously described Bivariate analysis of data was performed using the Statistical Package for Social Science version 19 software . Chi-square tests were used for comparison of categorical data. Statistical significance was evaluated at α = 0.05.S. aureus transmission in vehicles, namely crowdedness, significant hand-to-surface contact, particularly as vehicles become full with passengers, and the fact that there is no possibility for hand hygiene during and immediately after the use of the transport. In a previous study conducted in 2011 in Oporto, the second major city of Portugal (approximately 300 km north of Lisbon), we were able to isolate MRSA from 26% of the sampled buses that belonged to a different transportation agency Seventy-two out of the 199 (36.2%) buses that were sampled in Lisbon were contaminated with MRSA. Several factors are expected to contribute to Prior studies in other European countries have failed to isolate MRSA from public transport vehicles 2 = 5.169; p = 0.16) according to the sampling season, being 35% (21/60) in the spring, 29% (9/31) in the summer, 50% (24/48) in the fall and 32% (19/60) in the winter. Four months showed a contamination higher than the 36.2% observed for the total study: these were April (42%), October (67%), November (40%) and December (50%). Little is known about seasonal trends in the transmission of S. aureus within the community, but several authors have demonstrated seasonal variation in S. aureus infections, particularly skin infections, with a preponderance during the summer and fall In the present study, contamination of buses did not vary significantly  of the isolates belonged to three main clones .Clone ST22-IVh, the major clone found in the buses in Lisbon, corresponds to the international clone EMRSA-15, which accounted for 54% of the 629 MRSA isolates recovered in 2006 from 11 Portuguese hospitals scattered all over the country and therefore represented the predominant national nosocomial clone at that time mec IVa, which seem to enhance growth and survival of the clone and therefore make it well adapted to the community environment mec type . A recent study on the population structure of CA-MRSA in Europe showed that most (40%) of the isolates were related to USA300 mec type . These vThe third most prevalent clone in public buses in Lisbon, ST5-II, displays the characteristics of the New York/Japan clone which in 2006 was identified as the major clone in four hospitals located in Lisbon A most important aspect of our findings is the coincidence between the major clones present in hospitals and the same clones recovered from public buses circulating in the same area. Thus, the two more prevalent nosocomial clones, found in public buses, in the Lisbon study correspond to the main clonal types found in Lisbon hospitals in 2006 2 = 4.537; p = 0.033).The participating buses were assigned to 33 different routes, out of which seven do not pass near any hospital and the remaining 26 pass at a maximum distance of 200 m of the entrance of at least one hospital: a single hospital (n = 8), two hospitals (n = 6), three hospitals (n = 4), four hospitals (n = 3), five hospitals (n = 3), six hospitals (n = 1) and seven hospitals (n = 1). A total of eight bus routes served more than three hospitals and 14 bus routes presented MRSA contamination superior or equal to the overall prevalence of 36.2% found in this study, showing an association between the proportion of MRSA contamination and a bus route serving at more than three hospitals  were identical, we assumed that the four individuals were most probably self-contaminated. Moreover, the four individuals were students in clinical training in hospitals and therefore, as health care workers, they have a high risk of MRSA nasal colonization. We considered that 11 out of 575 individuals (2%) presented MRSA contamination on their hands. Molecular characterization distributed the 11 MRSA into three clonal types , correspFive of the 15 individuals showing MRSA on their hands were screened a second time within a week. Two of them showed once again MRSA contamination: (i) one of these individuals was a MRSA nasal carrier and the isolate was identical to the ones previously recovered from the nose and hands; (ii) the second individual was not a nasal MRSA carrier, travelled by public transportation on that day, and showed a different strain relatively to the isolate of the first screening (PFGE A1 instead of A10). These results seem to indicate that MRSA nasal carriage leads to frequent self-contamination of the hands. Moreover, contamination of the hands in non-nasal carriers seems to be transient and probably dependent on the daily public bus transportation.In summary, public buses in major cities in Portugal (Lisbon and Oporto) are a reservoir of HA- and CA-MRSA clones and may represent a mechanism for the spread of MRSA clones in the community. Given the importance of public transportation in most major cities, the results presented here should provide valuable insights to epidemiologists, infection control and environmental health professionals, to better understand the dynamics of MRSA in the community. The study should also serve as incentive to transportation agencies to introduce more effective procedures to reduce MRSA contamination.


Adherence to Internet-delivered lifestyle interventions using multiple tailoring is suboptimal. Therefore, it is essential to invest in proactive strategies, such as periodic email prompts, to boost re-use of the intervention.This study investigated the influence of content and timing of a single email prompt on re-use of an Internet-delivered computer-tailored (CT) lifestyle program.A sample of municipality employees was invited to participate in the program. All participants who decided to use the program received an email prompting them to revisit the program. A 2×3 (content × timing) design was used to test manipulations of prompt content and timing. Depending on the study group participants were randomly assigned to, they received either a prompt containing standard content (an invitation to revisit the program), or standard content plus a preview of new content placed on the program website. Participants received this prompt after 2, 4, or 6 weeks. In addition to these 6 experimental conditions, a control condition was included consisting of participants who did not receive an additional email prompt. Clicks on the uniform resource locator (URL) provided in the prompt and log-ins to the CT program were objectively monitored. Logistic regression analyses were conducted to determine whether prompt content and/or prompt timing predicted clicking on the URL and logging in to the CT program.P=.005) and after 6 weeks . Furthermore, participants who received an email prompt 2 weeks after their first visit logged in to the program significantly more often compared to participants receiving the prompt after 6 weeks . A trend was observed with regard to prompt content. Participants receiving a prompt with additional content were more likely to log in to the program compared to participants who received a standard prompt. However, this result was not statistically significant .Of all program users (N=240), 206 participants received a subsequent email prompting them to revisit the program. A total of 53 participants (25.7%) who received a prompt reacted to this prompt by clicking on the URL, and 25 participants (12.1%) actually logged in to the program. There was a main effect of prompt timing; participants receiving an email prompt 2 weeks after their first visit clicked on the URL significantly more often compared with participants that received the prompt after 4 weeks (odds ratio [OR] 3.069, 95% CI 1.392-6.765, The key findings suggest that boosting revisits to a CT program benefits most from relatively short prompt timing. Furthermore, a preview of new website content may be added to a standard prompt to further increase its effectiveness in persuading people to log in to the program. Internet-delivered lifestyle interventions applying computer-tailoring techniques -3 have rResearch on non-usage attrition has demonstrated an initial rapid decline in program use over the first few weeks ,21. DuriA substantial amount of Internet-delivered interventions use reactive strategies to achieve re-use, implying that a passive approach is used in which users themselves must undertake action to repeatedly benefit from the intervention content . HoweverMore specific evidence on the positive effects of the use of email to promote direct action stems from the field of e-marketing ,35. WithThis study aimed to assess the added value of periodic email prompts to boost revisits to an Internet-delivered CT lifestyle program. We aimed to answer 2 questions: (1) which prompt timing interval is most effective in boosting re-use of the Internet-delivered CT lifestyle program and (2) which prompt content is most effective in boosting re-use of the program? We addressed these questions among participants in an Internet-delivered CT intervention aimed at multiple health behaviors: increasing physical activity, increasing fruit and vegetable intake, smoking cessation, and decreasing alcohol consumption.This study was conducted in close collaboration with the Regional Public Health Service (RPHS) in the northern part of the Dutch region, Limburg. All people employed by the municipalities in this region were invited by the RPHS to participate in an Internet-delivered CT lifestyle program developed by our research group ,42. InviParticipants who logged in to the program to obtain CT advice were sent an email prompting them to re-use the program. Re-use was encouraged to allow participants to monitor their behavior change and to obtain iterative CT advice aimed at behavior change and relapse prevention. Furthermore, participants were offered an opportunity to obtain CT advice regarding an additional health behavior. Finally, re-use was stimulated to keep participants informed about new content added to the program website.A 2×3 design was used to test the effect of 2 factors: prompt content (standard and standard+) and prompt timing . The standard prompt (SP) contained a message reminding people about their previous visit to the program and invited them to re-use the program to monitor their progress and obtain additional feedback. The second version of the prompt (SP+) contained standard content complemented with a message alerting people to new content added to the intervention website. Participants received an email prompt after 2, 4, or 6 weeks. In addition to these 6 experimental conditions, a control condition was included, no prompt (NP). People allocated to the control condition did not receive an additional prompt and were only encouraged at baseline to re-use the program. To participate in the program, participants had to register by using a personal log-in code and password. Immediately after registration for the program, participants were randomly allocated to 1 of the 7 study conditions. Randomization occurred at the respondent level by means of a computer software randomization device. Data for the present study were collected from March to July 2011.People who were allocated to the experimental conditions of the study received an email prompting them to re-use the program. Periodic prompts should be distinguished from reminders, which are also often used in Internet-delivered interventions. Sending reminders is a technique that is used to increase response rates in online data collection by proactively stimulating participation among nonresponders and is used to prevent drop-out attrition ,43. PeriThe CT program integrated established CT programs tested and proven to be effective in randomized controlled trials for increasing smoking cessation, promoting the intake of fruit and vegetables, increasing the level of physical activity, and reducing the consumption of alcohol -48. The The program was embedded in a website designed for the current study. This website entailed general information considering a healthy lifestyle and the selected health behaviors. Furthermore, the website provided specific information regarding the project, contained a direct link to the CT program, and provided background information regarding the study and the research team. During the study, new information  was added to the website.Participants in the experimental conditions (SP and SP+) received an email prompting them to re-use the intervention. Participants had to take 2 steps to re-use the program in order to self-monitor their level of behavior change and obtain additional CT advice. The first was clicking on the URL provided in the email prompt. Clicking on the URL was tracked by referral ID codes that were integrated in the URL. The second step was logging in to the CT program after arriving on the program website. Log-ins were objectively monitored during a 2- week period and log-in dates were compared to baseline dates to determine revisits.To describe characteristics of program visitors, age, gender, and educational level of participants was assessed . Health behavior status consisted of information regarding the 5 key behaviors. Physical activity was measured by the Short Questionnaire to Assess Health-enhancing physical activity (SQUASH)  and guidGeneral descriptive statistics were calculated to describe characteristics of program visitors, as well as main findings concerning adherence to the public health guidelines. Baseline differences between the intervention groups were calculated using the Chi-square test for dichotomous and categorical variables and 1-way analysis of variance (ANOVA) for continuous variables. Finally, logistic regression analyses were conducted. There were 2 dependent variables: whether participants clicked on the URL  and whether participants logged in to the CT program . Prompt content  and prompt timing (dummy coded with 2 weeks as the reference category and 4 weeks as the reference category) and the interactions between these variables were used as predictors in the initial model of each dependent variable. Furthermore, because women and people with a higher educational level and age are more likely to use Internet-delivered lifestyle interventions ,58, age,26= 9.1, P=.17). Furthermore, there were no significant differences between the groups regarding educational level  and age . Overall visitors had a mean age of 50 years (SD 14.99) and most were medium  to highly educated . Regarding the 5 health behaviors included in the program, 12.9% (31/240) did not comply with the Dutch guidelines of at least 30 minutes of moderately intensive physical activity on at least 5 days of the week. With regard to fruit and vegetable intake, 50.8% (122/240) and 60.0% (144/240) did not adhere to the Dutch guidelines of at least 2 pieces of fruit and at least 200 gram of vegetables each day, respectively. Approximately 1 out of 10 participants indicated that they smoked , and 22.1% (53/240) did not comply with the Dutch guidelines for alcohol intake.In total, 240 participants visited the program, of which 73.3% (176/240) were female. Participants were randomly allocated to each of the 7 study conditions and a randomization check revealed that females were equally distributed  reacted to this email by clicking on the URL (step 1), whereas 25 participants (12.1%) actually logged in to the program (step 2). All results concerning clicking on the URL and logging in to the program are described per study group in There was no significant interaction between prompt content and prompt timing  with reg22 = 15.2, P = <.001). Participants who received an email prompt 2 weeks after their first visit, clicked on the URL significantly more often compared with participants that received the prompt after 4 weeks  and after 6 weeks . There was no significant difference in reaction to the email prompt between participants receiving the prompts after 4 weeks, compared with participants receiving the prompts after 6 weeks. Also, no main effects of prompt content could be detected . Participants receiving an email prompt 2 weeks after their first visit, logged in to the program significantly more often compared to participants receiving the prompt after 6 weeks . There was, however, no significant difference between participants receiving the prompts after 4 weeks compared with participants receiving the prompts after 2 or 6 weeks. With regard to prompt content, a trend was observed. Participants receiving a prompt with additional content were more likely to log in to the program compared to participants who received a prompt with standard content. However, this result was not statistically significant .With regard to logging in to the program, analyses of main effects indicated that there was a significant effect of prompt timing (χP<.001).Finally, all experimental groups were compared with the control condition (NP) that did not receive an additional email prompt to boost revisits to the intervention. Results from this analysis revealed that participants who received an email prompt containing additional content after 2 weeks were significantly more likely to log in to the program , but also to the proportion of log-ins to the program (step 2). Secondly, a trend was observed indicating that adding a preview of new website content to a standard prompt increased its effectiveness in persuading people to log in to the program. Finally, sending a prompt with additional content after a 2-week period significantly increased program log-ins compared to using a reactive approach in which no additional prompts were used.The effectiveness of using a relatively brief interval for sending a prompt may indicate that people tend to forget about the program. Therefore, the interval between engaging in a first visit and receiving an email prompt to boost re-use should be kept relatively short. Since the costs of sending a large number of email messages are relatively low , this stResults from the current study also show a trend toward the efficacy of adding additional content to a standard prompt. However, this trend was only present for logging in to the program (step 2) and not for clicking on the URL (step 1). A possible explanation for this effect on log-ins might be the repeated exposure to new website content. People who received an email prompt containing a preview of new website content were exposed to this website content twice: within the email prompt and on the program website. According to ease of processing theory , repeateopt-in email is used when receivers have agreed to the receipt of an email [With regard to clicking on the URL (step 1), significant effects of adding a peripheral cue to a standard prompt failed to occur. People were significantly more likely to respond to emails that were sent at a relatively short interval, irrespective of the email content. A possible explanation for this finding might be that participants consented to participate in the present study after being informed about the possibility of receiving an additional email to boost program use. Within the field of e-marketing, the term an email ,61. Resean email ,61. TherThe current study primarily focused on the effectiveness of using a single email prompt with regard to clicking on the URL and logging in to the program. Combined with results from our previous study , importaWe found that using relatively short prompt timing (2 weeks) resulted in more positive effects compared to using a longer time period. This effect not only referred to clicking on the URL, but also to the proportion of log-ins to the program. Although a trend was observed about the effectiveness of adding a preview of new website content to a standard prompt, this only referred to persuading people to log in to the program. The findings of this study underline the importance of sending an email prompt relatively shortly after a first visit to the program. Furthermore, it is important to further focus on prompt content because the addition of a peripheral cue may add to the prompt’s effectiveness.


Sc), which is the main component of prions, was efficiently incorporated into the intestine in suckling mice but not in weaned mice. Furthermore, suckling SCID mice lacking maternal antibodies showed decreased uptake of PrPSc into the intestine compared with suckling wild-type mice, while the lack of PrPSc uptake into the intestine of suckling SCID mice was rescued by the oral administration of IgG. These findings raise the possibility that the neonatal Fc receptor (nFcR), which contributes to the uptake of maternal antibodies into the intestine, plays a role in PrPSc incorporation into the intestine. The present immunohistochemical study further showed that the FcR blocker Z-ε-aminocaproic acid (ZAA) inhibited PrPSc incorporation into the intestinal villi of suckling mice, supporting the above mentioned concept. Therefore, our findings provide strong evidence that nFcR and maternal antibodies are involved in PrPSc incorporation into the intestine before the weaning period.Prion diseases are a family of neurodegenerative zoonotic foodborne disorders. Although prions can be transmitted orally, the mechanism by which prions are incorporated into the intestine remains unclear. Our previous studies have shown that an abnormal isoform of prion protein (PrP C (cellular prion protein), to a form with a high β-sheet content, PrPSc , that is protease resistant and infectious Prion diseases are a unique category of illness, the pathogenesis of which is related to conformational changes in the normal protein, PrPSc from the gut and into the lymphoid system during oral infection Sc was detected by immunohistochemistry in villous lacteals and the submucosal lymphatic system from 15 min to 3.5 h post-challenge and also in dendritic-like cells in the draining lymph nodes until 24 h post-challenge. This suggested a transepithelial pathway for prion entry through the mucosal epithelium rather than a pathway through M cells in Peyer's patches Sc passes through dendritic cells and accumulates in mesenteric lymph nodes, prior to invading neurons. On the other hand, in the M cell independent pathway, PrPSc is taken up by epithelial cell transport and directly accumulates in the enteric nervous system (ENS). The former is the most accepted pathway, whereas the latter was only suggested recently Sc was detected in the dome epithelium but most was incorporated through the villous epithelia of Peyer's patches. This indicated that uptake through the villi is important for the intestinal epithelial invasion of PrPScSc incorporated by suckling SCID mice lacking maternal immunoglobulins (Ig) Sc incorporated by suckling SCID mice was increased when immunoglobulin G (IgG) was administered orally together with PrPSc. It was therefore suggested that maternal immunoglobulins or the neonatal Fc receptor (nFcR), which is expressed on columnar epithelial cells and is responsible for taking up maternal antibodies into the body, play a role in the incorporation of PrPSc through epithelial cells Sc and IgG.Epithelial M cells are considered to be involved in the transmigration of PrPSc incorporation, the effect of the FcR blocker Z-ε-aminocaproic acid (ZAA)   [10] on Immunohistochemistry was applied to detect IgG using sheep anti-mouse IgG. IgG was detected in the villi in the group in which only IgG was administered . On the Sc was detected by immunohistochemistry using the T2 antibody. PrPSc was incorporated through the villous epithelium and detected in the lamina propria in the group in which only PrPSc was administered (Sc into the villi was significantly decreased in the group in which PrPSc was administered after ZAA treatment as well as in the group in which PrPSc and ZAA were administered at the same time (Sc-positive cells (cells per area) and ratio of PrPSc-positive cells (%). Inhibitory effects of 70.1% and 51.5%, respectively, were seen in the latter two groups  still remains unclear. Previous reports have shown that immune cells participate in the pathogenesis of murine prion diseases Sc was previously detected in the enteric nervous system (ENS) of an orally challenged rodent model of TSE, suggesting that PrPSc enters the body via the intestinal mucosa Sc after its ingestion involves the binding of PrPSc to the LRP followed by its internalization. It is considered that epithelial M cells might be involved in the transmigration of PrPSc from the gut and into the lymphoid system Sc has also been detected in villous epithelial cells and is resistant to degradation by gastric juices and intestinal enzymes because of its abundant stable β-sheet structure.PrPSc incorporated by suckling SCID mice lacking maternal immunoglobulins Sc was upregulated when IgG was administered together with PrPSc. Therefore, it was suggested that maternal immunoglobulins and nFcR play roles in the incorporation of PrPSc through epithelial cells. The nFcR binds to the IgG antibodies ingested in maternal milk and transports them through enterocytes to the systemic circulation of the newborn Sc infection.In a previous study, the amount of PrPSc and IgG after the blocking of nFcR. As described, the amount of IgG incorporated was decreased by ZAA treatment, and the degree of PrPSc incorporation in suckling CD-1 mice administered PrPSc and ZAA was significantly decreased compared with that in suckling CD-1 mice who were administered PrPSc alone. These findings support those of our previous reports and show that nFcR is associated with the incorporation of PrPScin vivo experiments using mice with ZAA before and after prion infection would be necessary to determine the effect of blocking Fc receptors on disease progression in their model.In the present study, we focused on the changes in the incorporation of PrPFifteen-day-old CD-1 mice  were housed in specific pathogen-free (SPF) conditions under an alternating 14 h/10 h light/dark cycle. The animals were given free access to standard laboratory food  and tap water and were treated in accordance with the procedures authorized by the Animal Experiment Committee of Nihon University College of Bioresource Sciences. This experiment was permitted by the guidelines for the care and use of laboratory animals approved by the College of Bioresource Science, Nihon University .To confirm the effect of ZAA (patent No.: WO2004/058747), a derivative form of ε-aminocaproic acid Sc. First, 15-day-old CD-1 mice (n = 3) were administered PBS containing ZAA (30 mg/ml). After 2 h, they were then administered 10 mg/ml emulsion of mouse brains infected with mouse-adapted scrapie (PrPSc) . In the second experiment, the same age mice were administered 10 mg/ml emulsion of mouse brains infected with mouse-adapted scrapie diluted with PBS containing ZAA (30 mg/ml). Then, the administration of PrPSc was repeated 3 h later, and mice were euthanized with ether at 1 h post-administration (p.a.) the.Two experiments were performed in order to investigate whether the Fc receptor inhibitor, ZAA, affects the incorporation of PrPAfter the mice had been euthanized with ether, their intestines were removed and fixed by immersion in PBS containing 4% paraformaldehyde for 2 h before being washed in PBS containing 6.8% sucrose. After dehydration in 100% acetone for 1 h, the tissue samples were embedded in resin  in accordance with the manufacturer's instructions and sectioned at a thickness of 4 µm.2 at pH 7.8 containing 0.01% trypsin for 10 min at 37°C and then quenched in 0.3% hydrogen peroxide in methanol for 30 min. For IgG detection, goat anti-mouse IgG antibody  was directly incubated at room temperature for 30 min. For detection, incubation with mouse anti-PrP monoclonal antibody  was performed at 37°C for 2 h followed by incubation with goat anti-mouse IgG antibody  for 30 min. Diaminobenzidine  was applied for 10 min, and then the sections were counterstained with hematoxylin for 1 min. The number of IgG positive cells in each microscopic visual field was counted at five random points in the villous epithelium. Cell counts are expressed as the mean ± SD of microscopic fields viewed at ×400 magnification. Intestinal epithelial cells were used to determine the intensity of infection, which was not known to the observer assessing the respective intestinal sections. And the inhibitory effect was calculated as the following formula.The resin sections obtained from the mice treated with IgG were pretreated with 0.1% CaClSc with ZAA treatment)/ ×100The inhibitory effect  =   and the inhibitory effect was analyzed by student's t-test. * p<0.05 **p<0.01.The statistical significance of differences compared to oral administration of IgG or PrP


Sc in Peyer’s patches (PP) of the small intestine of orally BSE infected cattle. In this follow-up study additional jejunal and ileal PP’s and ileocaecal-junction tissue samples from 1, 4, and 24 months post infection (mpi) were examined by mouse (Tgbov XV) bioassay. Infectivity was demonstrated in ileal PP’s 4 mpi and the distribution/extent of infectivity at 24 mpi was comparable to those seen at earlier time points, revealing no indication for a decline/clearance. These data are relevant for the definition of Specified Risk Materials in the context of the TSE legislation worldwide.Recently we have described the distribution of bovine spongiform encephalopathy (BSE) infectivity and/or PrP Sc). PrPSc can be distinguished from its cellular progenitor by its partial proteinase K resistance and hydrophobicity which lead to the formation of fibrils in-vivo and/or in-vitro. Classical BSE was transmitted by the oral uptake of infectious feedingstuff [Sc accumulations in the ileum [Sc up to an age of 24 mpi. This follow up study presented here therefore intended to clarify when BSE infectivity can be found in the small intestine at the earliest time point post infection and also focused on older cattle to prove whether the undulant pattern seen in IHC is reflected by varying infectivity levels. These data are important for a risk-based definition of the gut associated SRM which is needed in the currently ongoing discussion in the EU whether restrictions for the intestinal SRM tissues can be alleviated.Bovine spongiform encephalopathy (BSE) belongs to the group of transmissible spongiform encephalopathies (TSE) and is associated with the accumulation of a pathological isoform of a host-encoded glycoprotein, prion protein  were examined here. Anamnestic data are listed in Table Sc by a PTA immunoblotting method as described before [As described before ,13, 56 Sd before .Sc analysis and of the bioassay are shown in Table Results of the PrPInfectivity levels at the different localizations of the small intestine were unequal, with the highest levels in the ileal PP’s followed by the ileocaecal-junction and jejunal samples. For ileal PP’s (5/6) highest transmission rates (mostly between 66% and 100% of the mice affected) and shortest incubation periods  were observed in the bioassay. Only one animal  displayed sparse amounts of infectivity in this sample. More variable were the results obtained for ileocaecal-junction samples. At this location cow IT24 (24 mpi) carried infectivity levels comparable to those in the ileal PP’s, while two other cows  showed only low infectivity levels at this site, as indicated by low bioassay transmission rates  for most animals 24 mpi, and none in the animals that were sacrificed earlier. However, there was also one cow which was in the late preclinical state, IT 26 (24 mpi), where the jejunal PP’s yielded a bioassay transmission rate of more than 50% with an average incubation time of 440 days.Sc were seen in single follicles of the ileal PP’s at four mpi [Sc, these results support the theory that the latent period post exposure during which no detectable infection is present might be around two or three months.In this follow-up study we have mapped the exact temporal and spatial emergence and distribution of infectivity in the PP’s of the small intestine in orally BSE infected cattle. According to IHC results described previously ,11, infefour mpi . This isfour mpi . As the Sc at 24 mpi, since the extent of this accumulation varied at earlier time points with peaks at 8 and in particular 12 mpi and lows at 16 mpi respectively [Secondly we were interested in the amount and distribution of PrPectively . Howeverectively ,14. Thisectively -11,15.In summary, data presented here clearly showed that infectivity is not detectable in the small intestine of animals up to four months post experimental oral exposure with an extremely high dose. Moreover, the low amounts of infectivity detectable after the peak at 12 mpi as demonstrated previously, does not imply an irreversible clearance of the infectious agent from the gut over time, but is rather a time-dependent individual fluctuation, as a higher infectivity load is seen again at 24 mpi. Hence, the data presented here are important for a risk- based SRM definition.The authors declare that they have no competing interests.This study was designed by CF, BH and MHG, practically realized by CF, MK, ABB and the data were analyzed and the manuscript was written by CF and MHG. All authors read and approved the final manuscript.


Biomass is the energy source for cooking and heating for billions of people worldwide. Despite their prevalent use and their potential impact on global health, the effects of these fuels on lung biology and function remain poorly understood.We exposed human small airway epithelial cells and C57BL/6 mice to dung biomass smoke or cigarette smoke to compare how these exposures impacted lung signaling and inflammatory and proteolytic responses that have been linked with disease pathogenesis.in vitro exposure and siRNA studies demonstrated that biomass and cigarette smoke activated ERK to up regulate IL-8 and MMP-1 expression in human airway epithelial cells. In contrast to cigarette smoke, biomass also activated p38 and JNK within these lung cells and lowered the expression of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Similarly, in the lungs of mice, both biomass and cigarette smoke exposure increased macrophages, activated ERK and p38 and up regulated MMP-9 and MMP-12 expression. The main differences seen in the exposure studies was that mice exposed to biomass exhibited more perivascular inflammation and had higher G-CSF and GM-CSF lavage fluid levels than mice exposed identically to cigarette smoke.The Biomass activates similar pathogenic processes seen in cigarette smoke exposure that are known to result in the disruption of lung structure. These findings provide biological evidence that public health interventions are needed to address the harm associated with the use of this fuel source. In the developing world, it is estimated that air pollution from biomass smoke accounts for 2.2 to 2.5 million deaths annually in vitro and in vivo model of biomass exposure, this work detailed the biological mechanisms by which this exposure mediates lung damage. Moreover, comparative analyses were conducted with cigarette smoke in order to determine whether biomass activated pathogenic mechanisms linked to the development of COPD.In India, biomass accounts for approximately 90% of primary energy use broken down as wood in 56% of cases, dung in 21% and crop residues in 16% Cigarette smoke extract was prepared by bubbling the smoke of one cigarette  through 25 ml of PBS for ten minutes. The cigarette smoke extract was pH balanced to 7.4 and sterile filtered prior to use Escherichia coli strain 0111:B4 or 50 or 100 μM of naphthalene, which was analyzed since it is a primary component of biomass smoke Human small airway epithelial cells (SAECs)  were cultured and Western blot analysis for MAPK signaling and ELISAs to detect the level of MMP-1, MMP-9, TIMP1 and IL-8 were performed as previously reported in vitro cigarette exposure system. Instead of passing the smoke through PBS as in the in vitro system, the smoke produced from the grill was let into a specially designed chamber containing the mice, and the total particulate matter concentration in the chamber was regulated at 200 mg/m3  . To approximate the experience of those cooking with dung biomass, we opted for a short but more intense exposure. Food and drinking water were provided ad libitum. Mice were exposed to cigarette smoke in a specially designed chamber  for six hours a day at a total particulate matter concentration of 80 mg/m3 as per our established protocol 2 asphyxiation. Histological analysis of H&E stained slides were used to determine perivascular vascular inflammation (PVI) using a modified quantification schema For the mouse exposure studies, 8-week old C57BL/6  were exposed for 7 consecutive days to room air, cigarette smoke for 6 hours daily or dung smoke for 1 hour a day. The dung smoke was produced using the same experimental design as in our rements) Fig. 1Data are expressed as means ± S.E.M. We determined statistical significance by one-way analysis of variance for multiple group analysis using GraphPad Prism Software. Student t-tests (two tailed) were used throughout the study. All data sets are represented as mean +/− standard error.in vitro model is presented in ). High quantities of endotoxin have been reported within households that use dung biomass as a fuel . LPS, BSE and CSE treatment all significantly increased MMP-1 . In comparison, naphthalene had no effect on JNK but stimulated both ERK and p38 , both groups had similar inflammatory responses. Moreover, the overall number of macrophages within the lavage was significantly increased in the biomass and smoke-exposed mice  and these macrophages contained large vacuoles indicative of an activated state . These findings were not noted in the other groups of mice. Furthermore, both biomass and cigarette smoke-exposed mice exhibited perivascular lymphocytic inflammation (. To characterize the inflammatory and proteolytic responses produced by these exposures, we measured lung lavage cytokines and proteases within the lung lavage fluid of exposed mice. Both biomass and cigarette smoke increased IL-6, KC and IL-17 (Mice were exposed to biomass for 30 minutes twice a day for one week and compared to unexposed mice and mice that were exposed to one-week of cigarette smoke as per our standard protocol nd IL-17 Fig. 5A This study provides a detailed characterization the biologic effects of dung biomass exposure, a fuel source that is utilized by hundreds of millions of people worldwide Biomass activated ERK and p38, which are MAPK signaling proteins that play an important role in the development of chronic airway inflammation The activation of the distinct MAPK signaling pathways noted in this study could lead to pathologic changes from biomass smoke that are different from cigarette smoke related lung disease. In support of this, an intense, perivascular lymphocytic inflammation was observed in the lungs of the biomass-exposed mice. This is a finding reported to occur in human COPD in vivo studies however did not replicate the differential response of TIMP-1 to cigarette smoke and biomass. This is likely due to the fact that the in vivo analysis reflects the majority of changes from macrophage TIMP-1 obscuring the changes in epithelial cell production. Future studies will need to further evaluate the effect of biomass on the protease/anti-protease balance in the lung.Macrophages from biomass-exposed mice exhibited prominent vacuoles and carbonaceous material within their cytosol. When activated, macrophages produce high levels of MMP-9 and -12 This study fills a critical gap in the existing literature and provides new insights into the effects of biomass combustion on lung health. Based on our results, we assert that interventions that limit the intensity or duration of this exposure will have a major impact on global public health. Moreover, our research models could be utilized in intervention studies to evaluate the efficacy of improved stove technologies on respiratory health.


Glucagon hormone is synthesized and released by pancreatic α-cells, one of the islet-cell types. This hormone, along with insulin, maintains blood glucose levels within the physiological range. Glucose stimulates glucagon release at low concentrations (hypoglycemia). However, the mechanisms involved in this secretion are still not completely clear. Here, using experimental calcium time series obtained in mouse pancreatic islets at low and high glucose conditions, we propose a glucagon secretion model for α-cells. Our model takes into account that the resupply of releasable granules is not only controlled by cytoplasmic  Pancreatic islets of Langerhans, which play a crucial role in glucose homeostasis, are mainly formed by three types of exocytotic cells , the total amount of secreted glucagon in mice ranges from 30 to 40 picograms per islet (pg/islet) in one hour, and this amount is approximately twice the basal (non-stimulated) secretion that is seen at the high glucose concentrations typically used in these experiments Glucagon is released by Exocytosis by In this work, we propose a secretion model for All protocols were conducted following the regulations approved by the Animal Care Committee of the Universidad Miguel Hernández , according to national and European policies about ethics in animal research. Swiss albino OF1 mice (8–10 weeks old) were sacrificed by cervical dislocation and pancreatic islets were then isolated by collagenase digestion as previously described For imaging experiments, islets were placed on a perfusion chamber mounted on the microscope stage and attached onto poly-L-lysine treated coverslips for 10 min before commencing the experiments. Islets were then perfused at a rate of 1.5 ml/min with a modified Ringer solution containing (mM): 120 NaCl, 5 KCl, 25 NaHCO3, 1.1 MgCl2 and 2.5 CaCl2; pH 7.4, gassed with 95% O2 and 5% CO2. Following these methods, 2 different sets of The kinetic model for glucagon secretion, which describes The variables of the model are the number of granules in each of the 6 states schematized in In this equation, In equations (2)–(6), Since experimentally obtained oscillations reflect cytoplasmic Using To reproduce steady-state values of both the total number of granules and the basal secretion rate. Total number of granules for each To predict the experimentally reported secretion in response to  oscillations monitored in -cells of mice, in the presence of low glucose. At low glucose (0.5 and 1 mM), secretion is in the range 30–60 pg/islet/hr  The remaining parameters  using experimental All simulations were performed using the parameter values listed in In We have estimated the population of releasable granules at steady-state for both glucose conditions; this population includes all primed granules belonging to the releasable stage considered in our model . The popIndividual secretion rates induced by each of the 14  at  see . These rIn order to explore the impact of intracellular In The results shown in the preceding sections point toward the fact that oscillations favor secretion. To understand the reasons of this increase, we have first investigated the relationship between glucagon secretion and the frequency of It is indeed known that in physiological conditions, insulin and glucose both modulate glucagon release in lets see . In the In al cells . We alsoIn this study, we have developed a simple model that quantitatively reproduces the rates of glucagon secretion by Our modeling approach, however, emphasizes the importance of a specific regulation of secretion in Our results indicate that glucose may regulate glucagon secretion through two different and counter-acting actions in About the effect of insulin in islets at low glucose, we have observed a reduction of the mean ch islet , which ich islet . With thOverall, the present results emphasize the main role played by We conclude that under normal functioning, the


TBX5 and is characterised by cardiac and skeletal abnormalities. TBX5 is part of the T-box gene family and is thought to upregulate tumour cell proliferation and metastasis when mutated. We report the first clinical case of prostate cancer in an individual with Holt Oram syndrome.Holt-Oram syndrome is an autosomal dominant disorder which is caused by mutations of  TBX5 T-box transcription factor mutation. Prostate cancer is the most common male solid cancer and has a complex aetiology relying on interaction between both genetic and nongenetic factors. While it is known that the existence of one or more hereditary prostate cancer genes is supported through the existence of prostate cancer families, the identification of causative genes for prostate cancer has been elusive to date. While linkage studies on extended high risk families can compare genotypes between affected and nonaffected individuals within these high risk families, it is not currently possible to differentiate between those cases of sporadic versus inherited prostate cancer. One area of study in prostate cancer genetics is the T-box transcription family, which is also responsible for Holt-Oram syndrome. T-box factors are highly conserved transcription factors that act in cell cycle regulation and in the development of malignancy as well as in embryonic development. Their overexpression is thought to downregulate expression of cellular cyclin-dependent kinases which thus allows cells to bypass programmed senescence. This case outlines the diagnosis, management, and treatment sequelae of an individual with prostate cancer and a known A 41-year-old male received three-dimensional conformal radiation therapy (3DCRT) as monotherapy for an early stage carcinoma of the prostate. He had an opportunistic prostate specific antigen (PSA) which was raised at 8.9 ng/mL and on staging workup was found to have a cT2aN0M0, Gleason score 3 + 3 prostate adenocarcinoma. He had a background of Holt-Oram syndrome (hand-heart syndrome), an autosomal dominant condition characterised by upper limb and cardiac defects [Following multidisciplinary assessment, he was elected to have 3DCRT which was well tolerated without any Grade 3 or 4 urinary or gastrointestinal toxicity, and he is in biochemical remission with an undetectable PSA five years after completion of treatment. He has no enhanced normal tissue toxicity. TBX5 [TBX3, can cause absent or hypoplastic mammary glands and it is under investigation in breast cancer carcinogenesis. The TBX2 gene is deregulated in melanoma, breast, and pancreatic cancers where it suppresses senescence through the repression of cyclin-dependent kinase inhibitors, p19 and p21. Although this is the first case of prostate cancer reported in a patient with the TBX5 mutation, it has been demonstrated that T-box transcription factors have an important role in metastasis in murine models of prostate carcinoma [Holt-Oram syndrome is caused by mutations in TBX5 , a T-boxTBX5 , 4, withIn conclusion, our case demonstrates the importance of screening patients with rare genetic disorders for cancer and the growing knowledge of the genetic basis of prostate cancer.


CHRNA5-CHRNA3-CHRNB4 gene cluster on 15q25 has consistently been associated with smoking quantity, nicotine dependence and lung cancer. Recent research also points towards its involvement in cardiovascular homeostasis, but studies in large human samples are lacking, especially on the role of the gene cluster in blood pressure regulation.The CHRNA5-CHRNA3-CHRNB4 and systolic blood pressure (SBP), diastolic blood pressure (DBP), and body mass index (BMI) in 5402 young adults from the Northern Finland Birth Cohort 1966. We observed some evidence for associations between two SNPs and SBP and between six SNPs and BMI; the evidence for associations with DBP was weaker. The associations with the three phenotypes were driven by different loci with low linkage disequilibrium with each other. The associations appeared more pronounced in smokers, such that the smoking-increasing alleles would predict lower SBP and BMI. Each additional copy of the rs1948 G-allele and the rs950776 A-allele reduced SBP on average by −1.21  mmHg in smokers. The variants associated with BMI included rs2036534, rs6495309, rs1996371, rs6495314, rs4887077 and rs11638372 and had an average effect size of −0.38  kg/m2 per an additional copy of the risk allele in smokers. Formal assessments of interactions provided weaker support for these findings, especially after adjustment for multiple testing.We studied the associations between 18 single nucleotide polymorphisms (SNPs) in Variation at 15q25 appears to interact with smoking status in influencing SBP and BMI. The genetic loci associated with SBP were in low linkage disequilibrium with those associated with BMI suggesting that the gene cluster might regulate SBP through biological mechanisms that partly differ from those regulating BMI. Further studies in larger samples are needed for more precise evaluation of the possible interactions, and to understand the mechanisms behind. CHRNA5-CHRNA3-CHRNB4 gene cluster at 15q25 has consistently been associated with smoking quantity and nicotine dependence The CHRNA5-CHRNA3-CHRNB4 gene cluster and cardiovascular homeostasis in humans are scarce. Rana et al. observed in a twin study of 370 individuals that polymorphisms in the CHRNA5-CHRNA3-CHRNB4 gene cluster were associated with systolic blood pressure (SBP), circulating levels of catestatin (an endogenous antagonist of nAChR) and epinephrine. These results suggest that variation in CHRNA5-CHRNA3-CHRNB4 gene cluster, already implicated in smoking behavior, is also involved in the autonomic regulation of BP. The associations between variation in CHRNA5-CHRNA3-CHRNB4 and SBP persisted after adjustment for smoking, suggesting an effect independent of smoking. CHRNA3 increased susceptibility to develop peripheral arterial disease among smokers CHRNA3-rs1051730 interacts with smoking status in influencing body mass index (BMI). The allele that predisposes to heavier smoking was associated with a lower BMI in those who smoked but not in non-smokers. Animal studies exploring the role of nAChRs in cardiovascular risk factors exist but they have mainly focused on nAChR subunits α4β2 and α7. These studies have demonstrated the involvement of these subunits in central cardiovascular actions, such as the regulation of blood pressure (BP) and heart rate The potential involvement of these subunits in cardiovascular homeostasis is intriguing and could at least partly explain associations between smoking and BP observed in previous epidemiological studies. Smoking has been associated with BP in several studies with smokers having on average a lower BP than non-smokers CHRNA5-CHRNA3-CHRNB4 gene cluster on BMI and BP in large human samples is required. It is also of interest whether the effect is purely pleiotropic or if smoking acts as a mediator or a moderator of the associations between the gene cluster and BMI/BP, as suggested by some studies Replication of published results on potential pleiotropic effects of the CHRNA5-CHRNA3-CHRNB4 on BMI and BP and whether these effects are modified by smoking status. Our study adds further evidence to the previously reported association between variation at 15q25 and BMI in smokers. Moreover, ours is the first study, conducted in a large population-based birth cohort of over 5400 individuals, to suggest an interactive effect on BP as well.In the present study we have tested the effects of genetic variation within th week of gestation until adulthood, with the latest data collection being conducted at the age of 31 years. At that point, questionnaires on health, lifestyle and occupation were mailed to all cohort members alive with known address (N = 11541). The questionnaire was returned by 75% (N = 8767) of the individuals. Those living in the original target area or in the capital (Helsinki) area (N = 8463) were also invited to a clinical examination, to which 71% (N = 6033) participated. The study sample consisted of members from the Northern Finland Birth Cohort 1966 (NFBC1966). The cohort was initiated in 1965 by inviting all pregnant mothers from the two northernmost provinces of Finland with their expected date of delivery occurring in 1966 to participate. The cohort covered over 96% of all births in the target area in 1966 (N = 12055 mothers with 12058 live-born children). 2) was calculated from measured height and weight. SBP and DBP were measured twice with a mercury sphygmomanometer with a cuff size of 14 cm×40 cm in a sitting position from the right arm after 15 minutes of rest by trained nurses using a standardized procedure and ongoing quality control Phenotypic data were obtained from the questionnaire and the clinical examination at 31 years. Smoking was assessed from questions measuring current smoking behavior (during the past year) and smoking quantity. Based on those we formed a three-category variable as follows: 1) non-smokers (including never and former smokers), 2) light smokers (1–10 cigarettes/day), and 3) heavy smokers (>10 cigarettes/day). BMI  have been reported previously. P-value for Hardy-Weinberg Equilibrium >0.005, call rate >99.5% and no two SNPs were in LD of r2>0.2 with any other selected SNP. The data were further thinned such that 1 in 15 SNPs was selected for principal component analysis. Eigenvalue decomposition was performed using the eigen function in the statistical software R Principal components (PC) to control for potential population stratification in the subsequent association analyses were calculated from the 22 directly genotyped autosomes such that all SNPs had MAF >1%, CHRNA5-CHRNA3-CHRNB4 gene cluster and its surrounding area as in our previous publication For genetic data, we selected all available directly genotyped SNPs from the genome-wide data to cover the P-values for the interaction terms.We performed the analyses separately in non-smokers and smokers, and in all subjects together, including a term for gene-environment (SNP×smoking) interaction, too. To account for multiple testing, we performed parametric bootstrap for gene-environment interactions P-values are reported.Additionally, we performed a series of supplemental analyses. We examined dose-response effects by separating smokers into light and heavy smokers, and differences between never, former and current smokers were tested by separating the non-smoker group into never and former smokers. Also, we tested for possible gender×SNP interactions. The analyses were performed in Plink v1.06 P-values.Finally, we performed haplotype analyses to increase power and to refine the location of the susceptibility locus, because multiple SNPs may “tag” an untyped variant more effectively than a single typed variant Characteristics of the study sample are presented in 2 per an additional copy of the risk allele in smokers. The results from interaction models appeared to provide some support of the interactive effects observed in the stratified analyses; however, after adjustment for multiple testing the evidence for interactions was quite weak , but the results remained practically unchanged (data not shown).For SBP, the observed effects were slightly stronger in heavy smokers than in light smokers; e.g. for rs1948 the G-allele was associated with 0.97 mmHg lower SBP  in light smokers whereas in heavy smokers the corresponding figures were −1.50  . For DBPFor the SNPs on which we had observed evidence for associations in current smokers , the associations were attenuated in former smokers ,S5,S6. FSince the distributions of SBP, DBP and BMI differed by gender , we studP-value<0.05. Such results were observed only in smokers. In the single marker analysis we observed evidence for associations for SNPs from the first block and SBP, SNPs from the second block and DBP and SNPs from both blocks and BMI (The linkage disequilibrium (LD) plot of the region is displayed in  and BMI –5. The  and BMI , S11.In more detail, for SBP, and after adjustment for multiple testing, there was moderate evidence for a SBP-lowering haplotype consisting of the G-allele of rs1948 and A-allele of rs950776; the same alleles of the two SNPs that were associated with lower SBP in the single marker analyses. Instead, SBP-increasing haplotypes included the A and G alleles of the above-mentioned SNPs, respectively. The pattern for the alleles of the other SNPs included in SBP-associated haplotypes was not as clear-cut as it was for rs1948 and rs950776; the same alleles in different haplotypes could be associated either with increased or decreased SBP .The estimated effect sizes on SBP were bigger for many SBP-increasing haplotypes than for any of the SBP-lowering haplotypes or single SNPs in the single marker analysis . An incrRegarding BMI and the haplotypes from the first block, there was no strong evidence for associations after adjustment for multiple testing . In the CHRNA5-CHRNA3-CHRNB4 gene cluster and lower BP. We found weak evidence for associations between smoking-increasing alleles and lower BMI as well, although the distributions of BMI did not vary across different smoking strata. If same alleles predisposed to smoking and lower BMI, we would have expected to observe on average lower BMI in smokers, which was not the case in our data. However, we believe that the association between smoking and BMI is highly confounded by socio-behavioral factors, for which we did not control when comparing the distributions of BMI across different smoking strata, because addressing the associations between smoking and BMI/BP in detail was beyond the scope of the present study.The present study suggests that in addition to the previously presented hypotheses for the association between increased smoking and lower BP, there might be shared a genetic architecture behind as well. In the single marker analyses, weak evidence was found for associations between two polymorphisms from the smoking-associated CHRNB4 were associated with SBP, pointing towards β4 subunits being mostly responsible for these effects, whereas markers from CHRNA5 and CHRNB4 were associated with BMI, emphasizing the role of α5 and β4 subunits in affecting BMI. However, further studies are needed to locate the real causal variants and the receptor subunits responsible for the observed associations. For all of the six SNPs on which we found evidence for any association with BMI, the r2 with rs1948, the most strongly associated SNP with SBP, was <0.20 indicating that the SNPs associated with BMI and SBP seem to be nearly independent of each other. The haplotype association analyses also supported this finding since mainly haplotypes from block 1 were associated with SBP, haplotypes from block 2 with DBP, and haplotypes from both blocks with BMI. The haplotype analyses also provided further support for the two SNPs found to be associated with SBP in the single marker analyses; the haplotype GA consisting of rs1948 and rs950776 predicted lower SBP even after correction for multiple testing. For BMI, the findings from the haplotype analyses were less consistent with the findings from the single marker analyses.Different SNPs were associated with BP and BMI, and even with SBP and DBP. Mainly markers from The results from the additional, rather explorative analyses suggested that the effects of the SNPs on SBP would be stronger in heavy smokers than in light smokers. The associations between the SNPs and SBP/BMI were attenuated in former smokers, suggesting that the associations would weaken over time after cessation. In addition, there might be heterogeneity by gender in the associations. However, additional stratifications reduced the sample sizes resulting in very wide confidence intervals, and moreover, the interaction analyses provided no adequate support for these findings. Studies with larger sample sizes are needed to find further support for these suggestive results.We also tried to address the fact the possibility that the associations seen are due to residual confounding by smoking quantity. By adjusting the analyses in smokers by a continuous measure of cigarettes/day did not change the results obtained previously, suggesting that the associations seen are not due to residual confounding. However, in all of the analyses the measure of smoking was self-reported, possibly leading to underestimation of the true effect. For example, the availability and the use of cotinine levels might control better for the actual quantity of smoked cigarettes.CHRNB4 has previously been associated with heavy smoking in adulthood Of the SNPs that we found evidence for an association with BP, rs1948 in CHRNA5-CHRNA3-CHRNB4 gene cluster have previously been reported to be associated with BMI or SBP. SNP rs1051730 in CHRNA3 reported to be associated with BMI CHRNA3, reported to be associated with lower SBP in twins Only two SNPs from the CHRNA5-CHRNA3-CHRNB4 gene cluster and activity of the dorsal anterior cingulate-ventral striatum/extended amygdala circuit, which is an important circuit in modulating reward to drugs such as alcohol or smoking as well as food Co-morbidity between addictive behaviors of smoking, alcohol use and eating disorders has been reported 2 between the variants associated with SBP and BMI. This suggests that the CHRNA5-CHRNA3-CHRNB4 gene cluster regulates SBP through biological mechanisms that partly differ from those regulating BMI. One plausible mechanism is that the nicotinic receptors modulate SBP by influencing the autonomic function. In this regard, Rana et al. showed that polymorphisms in the region were associated with catestatin, a peptide regulating cardiac function and blood pressure. The catestatin peptide fragment of the endogenous catecholamine secretory vesicle protein chromogranin A (CHGA) acts as an antagonist of the nicotinic cationic channels and regulates catecholamine release. This led the authors to suggest that nAChRs may regulate BP via mediating release of catestatin. Indeed, the same variants associated with catestatin were also associated with SBP, and cathecolamine levels. The mechanisms by which the nAChRs regulate BP are also unclear. The genetic variants were still found to be associated with SBP even after adjustment for BMI suggesting a direct effect (i.e. not mediated via BMI) of these loci on SBP. In line with this idea, the genetic variants associated with SBP were different from those associated with BMI in our sample and they seemed to be nearly independent based on the low ret al. showed associations between polymorphisms in the gene cluster and SBP adjusted for smoking, but they did not test for interactive effects in their study In the present study the associations between variation in the gene cluster and BMI/BP appeared to be more pronounced in smokers, suggesting that smoking would be a modifier of these associations. However, after adjustment for multiple testing, there was no sufficient evidence for interactive effects. The shortage of evidence for interaction might be due to limited power of our study to detect gene-environment interactions. Previous studies have reported associations between polymorphisms in the gene cluster and BMI manifest only in smokers The analyses were performed in a population-based birth cohort including individuals of Northern European ancestry with a fairly homogeneous background. The study participants were of relatively young age (31 years) at the time of BMI and BP assessment, and as BP tends to increase with age, the effects could be stronger in older cohorts. In addition, age-varying associations are also possible et al.et al.However, replication is still required in populations of European ancestry, because although the sample size was moderately large compared to some of the previous studies, e.g. the 370 twins in the study for the gene cluster and SBP The replication of our suggestive results, and addressing further gene-environment, such as gender differences, or gene-gene interactions, would therefore require substantially larger numbers of individuals. Cohorts with certain biological markers will also be valuable in the search of specific pathways leading from nAChRs to cardiovascular risk factors.CHRNA5-CHRNA3-CHRNB4 is associated with cardiovascular risk factors and that smoking possibly acts as a modifier of these associations. Large collaborative efforts are needed to find further evidence for the associations, to test for their existence in other ancestral populations and age groups, and to understand the mechanisms by which the nAChRs affect BMI and BP.Our results suggest that variation in Table S1Estimated associations between variants in the 15q25 region and SBP according to smoking status  in the NFBC1966.(PDF)Click here for additional data file.Table S2Estimated associations between variants in the 15q25 region and DBP according to smoking status  in the NFBC1966.(PDF)Click here for additional data file.Table S3Estimated associations between variants in the 15q25 region and BMI according to smoking status  in the NFBC1966.(PDF)Click here for additional data file.Table S4Estimated associations between variants in the 15q25 region and SBP according to smoking status  in NFBC1966.(PDF)Click here for additional data file.Table S5Estimated associations between variants in the 15q25 region and DBP according to smoking status  in the NFBC1966.(PDF)Click here for additional data file.Table S6Estimated associations between variants in the 15q25 region and BMI according to smoking status  in the NFBC1966.(PDF)Click here for additional data file.Table S7Estimated associations between variants in the 15q25 region and SBP according to gender in the NFBC1966.(PDF)Click here for additional data file.Table S8Estimated associations between variants in the 15q25 region and DBP according to gender in NFBC1966.(PDF)Click here for additional data file.Table S9Estimated associations between variants in the 15q25 region and BMI according to gender in the NFBC1966.(PDF)Click here for additional data file.Table S10Estimated haplotype frequencies and estimates from association analyses (Block 1) for SBP and BMI in smokers in the NFBC1966.(PDF)Click here for additional data file.Table S11Estimated haplotype frequencies and estimates from association analyses (Block 2) for DBP and BMI in smokers in the NFBC1966.(PDF)Click here for additional data file.


To evaluate the cost-effectiveness of post-diagnosis dementia treatment and coordination of care by memory clinics compared to general practitioners’ care.A multicentre randomised trial with 175 community dwelling patients newly diagnosed with mild to moderate dementia, and their informal caregivers, with twelve months’ follow-up. Cost-effectiveness was evaluated from a societal point of view and presented as incremental cost per quality adjusted life year. To establish cost-effectiveness, a cost-utility analysis was conducted using utilities based on the EQ-5D. Uncertainty surrounding the incremental cost-effectiveness ratio (difference in costs divided by difference in effects) was calculated by bootstrapping from the original data.Compared to general practitioners’ care, treatment by the memory clinics was on average €1024 (95% CI: −€7723 to €5674) cheaper, and showed a non-significant decrease of 0.025 (95% CI: −0.114 to 0.064) quality adjusted life years. The incremental cost-effectiveness point estimate from the bootstrap simulation was € 41 442 per QALY lost if one would use memory clinic care instead of general practitioner care.No evidence was found that memory clinics were more cost-effective compared to general practitioners with regard to post-diagnosis treatment and coordination of care of patients with dementia in the first year after diagnosis.ClinicalTrials.gov NCT00554047 The quest for high quality, yet sustainable dementia care is becoming ever more challenging. Dementia is an important – and in numbers growing – cause of disability and burden of care and one of the diseases with the largest per capita healthcare consumption Recently we showed that there is no evidence of a difference in effectiveness, evaluated as quality of life of the patient and caregiver burden, between memory clinics and general practitioners with regard to dementia treatment and follow-up care The study was approved by the Medical Ethics Committee of the Radboud University Nijmegen Medical Centre. Both each patient and informal caregiver gave written informed consent before inclusion in the study.This study (the AD-Euro Study), was a pragmatic multicentre randomised trial with 12 months’ follow-up. Web based randomisation took place after baseline measurements. Participants (patient-caregiver pairs) were assigned for post-diagnosis dementia care to either the memory clinic or the general practitioner. Details have been published elsewhere The whole study ran from December 2007 until July 2010, with recruitment running from December 2007 until July 2009. Nine Dutch memory clinics recruited participants for whom the diagnostic work up resulted in a new diagnosis of dementia. Each patient had an informal caregiver. Patient-caregiver pairs were excluded when the patient lived in a nursing home, had a life expectancy of less than a year, or needed specific memory clinic care  that could not be given by general practitioners.The interventions consisted of care by either the memory clinic or the general practitioner. The memory clinic provided treatment and care coordination based on the specialist Dutch dementia guideline of the Dutch Institute for Healthcare Improvement After baseline measurements, follow-up measurements were made at six and 12 months. Measurements were made at the patients home by interview. Research assistants were instructed, both oral and in writing, how to conduct the interview to prevent differences between interviewers. An overview of the different outcome measures and when they took place has been published elsewhere To establish cost-effectiveness, a cost-utility analysis was conducted using utilities generated by the EuroQol instrument (EQ-5D) for both patient and caregiver Costs were calculated by multiplying volumes of resources by the cost price per resource unit . Much ofData were analysed on an intention-to-treat basis. We used descriptive statistics for baseline characteristics. To present confidence intervals surrounding the costs and effects we undertook nonparametric bootstrapping on the incremental costs and effectiveness with 1000 draws from the original sample. The incremental cost-effectiveness is represented visually by the incremental cost-effectiveness plane. The horizontal axis divides the plane according to incremental effects, whereas the vertical axis divides the plane according to incremental costs. The probability that an intervention is cost-effective varies according to the ceiling ratio. This probability is shown in the cost-effectiveness acceptability curve (CEAC), based on nonparametric bootstrapping We included 175 patient-caregiver pairs in the study; 87 were randomly assigned to the memory clinic group and 88 to the general practitioner group. Baseline characteristics of patients and caregivers were similar between the two groups . The aveWe evaluated 160 pairs  out of the 175 patient-caregiver pairs included in the study. Eleven pairs (four in the memory clinic group) dropped out because they considered further participation to be too burdensome. One caregiver died, one caregiver did not fill out the questionnaires, and one caregiver was not present during the measurements without giving any reasons, all in the general practitioner group. In one patient in the general practitioner group the diagnosis of dementia was changed just after inclusion and was the reason for the patient and caregiver to withdraw from the study.The average cost, cumulated over 12 months follow-up, was €22 035 (range €682 to €120 698) per patient in the memory clinic group. In the general practitioner group the mean cost per patient was €23 059 (range €674 to €78 721). This resulted in a statistically not significant difference between the two study-groups of €1024 (95% CI: −€7723 to €5674). The analyses showed a difference in QALYs of 0.025 in favour of the general practitioner group, which was also not statistically significant (95% CI: −0.114 to 0.064). Including the minimisation factors as covariates in the regression analysis left the difference in costs between the two study-groups (€2441 (95% CI: −€8152 to €3270)) and the difference in QALYs (0.029 (95% CI: −0.1 to 0.06)) unchanged. Also, adding comorbidity as covariate, left the difference between the two study-groups unchanged (difference in costs €3470 (95% CI −€9257 to €2316) and in QALYs 0.015 (95% CI: −0.1 to 0.07)). From the different cost variables  three vaThe incremental cost-effectiveness point estimate from the bootstrap simulation was € 41 442 per QALY lost if one would use memory clinic care instead of general practitioner care. The 95% confidence interval surrounding the incremental cost-effectiveness ratio (cost per QALY gained) in the bootstrap simulation ranged from −€1 221 001 (2.5 percentile) to €1 026 234 (97.5 percentile), using the percentile method. The scatter plot in The cost-effectiveness acceptability curve for the situation of our primary cost-effectiveness analysis  is shown in We found, applying a societal point of view, no evidence of a statistical significant difference in cost-effectiveness between memory clinics and general practitioners with regard to post-diagnosis treatment and coordination of care for patients with dementia at one year follow-up. A Bayesian interpretation of cost-effectiveness (the CEAC) also shows that cost-effectiveness between both modalities remains unresolved.Comparison of our results with others is difficult, because of the lack of similar studies. The study of Wolfs et al. showed that, in comparison with usual care, an integrated multidisciplinary diagnostic approach to dementia in a memory clinic setting increased the health related quality of life of dementia patients and was cost-effective Other studies compared cost of care of specific interventions – which could be part of post diagnosis dementia guidance – with alternatives We ensured a robust study design by using a randomised controlled trial and we carried out the economic evaluation from a societal perspective. By using a generic measure for quality of life (EQ-5D) we are able to compare our results with other interventions. This is important to interpret the cost savings for a QALY lost. The participation of nine different memory clinics, based in different settings , enhanced the heterogeneity among care setting and thus the generalisability of our study results. However, differences in healthcare systems and variability in dementia care between countries makes international generalisability of our results difficult. Furthermore it is argued that, especially when effects are expressed as QALYs in an economic evaluation, there is a risk of double counting. We based QALYs on the EQ-5D where productivity is no dimension. Therefore double counting seems less of a threat than if quality of life was measured on for example a VAS , ranging from best to worst imaginable quality of life. Further Krol et al. suggest that QALY measures such as the EQ-5D are insensitive to concerns regarding effects on income even when these are (explicitly) incorporated Another limitation that possibly may have affected the outcome of this study is the length of the follow-up period. Dementia is a disease that progresses over years, so an extended follow-up lasting several years would be preferable to the relatively short 12 month period we used. It is known that costs increase over time in case of a patient with dementia.The lack of difference in costs was unanticipated due to the assumption that primary care in general is cheaper. A possible explanation could be that our group of patients was in their first year after diagnosis with relatively mild dementia and therefore often could take care of oneself with relatively little help from others, both in the memory clinic and in the general practitioner group. However, this is a very topical subject as there is a strong international trend towards early diagnosis of dementia. Nevertheless, this means that these results cannot be generalised to more severely affected individuals. With further progress of the disease other needs will occur and it could be that in the long term a difference between the memory clinic and the general practitioner group could emerge.This study adds important data, which seem to point to a lack of difference in cost-effectiveness between memory clinics and general practitioners in the treatment and coordination of care for patients with dementia at one year follow-up. Together with former results that no evidence was found of memory clinics being more effective than general practitioners As our follow-up period was only one year after diagnosis, using a decision analysis modelling strategy to compare costs and effectiveness of the interventions in longer term should be a topic for future research.


Over the last decade, we have witnessed major advances in the understanding of the molecular basis of neonatal and infancy-onset diabetes. It is now widely accepted that diabetes presenting before 6 months of age is unlikely to be autoimmune type 1 diabetes. The vast majority of such patients will have a monogenic disorder responsible for the disease and, in some of them, also for a number of other associated extrapancreatic clinical features. Reaching a molecular diagnosis will have immediate clinical consequences for about half of affected patients, as identification of a mutation in either of the two genes encoding the ATP-sensitive potassium channel allows switching from insulin injections to oral sulphonylureas. It also facilitates genetic counselling within the affected families and predicts clinical prognosis. Importantly, monogenic diabetes seems not to be limited to the first 6 months but extends to some extent into the second half of the first year of life, when type 1 diabetes is the more common cause of diabetes. From a scientific perspective, the identification of novel genetic aetiologies has provided important new knowledge regarding the development and function of the human pancreas. Presentation of diabetes within the first year of life was first reported in 1789  but did Only a few years ago, type 1 diabetes (T1D) was considered the main form of diabetes presenting in infancy, and the term ‘neonatal diabetes’ (NDM) was reserved for diabetes presenting within the first 4-6 weeks of life [T1D is a T cell-mediated autoimmune disease that results from a selective destruction of the pancreatic insulin-producing β-cells and is the most common cause of diabetes in children, accounting for over 95% of cases . The disFOXP3 mutations, and not T1D, will account for most of these cases [The environmental triggers that initiate pancreatic β-cell destruction in T1D remain largely unknown. Pancreatic autoantibodies are a hallmark of the underlying autoimmune process and their presence long precedes clinical diagnosis of T1D ,13, but se cases .Almost two thirds of the infants diagnosed in the first 6 months were born small for gestational age, in contrast to 15% of those presenting between 6 and 12 months . On the According to the information displayed above, clinical presentation of polygenic autoimmune T1D within the first 6 months of life is rare. The majority of these patients will have a monogenic form of diabetes .Over the last few years, the age limit to consider a non-autoimmune, monogenic cause has changed from the first 30-45 days of life  to 3 monNDM is genetically heterogeneous, with at least 20 different causal genes identified to date table . The vasKCNJ11 or ABCC8 genes encoding the two subunits  of the ATP-sensitive potassium (KATP) channel of the β-cell membrane, prevent insulin secretion in response to hyperglycaemia and can cause both PNDM and TNDM [KCNJ11 mutations are more frequent, and most patients have PNDM rather than TNDM. In contrast, mutations in ABCC8 cause TNDM more frequently. Consistent with the expression of KATP channels in neurons [KCNJ11 mutations present with associated neurological symptoms [ABCC8 but are less frequent and usually milder  [ATP channel genes can transfer from insulin to high-dose sulphonylureas, with improved glycaemic control and decreased risk of hypoglycaemia [Activating mutations in either the and TNDM ,27,28. K neurons , about 2symptoms . The mos mainly) . There a mainly) ,25. Diab mainly)   are the second most common cause of isolated PNDM [INS mutations is similar to that of patients with KATP channel mutations, but diabetes tends to present slightly later [Heterozygous coding mutations in the proinsulin gene ted PNDM ,38,39,40ly later , even inly later ,39,41. SATP channel genes and the proinsulin gene (INS) account for at least 50-75% of infancy-onset permanent monogenic diabetes in outbred populations [EIF2AK3 being the commonest known genetic cause  are linked to 3 different abnormalities in an imprinted region on chromosome 6q24 ultimately leading to overexpression of a l allele ,47. The l allele  secondarhylation .Patients with 6q24 abnormalities are born with moderate intrauterine growth retardation  and usually develop severe non-ketotic hyperglycaemia during the first week of life ,50. DespZFP57 show an autosomal recessive inheritance, and hence the recurrence risk is 25% for siblings and almost negligible for the offspring of an affected patient.Genetic counselling for families with 6q24 TNDM depends on the underlying molecular mechanism. Uniparental disomy of chromosome 6 is generally sporadic and therefore the risk of recurrence in siblings and offspring is low. For paternal duplication of the 6q24 region, males have a 50% chance of transmitting the mutation and the disease to their children. In contrast, females will pass on the duplication, but their children will not develop the disease, which may recur instead in the next generation as their asymptomatic sons pass on the molecular defect to their own children. Methylation defects are usually sporadic, but mutations in KCNJ11 and ABCC8) encoding the two subunits  of the KATP channel of the β-cell membrane account for most of the remaining cases of TNDM [ATP channel mutations show milder intrauterine growth retardation and are diagnosed later, suggesting that the prenatal insulin deficiency is less severe. In addition, diabetes tends to remit later and relapses sooner [Activating mutations in any of the genes , both endocrine and exocrine lineages are involved, and the affected patient shows some degree of pancreatic hypoplasia and exocrine dysfunction, in addition to the associated extrapancreatic features secondary to the extrapancreatic expression of the respective gene [NEUROG3, NEUROD1, PAX6), pancreatic hypoplasia and exocrine dysfunction are lacking [IER3IP1, which cause NDM plus lissencephaly and other neurological abnormalities by inducing ER-associated apoptosis [When the mutated transcription factor is normally expressed early in development , and diabetes presented within the first week of life. If this is also negative, further testing may be guided by additional clinical criteria.Age at diabetes onset can be considered the cornerstone criterion since most infants diagnosed before 6 months will have monogenic diabetes . It has ffective  and therHNF1B and GATA6), cause autosomal recessive disorders  who developed diabetes slightly before 6 months of age.INS mutations followed by KATP channel genes testing should be considered. Apart from Wolcott-Rallison syndrome, late infancy-onset diabetes is exceedingly rare in other forms of syndromic monogenic diabetes.It is important to keep in mind that some infants presenting with diabetes after 6 months will also have monogenic diabetes, especially when pancreatic autoantibodies are negative. If there are no associated clinical features, screening for The advent of next-generation sequencing technologies brings the possibility of simultaneous testing for all known NDM genes in a single assay , and thiEarly insulin administration is acutely required in most infants with newly diagnosed diabetes to treat or prevent acute metabolic decompensation and to allow weight gain . CaloricInsulin can be provided by multiple daily injections or continuous subcutaneous infusion . It may KCNJ11 or ABCC8 can be successfully switched from insulin to oral sulphonylurea [www.diabetegenes.org. The required doses of sulphonylurea are usually high, especially in patients with neurological features, but tend to decrease over time irrespective of the mutation. Long-term follow-up has revealed persistent efficacy and safety [Most patients with activating mutations in onylurea ,33, withd safety . The onld safety  and staid safety .Patients with agenesis of the pancreas require pancreatic enzyme supplements in addition to insulin treatment.ATP channel mutation who are able to transfer from insulin injections to sulphonylurea tablets and achieve improved glycaemic control. More than 20 different causal genes have now been identified, with many new aetiologies discovered through a combination of homozygosity/sequencing analysis of candidate genes suggested from mouse knockout studies. Exome sequencing has revealed GATA6 haploinsufficiency as the most common cause of pancreatic agenesis, and the expectation is that genome-wide analysis by next generation sequencing will rapidly uncover additional new causal genes to assist in the diagnosis and clinical management of NDM.When a newborn or infant presents with diabetes, both clinicians and families face a highly complex situation. Reaching a molecular diagnosis confirms the genetic subtype, predicts the prognosis and possible development of extra-pancreatic features, and determines the risk of diabetes in future siblings/offspring. The most dramatic impact is for those patients with a K