Early identification of cardiovascular disease (CVD) risk is important to reach people in need of treatment. At-risk patients benefit from behavioral counseling in addition to medical therapy. The objective of this study was to determine whether enhanced counseling, using patient navigators trained to counsel patients on CVD risk-reduction strategies and facilitate patient access to community-based lifestyle-change services, reduced CVD risk among at-risk patients in a low-income population.2 and t tests.We compared clinical characteristics at baseline and 12-month follow-up among 340 intervention and 340 comparison patients from community health centers in Denver, Colorado, between March 2007 and June 2009; all patients had a Framingham risk score (FRS) greater or equal to 10% at baseline. The intervention consisted of patient-centered counseling by bilingual patient navigators. At baseline and at 6-month and 12-month follow-up, we assessed health behaviors of intervention participants. We used an intent-to-treat approach for all analyses and measured significant differences by χWe found significant differences in several clinical outcomes. At follow-up, the mean FRS was lower for the intervention group  than for the comparison group ; total cholesterol was lower for the intervention group  than for the comparison group . Intervention participants reported significant improvements in some health behaviors at 12-month follow-up, especially nutrition-related behaviors. Behaviors related to tobacco use and cessation attempts did not improve.Patient navigators may provide some benefit in reducing risk of CVD in a similar population. The Interheart study demonstrated that 90% of the population-attributable risk of a first myocardial infarction is due to modifiable risk factors . Many peEarly identification of CVD risk is important for treating at-risk people. The National Cholesterol Education Program guidelines recommend using the Framingham risk score (FRS) to identify people with an increased 10-year risk for coronary heart disease (CHD) events . The eviThe appropriate use of a risk score during a provider visit may involve recommendations for preventive lifestyle change in addition to medical therapy . HoweverPatient navigators have been effective for chronic disease prevention and management activities such as cancer screening and treatment, assessment of primary care services, and cardiovascular health promotion -17. The The objective of this study was to determine whether enhanced counseling, using patient navigators trained to counsel patients on CVD risk-reduction strategies and facilitate patient access to community-based lifestyle-change services, reduced CVD risk among at-risk patients in a low-income population.We used a quasi-experimental pre–post (baseline and 12-month follow-up) design to compare changes in clinical outcomes among intervention participants with changes among a comparison group drawn from the same patient population. We used a nonexperimental pre–post (baseline and 12-month follow-up) design to assess behavioral changes among the intervention participants only. We used an intent-to-treat approach for all analyses.We enrolled 486 intervention participants from 3 community health centers in the Denver Health and Hospital Authority (DHHA) from March 2007 through June 2009. We collected data on 480 comparison patients from 3 other DHHA community health centers during the same time. This study was reviewed and approved by the Colorado Multiple Institutional Review Board. All intervention participants verbally consented to participate.DHHA is an urban safety-net health care system that provides services to 25% of Denver residents, including 50% of Denver's children and a large proportion of the city's indigent, vulnerable, and racial/ethnic minority populations. Clinical care components of DHHA include a 500-bed hospital, the 9-1-1 medical response system for the city and county of Denver, 8 federally qualified community health centers, 13 school-based clinics, and Denver Public Health.The patient navigation model has been successful in facilitating care and improving clinical outcomes among cancer patients . We reviThe intervention consisted of a 1-hour counseling session, conducted by a patient navigator at a community health center, plus follow-up telephone calls. During the counseling session, navigators assessed participants' readiness to change behaviors and encouraged them to set goals. The navigator and participant discussed potential CVD risk-reduction activities, focusing on nutrition, physical activity, and smoking cessation. Navigators encouraged participants to continue or increase current activities , provided a free 3-month pass to local Denver County recreation centers, where participants could engage in exercise programs and bilingual nutrition classes, and referred smokers to the Colorado QuitLine, a free smoking-cessation program. Although all intervention activities were independent of any clinical services; the navigator encouraged follow-up with the primary care provider for ongoing clinical care.The navigators called participants at 1 to 4 weeks and 6 to 10 weeks after enrollment to assist with and encourage the chosen behavior changes or participation in intervention activities; additional calls were made within 6 months of enrollment. On average, each participant received four 15-minute calls.International Classification of Diseases, Ninth Revision, Clinical Modification [ICD-9-CM] [www.cdc.gov/nchs/icd9.htm]  ), blood active as having been seen at least twice during the previous 18 months; the most recent visit had to be within the previous 6 months. We excluded patients if they were pregnant or lactating; had a history of coronary artery disease, ischemic cardiomyopathy, myocardial infarction, peripheral vascular disease, symptomatic carotid artery disease, or abdominal aortic aneurysm ; or hadIntervention staff identified 1,425 potentially eligible participants from the registry during the 2-year study period; 506 people had inaccurate contact information, leaving 919 eligible for inclusion. On a monthly basis, staff sent an introductory letter explaining the intervention to approximately 38 patients. Patient navigators followed up by telephone within 2 weeks to assess interest in participation and invite participants to the 1-hour counseling session. A total of 486 patients verbally consented to participate before counseling began.Not all intervention participants were included in the final analysis. Three participants who experienced a CHD event after enrollment were not eligible. We defined a CHD event as a diagnosis of angina pectoris, myocardial infarction, coronary insufficiency , or CHD death (based on ICD-9-CM codes ); we verWe used the same eligibility criteria to select a comparison group of 340 patients from 3 nonintervention community health centers. We matched people in the comparison group to intervention participants by age, race/ethnicity, sex, and the month and year in which we identified the participant for inclusion in the registry; we used this group to evaluate the extent to which the changes in the clinical outcomes among intervention participants may have been related to the intervention. We did not contact or counsel people in the comparison group or assess their health behaviors.baseline as the date of enrollment for the intervention participants and date of record selection for the comparison group. In addition to the data collected to calculate the FRS at baseline and 12-month follow-up, we extracted the following clinical data from electronic medical records for both groups: weight, height, low-density lipoprotein (LDL) cholesterol, and CHD event.We assessed clinical characteristics at baseline and 12-month follow-up for both intervention and comparison groups. We defined baseline pick-up as pick-up at least twice before study enrollment and follow-up pick-up as pick-up at least twice after study enrollment. We calculated medication adherence as a rate: the number of days of medication possession (supply days minus gap days) divided by the number of days of medication exposure (supply days plus gap days).In addition, we collected information on use of medication for lowering blood pressure or cholesterol levels from DHHA pharmacy databases; we defined For certain clinical characteristics, we established dichotomous categories of "at goal" and "not at goal": FRS ; blood pressure ; total cholesterol ; HDL cholesterol ; and LDL cholesterol .We developed a questionnaire to guide motivational interviewing and assess behavioral outcomes after reviewing standardized instruments with demonstrated psychometric properties. The final versions included 30 questions designed to assess 13 measures of nutrition, physical activity , stages Navigators administered the questionnaire verbally, in English or Spanish, at baseline in person and at 6-month and 12-month follow-up by telephone; completion time averaged 30 minutes. For follow-up, navigators attempted at least 3 telephone calls before considering the participant lost to follow-up. At 12-month follow-up, navigators still attempted to contact participants considered lost to follow-up at 6 months. Among 340 participants eligible for 12-month follow-up, 222 (65%) completed at least 1 follow-up questionnaire at either 6 months or 12 months.2 test for proportional comparisons and the t test for continuous variables. For clinical outcomes, we compared changes from baseline to 12 months between the 2 groups. To decrease the possibility of selection bias, we used an intent-to-treat approach for all analyses.We generated descriptive statistics on baseline demographic and clinical characteristics for the intervention and comparison groups. We performed bivariate analyses and measured significant differences between the groups by using the χ2 analyses. We conducted all analyses by using SAS version 9.1.3 software .For behavioral outcomes, we assessed changes from baseline to follow-up among the intervention participants only. If the 12-month questionnaire was missing, we used either the baseline questionnaire or the 6-month questionnaire, whichever provided the value closest to the 12-month value. A series of questions determined the number of fruits and vegetables consumed per day and amount of physical activity per week; the goal for each corresponds to current recommendations. A series of questions and predefined algorithms defined the stage of change, which we dichotomized as precontemplation/preparation or action/maintenance. We trichotomized the frequency of consuming high-fat or high-calorie foods. We dichotomized other questions as yes or no. We assessed significant differences between baseline and follow-up in behavioral characteristics by using χ2), and the mean FRS was 15.5%. The comparison group was similar to the intervention group, except for income; only 34% of the comparison group had an income of 150% or less of the poverty level .The participation rate for the intervention was 53% (486/919). Mean age was 56 years . Most (66%) participants were Hispanic/Latino; 34% spoke Spanish only. Most (76%) had incomes of 150% or less than the federal poverty level. At baseline, almost half of participants smoked, almost half self-reported a family history of heart disease, more than half had hypertension or diabetes, most were overweight or obese , no interest (33%), and failure to keep their appointment with the navigator (33%). Refusers, compared with participants, were significantly more likely to be white non-Hispanic (34% vs 24%), English speakers (79% vs 66%), single (45% vs 35%), and to have a higher baseline total cholesterol level (202 mg/dL vs 192 mg/dL). We found no other demographic or clinical differences.At follow-up, the mean FRS was significantly lower for the intervention group than the comparison group; 11.8% of the intervention group was at goal for an FRS, compared with 3.5% of the comparison group . At follThe mean baseline rate of cholesterol medication adherence was 70.1% for the intervention group; it increased by 12.0% to 78.5% at follow-up. The baseline rate for the comparison group was 73.9%; it increased by 2.7% to 75.9% at follow-up. The increases in medication adherence between the 2 groups were significantly different. The mean baseline rate (69.1%) of hypertension medication adherence increased by 9.7% for the intervention group; the mean baseline rate (78.8%) for the comparison group increased significantly by 4.2%.There were 3 CHD events in the intervention group and 4 CHD events in the comparison group between baseline and follow-up.We found no differences in baseline demographic, behavioral, or clinical characteristics among participants who completed a behavioral questionnaire and those who did not.We found significant changes in 10 of 13 behavioral measures from baseline to 12 months . ParticiThis intervention, which used patient navigators to encourage CVD risk-reduction behaviors and connect patients with community-based lifestyle-change services, demonstrated improvements in some clinical and behavioral outcomes for people at risk for CVD. These findings provide evidence for using behavioral approaches to reduce CVD risk in addition to medical therapy.The most effective approach for CVD prevention is a combination of efforts that works at all levels of influence to create a social and physical environment that is supportive of healthy behaviors. Evidence supports the use of community-based approaches for reducing heart disease risk -26. IndiWhereas other successful programs , consisting of a more intensive intervention, achieved better reductions in cholesterol levels and other coronary risk factors , our intOur study had limitations. For the clinical analyses, lack of randomization increased the possibility of selection bias and confounding. Matching at-risk comparison group members with intervention participants attenuates this possibility; the demographic and clinical characteristics of the 2 groups were similar, except for income levels. The participation rate of 53% may reflect the population often served by safety-net institutions; this population, composed largely of low-income and racial/ethnic minority patients, may harbor distrust in clinical programs. Nevertheless, we observed minimal differences between intervention participants and people who refused to participate. Another possible limitation is that only 65% of the eligible participants completed a follow-up behavioral assessment. We used intent-to-treat analyses to avoid selection bias. We found no differences in baseline characteristics between participants who completed a follow-up assessment and those who did not. Because we did not assess behavior changes in the comparison group, we cannot rule out that behavior changes identified in the intervention group were caused by factors other than the patient navigators. Future studies will need to examine health behaviors of both groups to fully assess the effect of using patient navigators. Finally, because follow-up was only for 1 year, the study was not able to assess the long-term effect of the intervention on CHD events.Using patient navigators to provide individualized counseling, assistance in goal setting, and linkage to community resources, seems to help intervention participants achieve positive behavior change  and improve several clinical outcomes. This intervention potentially offers a simple way to enhance traditional clinical CVD risk-reduction services. Future studies should consider conducting more rigorous evaluations of the effect of patient navigators on changes in health behaviors.


In light of the limitations of the current case finding strategies and the global urgency to improve tuberculosis (TB) case-detection, a renewed interest in active case finding (ACF) has risen. The WHO calls for more evidence on innovative ways of TB screening, especially from low-income countries, to inform global guideline development. We aimed to assess the feasibility of community-based ACF for TB among the urban poor in Cambodia and determine its impact on case detection, treatment uptake and outcome.Between 9/2/2012-31/3/2013 the Sihanouk Hospital Center of HOPE conducted a door-to-door survey for TB in deprived communities of Phnom Penh. TB workers and community health volunteers performed symptom screening, collected sputum and facilitated specimen transport to the laboratories. Fluorescence microscopy was introduced at three referral hospitals. The GeneXpert MTB/RIF assay (Xpert) was performed at tertiary level for individuals at increased risk of HIV-associated, drug-resistant or smear-negative TB. Mobile phone/short message system (SMS) was used for same-day issuing of positive results. TB workers contacted diagnosed patients and referred them for care at their local health centre.In 14 months, we screened 315.874 individuals; we identified 12.201 aged ≥15 years with symptoms suggestive of TB; 84% provided sputum. We diagnosed 783, including 737 bacteriologically confirmed, TB cases. Xpert testing yielded 41% and 48% additional diagnoses among presumptive HIV-associated and multidrug-resistant TB cases, respectively. The median time from sputum collection to notification (by SMS) of the first positive (microscopy or Xpert) result was 3 days (IQR 2–6). Over 94% commenced TB treatment and 81% successfully completed it.Our findings suggest that among the urban poor ACF for TB, using a sensitive symptom screen followed by smear-microscopy and targeted Xpert, contributed to improved case detection of drug-susceptible and drug-resistant TB, shortening the diagnostic delay, and successfully bringing patients into care. Despite the progress made in the last decade, Cambodia remains one of the countries with the highest tuberculosis (TB) burden in the world TB tends to concentrate in poor and marginalised communities who face many barriers to access health services such as lack of awareness, competing priorities for time and money, disconnection with health services due to lack of regular service and experienced personnel et al. on routine screening for HIV-associated TB before antiretroviral therapy Early detection of TB requires appropriate diagnostic tools. In many high burden countries smear microscopy remains the cornerstone of diagnosis. More sensitive and rapid diagnostics have become available but an affordable and accurate point-of-care test for TB is still lacking This study was undertaken to evaluate the feasibility of implementing community-based ACF using fluorescence microscopy and targeted Xpert in poor urban settlements of Phnom Penh, and determine its impact on TB case detection, treatment uptake and outcome.Ethical approval was granted by the National Ethics Committee for Health Research (Cambodia), and the Institutional Review Board at the Institute of Tropical Medicine and the Ethics Committee of the University Hospital, in Antwerp (Belgium). We also obtained permission from the national TB programme, the Municipality of Phnom Penh and the local health authorities. All participants provided written informed consent.Cambodia, a low-income country in South-east Asia, has an estimated TB prevalence  of 817/100.000 population - one of the highest in the world - and an incidence of 411/100.000 population Health services are organized into operational health districts. Each health district has a referral hospital and covers a number of health centres. Health centres provide basic care including TB services. Sputum smear microscopy and (if available) chest radiography are done at the referral hospital. Besides the four district hospitals, there are five tertiary hospitals in Phnom Penh providing care to patients from all over the country.Through a grant obtained from the TB REACH initiative (wave 2), the Sihanouk Hospital Center of HOPE conducted door-to-door screening for active TB in poor urban settlements of Phnom Penh from 9 February, 2012 to 31 March, 2013. We targeted communities with a presumed high prevalence of undiagnosed TB and/or restricted access to TB services. These included a heterogeneous group of slum dwellers, dump-site communities, migrants, factory workers, and displaced populations, the majority living in informal temporary or unstructured settlements. We selected the communities through purposeful sampling after consultation with health managers and municipal authorities. Community leaders, who were willing to participate, then identified the poorest or most hard-to-reach. The study area comprised 372 communities - scattered over the whole of Phnom Penh - with an estimated total population of 346.000. The study was embedded in the existing health services. It involved all levels of TB care of the four operational health districts of Phnom Penh providing services through 14 health centres, three referral operational district hospitals, and one tertiary level hospital .Key components of the intervention were:Trained TB workers, assisted by community health volunteers performed door-to-door visits to screen all available household members for symptoms suggestive of TB, defined as any cough, unintentional weight loss, fever, night sweats or haemoptysis. We relied on hetero-anamnesis for household members not at home at the time of the interview. If symptoms were reported, a new visit was arranged. If no symptoms were reported or the individual did not show up at the arranged visit no further attempts were made. All consenting adults aged 15 or more with a positive TB symptom screen (any of the above) were interviewed using a standardised questionnaire in the local language. Cases detected during the survey who had already started TB treatment were not considered as actively detected cases.The national TB programme recommends all symptomatic individuals to be evaluated with three sputum specimens. TB workers collected the specimens at home according to the following scheme: spot-spot-morning (SSM). Patients were instructed on how to produce a good quality sputum specimen following a standard operating procedure As per national policy, microscopic examination of three smears was done for all patients. At the time of protocol development, other than the rapid implementation guide for Xpert Light emitting diode (LED)-based fluorescence microscopy  was introduced at three referral hospitals and one GeneXpert MTB/RIF platform  was acquired at the tertiary referral laboratory. Direct examination for acid-fast bacilli was done with LED fluorescence microscopes using auramine O-staining and potassium permanganate counterstaining. Except when fresh specimens were needed (for Xpert and/or culture), smear preparation was done at the health centres; followed by smear staining and reading at all three referral laboratories.Plus assay  was requested to rapidly confirm isoniazid and rifampicin resistance at the laboratory of the Institut Pasteur de Cambodge.Xpert testing was done as per manufacturer's instructions. A single Xpert assay was done on a random spot or morning specimen. Inconclusive results were repeated once. For rifampicin-resistant cases on Xpert testing, a MTBDRMycobacterium tuberculosis, conventional proportion method DST  was performed. A para-nitro-benzoate test was included in the DST procedure.A single culture was performed for all patients with presumptive HIV-associated or MDR-TB. Culture and drug-susceptibility testing (DST) were done on conventional solid medium  following standard operating procedures of the laboratory. Cultures were considered negative if no growth was obtained after 8 weeks. Specimens yielding growth (any number of colonies) were subjected to MPT64-based species confirmation as per manufacturer instructions . If growth yielded All laboratories participated in external quality assurance for smear microscopy through the national TB programme. The Sihanouk Hospital Center of HOPE laboratory also participated in annual DST rounds by the Institute of Tropical Medicine (Antwerp) and passed a validation test for Xpert by the manufacturer.As soon as available, laboratory technicians issued positive results from smear microscopy or Xpert by short message system (SMS) to the respective TB workers, who  contacted the patient either directly or via the community health volunteer. Smear- or Xpert-positive TB patients were referred to the health centre for initiation of TB treatment at their earliest convenience. Written results were distributed to the TB worker at least once a week, including negative results. When smear microscopy was negative but TB symptoms persisted, individuals were advised to contact the TB worker or community health volunteer. They would then be asked to submit two more sputum specimens (for repeat smear and Xpert), and referred for further diagnostic work-up including chest radiography as per national policy.First-line anti-TB drugs were started for all bacteriologically-positive cases as well as for individuals with a high clinical suspicion of smear-negative or extra-pulmonary TB according to the DOTS-strategy TB workers and community health volunteers actively tracked patients who dropped out after diagnosis, or interrupted treatment and encouraged them to initiate or complete TB treatment. This could require several (up to three) visits. To ensure active engagement in care, the most convenient treatment modality (community- or ambulatory-DOTS), was discussed.Treatment outcomes were abstracted from the health centre's treatment registers in the first place. If not found, TB workers enquired the patient or his/her relatives by phone or on house visit.We newly recruited all TB workers, some of (para-)medical background, and trained them on TB diagnosis and treatment as well as data collection. They received a mobile phone, an ice box and a back pack containing data collection tools and sputum cups. Community health volunteers - lay members, some of whom had previously participated in health projects - were assigned by the village leader. They accompanied and assisted TB workers on their rounds. Health centre TB staff were offered a refresher course on smear preparation. LED fluorescence microscopy was introduced at three referral laboratories. Laboratory personnel attended a one-week training by the national tuberculosis programme. To ensure quality microscopy services the Sihanouk Hospital Center of HOPE provided continued training and on-site support for laboratory technicians in the district hospitals, and monthly quality control checks on a random selection of ten smears. A core team from Sihanouk Hospital Center of HOPE met weekly to address issues regarding coordination, clinical or laboratory work, research, and data management and evaluate the progress. Separately, all field TB workers were offered the opportunity to provide feedback on their activities during weekly meetings with the coordinating team. The coordinating team and the operational health district's TB staff also met quarterly.All TB workers received an appropriate salary and travel allowance. All community volunteers and health district personnel received a small incentive (US$ 15–30/month) to compensate for the extra work delivered to the project.M. tuberculosis detected by Xpert or isolated by culture were considered bacteriologically-proven TB Smear-positivity was defined as at least one smear containing at least one acid-fast bacillus Data were entered in an Access database. Verification of original data  was done monthly for a random sample of ten TB patient files. Error queries were run weekly to check for missing data and resolve inconsistencies.Patients' demographic and clinical characteristics were described in terms of percentages, medians and interquartile ranges (IQR). We calculated frequencies, proportions and 95% confidence intervals (95% CI). A two-sided p-value<0.05 was considered significant. All analyses were performed using STATA software version 10.0 .Over 14 months, a team of 37 TB workers assisted by 372 community health volunteers screened a population of 315.874 (out of an estimated 346.000) for TB. Among individuals providing a sputum specimen (n = 10.301) 7,2%  had bacteriologically-positive TB. However, this ranged from 12,5%  among the presumptive or confirmed HIV-infected patients, 10,0%  among individuals with risk factors for MDR-TB and 6,5%  in the group without any of these risk factors. The median age of patients diagnosed with bacteriologically-positive TB was 47 years (IQR 32–60); 55,4% were men.Besides the 737 bacteriologically-confirmed TB cases, we diagnosed 46 “clinical” TB cases without confirmation (32 pulmonary and 14 extra-pulmonary TB) totalling up to 783  adult TB diagnoses .Considering all 315.874 individuals screened, overall TB prevalence was 339/100.000 (95% CI: 319–360) including 274 patients already on TB treatment and 13 children diagnosed with TB.Of the 737 bacteriologically-confirmed TB cases, the majority  could have been diagnosed by a positive smear only. Of the 10.301 presumptive TB patients whose sputum was analysed 663  had a positive smear; 48,7% of which were graded 1+ or more.Overall 61 additional TB cases were identified by Xpert and 13 by culture.The median time from sputum collection to the first positive result either by smear microscopy or Xpert was 4 days (IQR 2–7). Due to clerical errors only a limited number of SMS data  from a single laboratory were available. Positive results were sent by SMS at a median of 3  days after sputum collection, whereas written results (including positive and negative results) were available at a median of 5 days (IQR 2–8).Twenty per cent experienced a treatment delay of more than one week. Another 21  TB patients never started treatment despite several counselling visits. This included two patients who died one month after diagnosis but who had not yet started treatment.The overall treatment success rate of a first-line TB regimen was 81,4% (n = 632/776). Four individuals failed treatment, 18  died, 59  were lost to follow-up (including 38 who interrupted treatment and 21 who never started), and 63  were not evaluated because they were transferred out and/or treatment outcome was not reported. Of the eight patients with rifampicin resistance detected on Xpert, all but one were successfully started on a standardised second-line regimen. One subsequently died on treatment; other outcomes are pending.Case notifications in Phnom Penh increased from 4.073 to 4.561 – a 12% increase - for all forms of TB in the five quarters preceding ACF implementation (quarter four of 2010 plus 2011) compared with the intervention period (2012 and first quarter of 2013). Case notifications of bacteriologically-confirmed TB increased from 1.610 to 2.075 – a 29% increase - over the same period of time From February 2012 to March 2013 we performed door-to-door active TB screening in poor urban settlements of Phnom Penh reaching a population of 315.874. We diagnosed 783 TB cases – 737 of which were bacteriologically-confirmed - within a median of three days for smear- or Xpert-positive disease. Overall 95% begun TB treatment and 81% obtained a successful treatment outcome; less than 3% failed to start.The effectiveness of community-wide active TB screening is widely debated for various reasons: the high number of individuals needed to screen, the risk of false-positive TB diagnoses and the unacceptable number of patients failing to start treatment et al.et al. have shown the important role of community health workers by improving access to TB diagnosis and care in enhanced case finding in rural Ethiopia The latest national prevalence survey in Cambodia revealed a substantial burden of undetected TB, with TB prevalence rates more than twice as high as TB case notification rates particularly among persons aged more than 45. It also showed that the symptom screening used - cough >2–3 weeks or haemoptysis - had poor sensitivity: more than 70% of the smear-negative culture-positive TB cases were “asymptomatic” et al. also reported substantially higher performance characteristics for Xpert compared with microscopy (63% vs. 18% sensitivity) Xpert clearly outperformed smear microscopy in the few studies reporting on its use in ACF To achieve an impact on patients' outcomes and TB transmission, active screening for TB ideally engenders improved case detection, shortened diagnostic delays and earlier treatment initiation Second, we were able to issue results for bacteriologically-confirmed TB within a median of three days from sputum collection, which is a substantially shorter turn-around-time than the generally reported 7–10 days delay (community experience). The use of mobile phone/SMS contributed to expediting the time to diagnosis, as did other interventions such as facilitation of specimen transport, and not at least the active network of community workers, health centre staff and laboratory technicians. While we should aim for same-day issuing of results Third, the impact of ACF is thwarted when improving case detection without ensuring treatment initiation and completion. Unlike reports from South-Africa and India Taking into account that our population was highly mobile and hard-to-reach, the 2,6% initial lost to follow-up compares favourably with the 32% initial lost to follow-up in India Successful treatment outcomes of 81% are consistent with findings from ACF-projects in Cape Town Although not without challenges, door-to-door screening in our communities was operationally feasible and effective. Different interventions for community TB screening have been described such as ACF through home visits Although our study has several strengths such as the large-scale implementation of community-based ACF embedded in a well-functioning national TB programme, low initial lost to follow-up rate and high treatment success, it also has limitations. Almost 50% of the household members in the communities were not available for interview, mostly because of work, and symptom screening was done through a proxy. This may have led to an underestimation of presumptive or real TB cases since no further investigations were done if an individual reportedly had no symptoms.The current strategy resulted in an under-diagnosis of smear-negative TB: 6%, in contrast with 40% of smear-negative or extra-pulmonary TB cases notified annually to the national TB programme. The improved sensitivity of fluorescence microscopy and Xpert only could not explain this finding. Targets and incentives drove the case detection of bacteriologically-confirmed TB, likely overlooking presumptive TB cases who were unable to provide sputum or smear-negative but did not return for further work-up. A more comprehensive diagnostic algorithm including screening with CXR and ensuring diagnostic work-up of non-confirmed symptomatic individuals is recommended.In 9,5% of the smear-positive patients with prior TB non-tuberculous mycobacteria grew on culture (and Xpert remained negative), suggesting that the concern of false-positive TB diagnosis (if only smear microscopy is used) is justified in certain subgroups. Further research on the confounding role of underlying lung disease on TB diagnosis in ACF is warranted.Although our study is one of the few large-scale ACF studies using targeted Xpert (and culture), the majority of TB cases was diagnosed by smear microscopy. Xpert and culture were performed for high-risk patients only on randomly selected specimens with possible different bacterial load. Hence, a discussion on sensitivity and specificity of Xpert and culture in ACF is beyond the scope of this study.No formal cost-effectiveness study was done. ACF is a more resource-intensive method of case detection, and its cost-effectiveness, even in high prevalent settings, needs to be assessed In conclusion, community-based ACF through community health workers, with targeted use of Xpert along smear microscopy proved feasible and effective in poor urban settlements of Phnom Penh. It allowed these communities to timely access TB diagnostic services; it identified a substantial proportion of infectious cases and a flexible patient-centred treatment approach ensured a high treatment success rate. Longer term follow-up data are needed to evaluate the impact on TB transmission. Community-wide TB screening could be considered as a complementary strategy to passive case detection in similar TB-endemic settings.


Ceratocystis polonica.NB-LRR resistance proteins are involved in recognizing pathogens and other exogenous stressors in plants. Resistance proteins are the first step in induced defence responses and a better understanding of their regulation is important to understand the mechanisms of plant defence. Much of the post-transcriptional regulation in plants is controlled by microRNAs (miRNA). We examined the expression of five Norway spruce miRNA that may regulate NB-LRR related transcripts in secondary phloem (bark) of resistant Norway spruce after wounding and inoculation with the necrotrophic blue stain fungus PaChi4, PaPAL and PaPX3 indicative of an effective induced host defence response. There were minor local and systemic changes in the expression of five miRNAs and 21 NB-LRRs between healthy and treated plants. Only five putative NB-LRRs  showed significant increases greater than two-fold as a local response to C. polonica. Of all NB-LRRs only PaLRR3, the most highly differentially regulated NB-LRR, showed a significant increase also due to wounding. The five miRNAs showed indications of an initial local and systemic down-regulation at day 1, followed by a later increase up to and beyond the constitutive levels at day 6. However, the initial down-regulation was significant only for miR3693 and miR3705.The plants of this clone recovered from both the pathogen inoculations and wounding alone. We found local and systemic induction of the resistance marker genes PaLRR3. The minor expression changes observed both for the followed miRNAs and their predicted NB-LRR targets suggest that the expression of most NB-LRR genes are maintained close to their constitutive levels in stressed and healthy Norway spruce plants.Overall, local and systemic expression changes were evident only for the established resistance marker genes and  Arabidopsis resistance gene FLS2 coding for a LRR-kinase that recognize the PAMP in bacterial flagellin [The ability of individual plants to recognize pathogens depends on the plant’s complement of resistance genes ,2. Plantagellin , that caagellin . Host plagellin ,5.The most prevalent class of disease resistance proteins involved in pathogen recognition are NB-LRRs ,6,7. NB-Arabidopsis, suggests that NB-LRR-encoding gene expansion is a mechanism to compensate for the longer generation time in woody perennials [Populus trichocarpa genome, and this is at least twice the number found in Arabidopsis. NB-LRRs are also found in gymnosperms such as pine, where they have been tightly linked to resistance to e.g. white pine blister rust Cronartium ribicola[Pinus monticola, where Liu and Ekramoddoullah found a large number of TIR-NB-LRRs as well as 61 transcripts of the CC-NB-LRR subfamily [The extensive recent R-gene duplications that have taken place in the perennial angiosperms grapevine and poplar compared with annuals such as rice and ennials . Kohler ennials  detected ribicola,14. Mostbfamily ,14. The Ceratocystis polonica (Siem.) C. Moreau, is a virulent associate of the Eurasian spruce bark beetle Ips typographus L. [C. polonica is able to kill the host Norway spruce, and is considered the causal agent of tree death rather than the beetles that vector it during bark beetle mass outbreaks. Norway spruce trees pre-treated with mechanical wounding are much more resistant than untreated trees to subsequent challenge inoculation with this necrotrophic blue stain fungus [C. polonica inoculation [C. polonica is likely very ancient and this pathogen is thus likely highly co-evolved and well adapted to its host. This raises the possibility that there is a highly refined suite of NB-LRR type host receptors and ETI related responses involved. However, it is not known to what extent NB-LRRs are involved in pathogen recognition and wound associated responses in spruce.The necrotrophic blue stain fungus phus L. . The pat fungus . Similarulation . The intPaPX3 has a general stress-induced function and is upregulated in Norway spruce bark in response to infection by the root rot fungus Heterobasidion parviporum and wounding [PaPAL) also has a key role in lignin and phenylpropanoid formation in gymnosperms such as spruce and in angiosperm plants [PaChi4 has proven to be a particularly useful marker for local and systemic defence response in both Norway spruce and Sitka spruce [Recognition of the pathogen and other stresses induce signalling pathways that again cause expression of the downstream genes such as the ones coding for pathogenesis related proteins and enzymes in the phenylpropanoid pathway. Genes regulating lignin metabolism are important for angiosperms resistance to fungal pathogens  as plantounding . Phenyla plants -22. In N spruce -25.Arabidopsis[Pinus taeda miRNAs have been associated with the fusiform rust gall disease [MicroRNAs (miRNAs) that are involved in plant innate immunity triggered by pathogen associated molecular patterns (PAMPs) have been identified in abidopsis. miRNAs abidopsis,28. In ldisease . The impin silico, but the exact number of these genes in conifers such as Norway spruce and other gymnosperms is unknown. Here we follow the differential expression of 21 NB-LRR related genes and five putative miRNAs targeting these genes, as well as the defence response markers PaChi4, PaPX3 and PaPAL in Norway spruce over a spatial and temporal gradient. Expression profiling of these key genes will clarify whether transcriptional regulation of NB-LRRs or miRNAs is a key feature in the local and systemic resistance responses in Norway spruce, and reveal if there is a differential host response to wounding and pathogen infection.NB-LRRs have been identified in spruce and there is now a wealth of available transcript sequences that makes it possible to identify NB-LRRs PaPX3 and the class IV chitinase PaChi4 increased significantly both locally and systemically in the bark following C. polonica inoculation and wounding  [PaLRR25, PaLRR26, PaLRR27, PaLRR28 and PaLRR29, respectively  , and bas705 , anPaLRRs 2, 5, 6, 7, 8, 11, 12, 13, 17, 18, 19, 25, 26, 27, 28 and 29; Figures PaLRR1 PaLRR3, PaLRR14, PaLRR15 and PaLRR16) showed more than two-fold significant changes between control and treatments , so here a value of 1 equals 2 Figure . The rapC. polonica. The plants seemed to recover quickly from the treatments, confirming this clone’s high level of resistance. Most of the NB-LRRs we studied remained close to their constitutive levels in both healthy, wounded and pathogen inoculated Norway spruce. However, PaLRR3 (a CC-NB-LRR type resistance gene) showed extensive local and systemic increase in response to wounding and inoculation. We found a rapid drop in miRNAs putatively targeting NB-LRRs, but the change was significant only for two miRNAs and there was no corresponding change in their putative NB-LRR targets.This work is the first attempt to survey the diversity of NB-LRRs in spruce and to follow the expression pattern of miRNAs putatively targeting resistance genes in plants. Expression of host defence marker genes was induced both locally and systemically in response to both wounding alone and inoculation with the pathogen Populus tree species [PaPalPaPX3 and PaChi4, but also some NB-LRRs-like transcripts and two putatively regulatory miRNAs, are differentially regulated locally and systemically following fungal inoculation and wounding. However, this is overshadowed by the main finding that most NB-LRRs appear to be under very strict transcriptional control and are maintained at low and stable constitutive levels in both healthy and stressed plants. The maintenance of stable low levels of most NB-LRRS might be related to the cost of preserving these large proteins and associated signalling pathways, and the fact that even low constitutive levels may be sufficient to respond effectively to foreign invaders.Surprisingly little is known about how NB-LRRs and related genes are regulated after pathogen attack and other stresses in long lived plants such as trees, and this even includes the well-studied species . Previouspecies ,29, and Arabidopsis genome [Populus[Pinus taeda (L.) [Cryptomeria japonica[Pinus lambertiana and western white pine [http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=spruce). In silico analysis of these confirms that spruce has a reasonably large repertoire of defence and resistance-like genes, and in our search we found all the major subgroups of LRR genes, except the BED-finger LRR protein genes reported from Populus[NB-LRR genes appear to be abundant in the conifer Norway spruce, but since we do not have the genome sequence of Norway spruce we cannot at this time determine the true number of resistance genes present. We found more than 200 LRR-containing resistance-like genes in the available spruce databases, and this is similar to other plant species. More than 160 NB-LRR-encoding genes have been identified in the Col-0  genome , 250 ful genome , and so [Populus. Among c[Populus,14. Liu [Populus proposedda (L.) , Japanes japonica, Norway  japonica, sugar pte pine ,35. Therm Populus.PmTNL1 is a resistance gene linked to partial resistance to white pine blister rust C. ribicola. The PmTNL1 transcript is expressed at low basal levels in different tissues. Expression remained unchanged during compatible and incompatible interactions with C. ribicola at the early stages post-inoculation, but in later stages of the interaction higher levels were found in symptomatic plants [PaLRR15, as well as for PaLRR1PaLRR14 and PaLRR16, and most notably for the CC-NB-LRR class PaLRR3. However, most of the TIR-NB-LRRs and other NB-LRRs that we studied remained at stable levels following wounding or inoculation. This agrees with previous studies showing that resistance genes such as NB-LRRs typically are expressed constitutively at low basal levels and do not increase notably after pathogen challenge [Populus. Kohler and coworkers, examining the diversity of NB-LRR genes in the Populus trichocarpa genome, detected only 34 of 400 known NB-LRR homologues from rust-infected and non-infected leaves using a whole-genome oligoarray, and none of the 34 NB-LRRs showed an altered expression two days post-inoculation [NB-LRRs have been linked to resistance in conifers. In white pine the TIR-NB-LRR homologue  plants . We saw allenge -37. Simiulation . Our resulation .PamiR950, PamiR951, PamiR3693, PamiR3697 and PamiR3705, their NB-LRR candidates PaLRR25 to 29, and the downstream defence induction markers in Norway spruce. However, it was expected that the rapid and statistically significant down-regulation of PamiR3697 and PamiR3705 would be mirrored by greater expression of their putative NB-LRR targets. There could be several explanations for the lack of a clear correspondence between the expression of miRNAs and their predicted NB-LRR target sequences; (1) the change in miRNA levels detected here may simply be too small to have a significant impact on NB-LRR targets, (2) the miRNAs may act preferentially on other NB-LRR paralogues than the ones identified here, (3) the putative NB-LRR targets could be also the origin of the miRNAs, complicating matters even further, (4) the expression of the NB-LRR target genes may be under tight control of positive and negative regulators that bind their promoters, thus overriding the effect of miRNAs, or less likely (5) that these miRNAs might inhibit the translation of NB-LRRs and not cause breakdown of the RNA transcripts. The most likely explanation is probably that the small changes we detected in miRNA levels work in concert with regulatory changes by transcription factors at the promotor of these genes in order to maintain stable NB-LRR transcript levels. It is also possible that we should have looked at changes in miRNAs and their NB-LRR targets at a much earlier time point after treatments than day 1 to 6, and preferentially have focused only on the host cells in the immediate vicinity of the inoculation/wounding site. However, our combined results do not rule out that miRNA might be involved in local and systemic defence response in Norway spruce, but show that a much larger set of small RNAs (extending to other targets than NB-LRRs) must be studied in combination with transcriptome and degradome studies of all targets by way of latest generation sequencing methods.Our transcriptional data are not sufficient to establish a clear link between the expression of the miRNAs Specific cell types in a tissue may have specialized roles in pathogen recognition and systemic defense signaling that are not resolved when collecting samples from large sections of tissues such as bark. The bark of a conifer contains specialized tissues  with specialized cells that are organized in a regular pattern , such asTwo conclusions can be drawn from our data. First, most of the NB-LRRs in the gymnosperm Norway spruce are maintained at low constitutive levels, suggesting that basal levels of these gene products are sufficient to serve their putative resistance gene function as receptors (or translocators) of external challenges such as a pathogen. Thus, the NB-LRRs’ role in resistance may be to be constitutively present to detect pathogen effectors (directly or indirectly), and not to be differentially regulated following a challenge ,26,35-37Secondly, in this first study of putative NB-LRR targeting miRNAs we observed relatively minor changes in expression of miRNAs and their predicted NB-LRR targets. This does not necessarily rule out a role for miRNAs in resistance, but suggests that they may, in concert with other regulators, contribute to the tight constitutive expression of NB-LRRs needed to detect external challenges.Heterobasidion parviporum and Ceratocystis polonica[-2 s-1 light conditions. The plants were inoculated and wounded at the time when the leader shoot had extended (actively growing) and well prior to any new bud formation. The plants were well-watered and provided with sufficient fertilizers throughout the experiment [The experimental material consisted of 21 identical 2-year-old saplings generated by somatic embryogenesis from a single Norway spruce clone. This clone (AL15886-B10) was derived from a single seed from a full-sib cross (♀ #2650 × ♂ #2707) and the saplings were produced at the Biri Nursery and Seed Improvement Centre, Norway . AL15886 polonica. After h polonica the geneeriment .C. polonica (isolate no.NISK 93–208:115) (Cp) or wounded (W) on the stem. Plants were inoculated 5 cm above the ground by cutting a ~5 mm wide bark flap using a scalpel. A small amount of malt agar  containing actively growing mycelium was placed underneath the bark flap, the flap was pressed firmly against the stem, and the wound was sealed with parafilm. Wounding was done in the same way, but using malt agar without fungus. Unwounded control plants (C) were also wrapped with parafilm at the corresponding position of the stem. Local and more distal samples of bark (1 cm long) were harvested 1, 3 and 6 days after treatment from at least two ramets per treatment and time point. Samples were quick-frozen in liquid nitrogen and stored at −80°C until analysis. To distinguish between local and systemic gene expression local and distal samples were analysed separately. The local sample (W1/Cp1) included the inoculation/wounding site and extended ca 0.5 cm above the upper margin of the wound. The distal sample (W2/Cp2) included the entire bark region 2–3 cm above the inoculation/wounding site. The unwounded controls were sampled at the corresponding positions.Saplings were inoculated with Picea (taxid: 3328) sequences at the National Centre for Biotechnology Information (NCBI) database using BLASTP algorithms. The conserved domain was derived from a range of NB-LRR gene sequences available from NCBI GenBank (http://www.ncbi.nlm.nih.gov/). All the unique sequences we obtained were verified using the NCBI blast server with BLASTP searches against the NCBI GenBank and the Conserved Domain Database (CDD) [http://compbio.dfci.harvard.edu/cgi-bin/tgi/gireport.pl?gudb=spruce) using the keyword “LRR” to select a list of unigenes temporarily annotated as NB-LRR motif-containing genes.Initial searches for NB-LRR genes were performed using the NB-LRR family conserved catalytic domain to probe the e (CDD) . Gene moWe conducted phylogenetic and molecular evolutionary analyses for translated amino acid sequences of the selected NB-LRR gene models using the MEGA4 software . The evoPaLRR25PaLRR26PaLRR27PaLRR28 and PaLRR29 (table s1.2). These miRNAs had all the in silico characteristics of microRNA identified in an earlier study of miRNAs involved in epigenetic regulation and climatic adaptation in Norway spruce [We identified five miRNAs putatively targeting the NB-LRR transcripts 9 table s.2. These2 throughout the grinding process. Total RNA  was extracted from 100 mg of the pulverized samples using the Master Pure™ RNA Purification Kit  following manufacturer recommendations and stored at −80°C until further use. RNA was quantified using a micro-volume spectrophotometer .Bark samples (100–150 mg) were ground first in liquid nitrogen using a mortar and a pestle and then transferred to 2 ml eppendorf tubes for fine grinding in a Retsch 300 Mill  for 1.5 min. The equipment and tissue samples were kept chilled with NPaChi4, PaPAL and PaPX3 are previously verified [PaAct). In our previous work the PaAct transcript has proved to be the best endogenous reference for RT-PCR in Norway spruce as it gives the better stability index when compared to alphaTubulin (PaTub), Polyubiquitin (PaUbq) and Glyceraldehyde-3-phosphate dehydrogenase (PaGAPDH) [PaAct was a stable reference we compared the stability of PaAct expression levels in all the day one samples against PaTub and the translation initiation factor (PaTif) and found that using PaAct alone as endogenous reference for RT-PCR was equally significant as using the combination of the three references PaActPaTub and PaTif (data not shown).Gene specific primers were designed using Primer3  with theerified . Total RaGAPDH) . To furtAbsolute quantification was performed using the 7500-system SDS software and data were further processed in MS Excel.Transcript abundance of the selected miRNAs was determined with relative real-time RT-PCR as described by Yakovlev et al. 2010). cDNAs were synthesized from 600ng of small RNA using the NCode miRNA First-Strand cDNA Synthesis Kit  following the manufacturer’s recommendations. Real-time RT-PCR amplification was performed using NCode SYBR GreenER miRNA qRT-PCR Kit  in a 25μl reaction volume, using 2μl of a diluted cDNA solution as template and 200 nM of each primer. Reactions were conducted on the 7500 Real-time PCR System  using the Invitrogen recommended cycling conditions. After PCR, dissociation curves were made to verify the specificity of the amplification. There were two technical and two biological replicates for each sample. All miRNA expression levels were normalized to the geometric mean of three selected ribosomal and transfer RNA genes. Forward primers were designed based on mature miRNA sequences. If Tm of the mature miRNA was&60°C, it was adjusted by adding Gs and Cs to the 5-end and/or the 3-end of the miRNA sequence. To verify the specificity of the miRNA amplification we analysed several PCR samples for each miRNAs on 2% agarose gels with ethidium bromide (EtBr) visualization of bands. Reverse primers was supplied with the NCode miRNA First-Strand cDNA Synthesis Kit . The miRNA primers used are listed in Additional file 010. cDNAThe relative ddCT quantification method based on the critical cycle threshold values was used for the miRNAs, using the 7500-system SDS software. Data were further processed in MS Excel.Relative transcript amounts were calculated from raw data obtained from real-time PCR in the form of critical threshold cycle values (dCt) for the miRNA data as described in . For theThe authors declare that they have no competing interests.CGF and PK conceived and designed the study, IAY, PK, CGF and NY performed the experiments and collected the data, HK, IAY, CGF, PK and NY performed the data analysis. CGF, PK, HK, IAY, HS and NY drafted the manuscript. All authors read and approved the final manuscript.Table s1.1 Selected NB-LRR family genes in Norway spruce analysed by real time qRT-PCR.Click here for fileTable s1.2 The additional selected 5 NB-LRR family genes in Norway spruce putatively regulated by specific miRNAs and analysed by real time qRT-PCR. Table s2. Selected specific miRNAs putatively targeting NB-LRR family genes .Click here for fileSupplement 3. Phylogenetic tree and cluster description of the selected NB-LRR gene models (full ORF amino acid sequences) based in the spruce ESTs available in the NCBI Databases. The phylogenetic tree was constructed using MEGA4 as detailed in Materials and Methods.Click here for fileFigure s4. Gel showing primer test to identified NB-LRRs in Norway spruce using primers designed from in silico studies of exiting spruce sequences in sequence databases. Only primer pairs giving a single band was further used for real-time RT-PCR. Therefore, NB-LRRs 22, 20, 10, 8 and 4 were not studied by qRT-PCR.Click here for file


Deep brain stimulation (DBS) has been proposed as an alternative to ablative neurosurgery for severe treatment-resistant Obsessive-Compulsive Disorder (OCD), although with partially discrepant results probably related to differences in anatomical targetting and stimulation conditions. We sought to determine the efficacy and tolerability of DBS in OCD and the existence of clinical predictors of response using meta-analysis.We searched the literature on DBS for OCD from 1999 through January 2014 using PubMed/MEDLINE and PsycINFO. We performed fixed and random-effect meta-analysis with score changes (pre-post DBS) on the Yale-Brown Obsessive Compulsive Scale (Y-BOCS) as the primary-outcome measure, and the number of responders to treatment, quality of life and acceptability as secondary measures.Thirty-one studies involving 116 subjects were identified. Eighty-three subjects were implanted in striatal areas—anterior limb of the internal capsule, ventral capsule and ventral striatum, nucleus accumbens and ventral caudate—27 in the subthalamic nucleus and six in the inferior thalamic peduncle. Global percentage of Y-BOCS reduction was estimated at 45.1% and global percentage of responders at 60.0%. Better response was associated with older age at OCD onset and presence of sexual/religious obsessions and compulsions. No significant differences were detected in efficacy between targets. Five patients dropped out, but adverse effects were generally reported as mild, transient and reversible.Our analysis confirms that DBS constitutes a valid alternative to lesional surgery for severe, therapy-refractory OCD patients. Well-controlled, randomized studies with larger samples are needed to establish the optimal targeting and stimulation conditions and to extend the analysis of clinical predictors of outcome. Obsessive-compulsive disorder (OCD) is characterized by the presence of upsetting, persistent thoughts, images, or impulses, which are experienced as intrusive and senseless (obsessions) and/or excessive repetitive behaviors or mental acts (compulsions) intended to neutralize the anxiety induced by the obsessions . OCD hasSo, the goal of the current meta-analysis was 1) to systematically record the treatment effects of DBS in severe therapy-refractory OCD patients, and 2) to identify any clinical variables associated with a better response to this therapeutic approach.We performed a comprehensive PubMed/MEDLINE and PsycINFO search from January 1999 through January 30, 2014, including the following terms: “deep brain stimulation” or “DBS” in association with “obsessive-compulsive”, “obsessive-compulsive disorder” or “OCD”. These words were searched as key words, title, abstract and Medical Subject Headings. Reference lists from retrieved reports were reviewed for additional relevant studies.Candidate studies–judged on the basis of their title and abstract–had to satisfy the following criteria to be eligible for inclusion in this review: 1) human studies assessing the efficacy of DBS on OCD according to changes on the Yale-Brown Obsessive Compulsive Scale (Y-BOCS) scores or percentage of responders defined by standardized criteria; 2) subjects aged 18–75 years with a diagnosis of OCD according to the Diagnostic and Statistical Manual of Mental Disorders IV ; 3) stud- reviews of DBS use for OCD not providing results from de novo patients- discussions of ethical issues related to DBS- articles focused on biological correlates of DBS use in OCD–neuroimaging, electrophysiological or neuropsychological changes after DBS- articles focused on the secondary side effects of chronic DBS use in OCD or effects observed during acute stimulation programming- articles focused on other indications of DBS different from OCD- articles focused on neurosurgical issues related to DBS implantation for OCD- studies on animal models of DBS use in OCDExclusion criteria:- sample characteristics: age, gender, age at onset of OCD, duration of OCD, OCD symptom dimensions.- DBS-related: brain target, lead model, duration of stimulation.- study-related: single or double-blind; sham-controlled; parallel or crossover designs- primary outcome measure: score changes (pre-post DBS) on the Y-BOCS- secondary outcome measure: number of responders to treatment based on standardized criteria (> 35% reduction in post-treatment Y-BOCS scores) and changes on quality of life (QOL) measures- acceptability of treatment: overall dropout rates and side effectsData were recorded as follows:Q statistics and I2 index [Analyses were performed using the statistical software R3.0.1  with met2 index . Values 2 index . StudentFlow of information according to PRISMA statement, study selection and reasons for exclusion are provided in Thirty-one studies were included in this meta-analysis , 20–46, I2 = 96.4%)  and I2 index (I2 = 0%) suggested that the low sample sizes did not permit a correct estimation of heterogeneity . A fixed effect model estimated the global percentage of responders at 60.0% (95% CI = 49.0% to 69.0%). The random effect model detected identical results due to the low sample size of the studies. Results for Information on the impact of DBS on quality of life was available for 29 patients , 47. Goo2 = 0.06, p = 0.8).Patient-level data were available for 45 patients who received DBS in striatal areas–including VC/VS, ALIC, NAc and NC–and 21 patients who were implanted in the STN. No significant differences were detected between the two targets in terms of the percentage of reduction in Y-BOCS scores  or in percentage of responders . Current age was not correlated with percentage of Y-BOCS score reduction . No significant differences were detected between males and females in percentage of Y-BOCS score reduction   or gender  .Responders to DBS reported a significantly older age at onset of OCD than non-responding patients . A tendency was detected for a significant positive correlation between age at onset of OCD and percentage of Y-BOCS scores reduction after DBS .No significant differences in years of OCD duration prior to DBS implantation were detected between responders and non-responders .No significant differences between responders and non-responders to DBS were detected for the presence of aggressive/checking symptoms , contamination/cleaning , symmetry/ordering , hoarding , and somatic obsessions and compulsions .Responders more frequently reported obsessions and compulsions of sexual/religious content than non-responders  .Five patients dropped out from DBS without completing the planned period of stimulation, representing 4.7% of the implanted patients. Two of these cases were in the early study by Nuttin et al. ; both paThe aim of this study was to measure the response to DBS in severe treatment-resistant OCD patients using meta-analysis. The data available from 116 subjects produced a global percentage of Y-BOCS score reduction of 45.1% and a global percentage of responders of 60.0%. Better response to DBS was associated with older age at OCD onset and with the presence of sexual/religious obsessions and compulsions. No significant differences were detected in the percentage of responders or in Y-BOCS score reduction between patients who received stimulation of striatal areas and those with STN implanted electrodes.These results confirm that DBS appears to have an efficacy comparable to that reported for capsulotomy or cingulotomy, ablative techniques after which 64% and 56% of patients respectively are rated as significantly improved , 49. NevMost published studies focus their attention on symptom reduction after DBS and scarce data is available for changes on quality of life in these highly-resistant and chronic severally ill patients , 37, 47.Distant DBS effects on abnormal neural connectivity in the cortico-striato-thalamo-cortical circuit involved in OCD might explain why stimulation of different brain regions finally achieves similar percentages of improvement. Stimulation of STN has been reported to decrease OFC and mPFC metabolism as well as ACC activity  while stBetter response to DBS was associated with older age at OCD onset. Age of onset has been postulated as an important marker for subtyping OCD. Early age of onset patients show more severe forms of OCD, poorer prognosis for pharmacological treatment, higher familial aggregation of both OCD and tic disorders, and a higher specific comorbidity pattern mainly with ADHD, Tourette’s syndrome and bipolar disorder , 54. A fAccording to the results of this meta-analysis, the presence of sexual/religious obsessions and compulsions was associated with a significantly better response to DBS. Recent studies have associated this OCD clinical dimension with specific brain functional connectivity patterns: patients with more sexual/religious obsessions demonstrated relatively greater connectivity between the ventral caudate and the middle and anterobasal insular cortex than patients with other symptom dimensions as well as healthy controls in a study addressing alterations of ventral corticostriatal functional connectivity in OCD . Since FThe present manuscript has a number of limitations. First, the small sample sizes in the studies included complicate the assessment of inter-study heterogeneity. The studies were heterogeneous in terms of anatomical targeting, electrode design and stimulation parameters. This makes the comparison between studies difficult, and reflects the fact that DBS for OCD is a tool that is still under development. Second, we decided to address the response to DBS in all available patients worldwide instead of restricting our analysis to double-blind sham-controlled studies, since only six of the published studies (with only 45 subjects) reported this design in their methodology. Moreover, even in this small body of studies, the duration of active and sham periods was not easily comparable since it lasted from minutes  to threeAlthough the number of severe OCD treatment-resistant patients treated with DBS is still low, and optimal targeting and stimulation parameters are still under debate, the results of this meta-analysis confirm that DBS constitutes an alternative to ablative surgery for this group of extremely ill patients and presents an acceptable adverse effect profile. Further well-controlled randomized studies in larger samples are needed to confirm and extend our findings on clinical predictors of response, and thus to improve both patient selection and response rate.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


Arabidopsis through the manipulation of the centromere-specific histone 3 variant, CENH3. Their approach, which involved extensive modifications to a transgenic CENH3, held promise of being translated to crop species, and has been successfully employed in maize (see cenh3 null by CENH3 from closely related plant species can result in plants that are fertile but haploid-inducing on crossing by CENH3 wt plants- suggesting that introgression of alien CENH3 may produce non-transgenic haploid inducers. Similarly, a remarkably wide variety of point mutations in CENH3, inducible by chemical agents, have recently been shown to result in haploid induction on crossing by wild-type CENH3 plants. These CENH3-variant plants grow normally, are fully fertile on self-pollination, and may be present in existing mutagenized collections.True-breeding lines are required for the development and production of crop varieties. In a classical breeding approach these lines are obtained through inbreeding, and often 7–9 generations of inbreeding is performed to achieve the desired level of homozygosity, over a period of several years. In contrast, the chromosomes of haploids can be doubled to produce true-breeding lines in a single generation. Over the last century, scientists have developed a variety of techniques to induce haploids and doubled haploids, though these techniques apply only to particular crop varieties. aize see . Refinem Genliseatuberosa) (Paris japonica)   and 160 aponica)  to fit iaponica) . The pataponica) . This seDrosophila, CENP-A in mammals and Cse4 in yeast; Saccharomyces cerevisiae) this histone variant does have sequence-specific DNA binding affinity (and so centromeres will form at specific DNA sequences). Thus in the majority of eukaryotes studied, centromere positioning appears to be an epigenetic, rather than sequence-based, phenomenon  recent findings have demonstrated that these features are not essential to centromere localization; some eukaryotes lack long tandem arrays entirely, some have different repeat arrays on different chromosomes, and neocentromeres can form at unique sequences . Rather enomenon .CENH3, like other histones, carries an N-terminal tail  and C-terminal Histone Fold Domain (which interacts with DNA and other histones to form the nucleosome). Unlike conventional histones, CENH3 is rapidly evolving. The N-terminal tail of CENH3 barely alignable even among closely related species whereas the histone fold domain is relatively well conserved .CENH3 cause chromosome non-disjunction and cell cycle arrest at mitosis  in CENH3-modified lines may result from errors in reloading of CENH3 at centromeres derived from CENH3-modified lines. Although the reloading process has been extensively investigated in animals and fungi , we are Doubled haploids- plants carrying a genome derived from a single (haploid) gamete- provide enormous timesaving in the production of true-breeding lines, literally reducing the time to acceptable levels of homozygosity by an order of magnitude. Haploid lines, and “haploid genetics” can accelerate a variety of genetic constructions and is useful in the research laboratory also . The spocenh3–/– Arabidopsis null mutant, when complemented by altered version of CENH3, could induce haploids. A remarkably effective haploid inducer, termed “green fluorescent protein (GFP)-tailswap,” carried GFP fused to the N-terminal tail domain of an H3 variant (At1g13370), which then replaced the N-terminal tail of CENH3 . They found, surprisingly, that the CENH3 mitotic functionality was maintained in spite of the “tailswap,” though meiotic function was compromised- the plants were almost entirely male sterile, with some female sterility also. On self-pollination, GFP-tailswap produces normal diploid seeds . However, when crossed as a female by plants carrying wild-type CENH3, GFP-tailswap produces a remarkable 25–45% paternal haploid progeny. The maternal (GFP-tailswap) genome is lost in these haploids  further degrades the strength of the resulting centromere.The initial findings with Arabidopsis, is a haploid inducer, but otherwise fully functional. The Arabidopsis null mutant can also be complemented  by CENH3 from a wide range of angiosperms .The addition of adducts such as GFP, and changes in the sequence of CENH3, can act additively to affect protein function. For example, as described above, GFP-CENH3, in iosperms , includiCENH3 (Cse4) was able to complement a human defect in CENH3 (CENPA) function  did not target to A. thaliana centromeres. In the same study it was shown that GFP tagged versions of CENH3 from Brassica rapa and Z. mays were able to localize to A. thaliana centromere, but were unable to complement A.thaliana cenh3 null function. This result was surprising given ability of the yeast protein to complement the human defect, which suggests that CENH3 function is highly conserved across kingdoms. This discrepancy was explained when B. rapa, Lepidium oleracuem, Vitis Vinifera, and Z. mays found that all of these untagged proteins were able to complement A. thaliana cenh3 null function. In another intriguing note, when the GFP is attached C-terminally to the AtCENH3 protein, it is able to localize to the centromere but unable to complement cenh3 null function in Arabidopsis  though some aneuploids were reported. Thus the sequence of the N-terminal tail affects the “strength” of CENH3 in a competitive cross. The difference in sequence between the N-terminal tails of L. oleraceum and that of A. thaliana is substantial- they are of similar lengths but differ at 29/82 positions . Similarly, the N-terminal tail of histone H3.3  has very little similarity to that of CENH3, but it complements CENH3’s mitotic and meiotic functions and is, in the absence of the GFP tag, a weak haploid inducer. Whether subtle differences in the N-terminal tail- for example, single amino acid substitutions- can possibly affect the “strength” of CenH3 remains to be determined.The Arabidopsis CENH3) induced haploids , while P102S and G173E appeared to be fully competitive with wild-type CENH3. Aneuploid progeny were also obtained from among the progenies of these plants. Because these mutations could be obtained in other crop plants by ethyl methane sulfonate (EMS) mediated mutagenesis (the sites tested were conserved among crops), this discovery suggests that non-transgenic haploid inducers could be induced- and may already exist, undetected- in EMS mutagenized crop species.The tail region of CENH3 is rapidly evolving in both length and sequence whereas the histone fold domain is well conserved. This suggests that subtle changes in the HFD might have substantial effects on its activity. Recently, it has been shown that point mutations resulting in single amino acid substitutions induced uniparental genome elimination, resulting in haploidy or aneuploidy, with retention of the wild-type chromosomes . RemarkaGFP-tailswap crosses in Arabidopsis  chromosomes were indeed the most commonly encountered class of aneuploids. Because the analysis was sequence-based, and the crosses were between two different ecotypes, the parental origin of each chromosome could also be scored. In all cases the supernumerary chromosomes were derived from the GFP-tailswap  parent. The second most commonly encountered class of aneuploids were diploids carrying an additional truncated chromosome (again GFP-tailswap-derived). In a very small fraction of aneuploids, during the process of genome elimination, a chromosome from the GFP-tailswap parent undergoes “shattering,” revealed by copy number discrepancies within a single supernumerary chromosome. The investigators observed the encapsulation of chromatin into micronuclei, and hypothesize that one or more laggard GFP-tailswap chromosomes left behind during segregation can be compartmentalized into micronuclei, which occasionally rejoin the nucleus proper. At some point during this excursion, the chromosomes undergo endonucleolytic degradation (referred to as “shattering”) and subsequent rejoining, resulting in variety of intra- or interchromosomal rearrangements. Retention of– or fusion to- a centromeric region would result in heritability. Meiotically heritable mini- and interchromosomal rearrangements were observed and confirmed cytologically. The resulting mini-chromosomes are potentially of value for plant breeding, if they can be designed to carrying a one or more useful traits in a single, simple package that can be easily transferred between varieties.Analysis of these putative aneuploids revealed that plants carrying 2GFP-tailswap by plants homozygous for a null mutation in Lig4 resulted in an increased frequency of haploids (doubling from 40% of progeny to 80%) at the expense of both diploid and aneuploid progenies. This suggests that both aneuploids and diploids experience LIG4-dependent chromosome rescue. This in turn suggests that the GFP-tailswap-derived chromosomes in diploids may have undergone extensive rearrangement and/or point-like mutations that are undetectable via the phenotypic screen employed to collect aneuploids for sequence-based characterization.The source of the double strand breaks that result in shattered chromosomes is unclear. Breaks might be induced by prolonged exposure to the cytoplasmic environment, or through the deregulation of replication vs. repair in micronuclei. Rearrangements could theoretically be generated either via non-homologous end joining or through ectopic homologous recombination (HR) of repetitive DNAs. LIG4 status of the GFP-tailswap  parent has no effect on HI frequency; the observed LIG4-dependent rescue of aneuploids and diploids depended entirely on the (wild-type CENH3) paternal genome. A potential explanation for this phenomenon is that chromosomes- even chromosomes that will eventually produce diploids- are frequently being rescued into what is essentially a haploid, paternal nucleus, and that the (perhaps extranuclear) GFP-tailswap-derived genome is not effectively expressed during this process. In mammalian systems, it has also been shown that unrepaired DNA damage increased the frequency of uniparental genome elimination  that complement endogenous cenh3-1 null do not display seed death on self-pollination. Though GFP-tailswap is nearly sterile, with poor seed set per silique, those seeds that are produced are plump and healthy. In contrast, when these lines are hybridized to wild-type plants they display substantial seed death, and the frequency of death is positively correlated with the frequency of HI.A common feature observed in all CENH3 mediated haploid inducing systems is seed abortion  in the HI cross. All variants of CENH3 . Euploidy of both the zygote and endosperm are important for seed development; defects in chromosomal balance in either tissue type could result in seed death. It makes sense that loss of the HI genome would occur in both the embryo and endosperm, but this is not necessarily true, as CENH3 loading and active unloading rates are developmentally specific . Thus thGFP-tailswap. This marker is expressed in both embryo and endosperm, and although endosperm tissue is limited in the mature Arabidopsis seed, the marker can be visualized through the seed coat of mature, dried seeds in both the seedling (where it is highly expressed) and in the residual endosperm. Seeds derived from GFP-tailswap p23S:GFP HI crosses fell into two phenotypic classes: fully GFP+ (endosperm vs. embryonic expression was not distinguished in this class), and mottled GFP expression . Mottled seeds always gave rise to haploid  seedlings, while fully green seeds produced diploids or aneuploids. Perhaps the most significant result, aside from the success of the marker for identification of haploid seeds, is that there were no GFP-free seeds, suggesting that fertilization events resulting in paternal “monoploid” endosperm are either very rarely produced (both copies of the HI-inducer’s genome are rarely lost) or generally lead to seed abortion, or particularly lead to abortion of haploids.In an effort to facilitate screening of haploids at seed level, Hordeum vulgare × Hordeum bulbosum, the H. bulbosum genome is lost during embryogenesis . In their visualization of anaphase mitoses in early embryos, the authors found that lagging chromosomes were, unsurprisingly, bulbosum-derived. More notably, these laggards lacked detectable CENH3, which is easily observed via immunolocalization in normal barley chromosomes. This indicates that there is-minimally- a failure of CENH3 to reload onto the bulbosum chromosomes, rather than (or perhaps in addition to), a failure of more distal centromeric components to recognize the bulbosum-derived centromeres.In a classical barley HI system involving crosses between ogenesis . Sanei ebulbosum chromosomes due to dilution (with a failure to reload) after replication, or due to active unloading of CENH3 in the zygote. Interestingly, Arabidopsis- that neither CENH3 transcripts nor CENH3-GFP (tagged at the C terminus) were detectable in the Arabidopsis egg cell. Although the authors observed localization of CENH3-GFP to sperm centromeres, this mark was rapidly lost after fertilization of wild-type eggs, prior to further cell division, in what is apparently the active removal of this mark. Localization of CENH3-GFP was visible later in embryogenesis. This very provocative observation raises the possibility that CENH3 is not present in the zygote. If this were true for CENH3, as well as for CENH3-GFP, this observation would challenge the model that centromeres are determined simply by the presence or absence of CENH3. It remains to be determined, however, whether wild-type CENH3 is similarly actively depleted from the zygote. CENH3-GFP may behave differently than CENH3; it does not, for example, complement the cenH3–/– mutation .Single amino acid substitutions obtained through point mutations generated by EMS is currently accepted as non-GMO under both interpretations of the law. Therefore, under existing regulations, chemically-induced point mutations of Arabidopsis, CENH3-mediated genome elimination needs to be tested in other crop species– its application to maize, described in this volume (CENH3 knockouts in other species. The recent development of efficient CRISPR-based gene targeting conveniently addresses this issue. GFP-tailswap is a simple and straightforward approach for plants with well-annotated and sequenced genomes. The alternative approaches to CENH3-mediated HI may also be employed. Non-GMO point mutants can be obtained by EMS mutagenesis, and may already be present in existing populations. The recent findings of complementation and HI by CENH3 from related species (Haploid induction technologies- and CENH3-based HI is only one among many- significantly reduces the time and labor required to derive new true-breeding varieties. Within the short period of its discovery, CENH3-mediated genome eliminations have also been shown to be useful in trait mapping , reverses volume  is very  species  offers aThe mechanism, through which CENH3 modification affects competitive crosses, without affecting self-pollinations, mitosis, or meiosis, remains a mystery. The elucidation of this process will not only provide insight into CENH3 function and the mechanism of chromosome assortment, but may reveal additional targets that will, upon modification, provide the next generation of haploid inducers.This review was written and edited by both authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JK and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


The receptor tyrosine kinase Ret (c-Ret) transduces the glial cell line-derived neurotrophic factor (GDNF) signal, one of the neurotrophic factors related to the degeneration process or the regeneration activity of motor neurons in amyotrophic lateral sclerosis (ALS). The phosphorylation of several tyrosine residues of c-Ret seems to be altered in ALS. c-Ret is expressed in motor neurons and in the enteric nervous system (ENS) during the embryonic period. The characteristics of the ENS allow using it as model for central nervous system (CNS) study and being potentially useful for the research of human neurological diseases such as ALS. The aim of the present study was to investigate the cellular localization and quantitative evaluation of marker c-Ret in the adult human gut. To assess the nature of c-Ret positive cells, we performed colocalization with specific markers of cells that typically are located in the enteric ganglia. The colocalization of PGP9.5 and c-Ret was preferentially intense in enteric neurons with oval morphology and mostly peripherally localized in the ganglion, so we concluded that the c-Ret receptor is expressed by a specific subtype of enteric neurons in the mature human ENS of the gut. The functional significance of these c-Ret positive neurons is discussed. The blocking was repeated for a second time. The sections were incubated with the primary antibody solution for 30 minutes followed by rinsing in PBS, for 3 minutes. The visualization was made by incubating with an Envision peroxidase-based visualization kit (Dako K5007) over a period of 30 minutes, washed twice in PBS, for 3 min according to manufacturer's directions. To confirm the presence of immunocomplexes, 3,3′-diaminobenzidine was used as chromogene and hydrogen peroxide as substrate. The samples were washed twice in distilled water, contrasted with Mayer's haematoxylin for 7 minutes, washed in tap water for 15 minutes, dehydrated in a graded series of ethanol, cleared in xylene, and coverslipped with DPX. Digital microscope images were captured by means of an Olympus BX 51 microscope and analyzed using public domain image software: ImageJ: Image Processing and Analysis in Java available from The immunolabelled neurons were quantified in a random intestinal sample of 3 cm in length from each patient. 30 areas per region were studied from each sample. The total number of ganglia  and the total number of the c-Ret positive cells, compared to total morphologically recognized neurons, were counted.Cells were counted by two independent investigators. If the cell count differed by 10%, a third investigator then analyzed the samples. The final cell count was the mathematical average of the independent cell counts. To minimize errors, only those neurons in which the nucleus was clearly visible were confirmed as positive controls.The expression and distribution of proteins were studied by the indirect immunofluorescence staining method for anti-Ret monoclonal antibody , polyclonal rabbit anti-glial fibrillary acidic protein , polyclonal rabbit anti-PGP 9.5 , polyclonal rabbit anti-human c-Kit , goat anti-mouse IgG (HL) Alexa Fluor 488 , and goat anti-rabbit IgG (H+L) Alexa Fluor 546 .The antibodies were diluted in Dako (S2022) diluent. Tissue sections were deparaffined in xylene (10 minutes twice) and rehydrated in a graded ethanol series up to distilled water. Before all assays, for heat-induced antigen retrieval, the samples were treated during 6 minutes in an 800 W microwave oven with 10% citrate buffer (Dako S2031) in distilled water and at 360 W for 5 additional minutes. After washing three times with PBS, for 5 minutes, the sections were treated with blocking buffer  for 20 minutes. The blocking was repeated for a second time. Sections were incubated with the primary antibody solution for 20 hours followed by three rinses in PBS for 5 minutes. The visualization was made with Alexa Fluor for 90 minutes, and then the sections werewashed in PBS three times for 10 minutes and incubated with 5 µM of DRAQ5  to counterstain the nuclei. Finally, cover slips were then mounted in Mowiol Sigma-Aldrich, Deisenhofen, Germany) and cell staining was documented using Leica TCS SL Laser Scanning Confocal Microscope.t-test. P < 0.05 was considered significant (*), P < 0.01 very significant (**), and P < 0.001 extremely significant (***).Data are expressed as mean ± SEM. Differences were analyzed by Student's Intense c-Ret immunoreactivity was detected in all cases studied of adult human intestine. c-Ret immunoreactivity was positive in the myenteric and submucosal ganglia, although significant differences between them were found. In the myenteric ganglia, only a few cells inside the ganglia were labelled , while iWe performed a double immunofluorescence technique to assess coexpression of c-Ret/PGP9.5, c-Ret/GFAP and c-Ret/c-Kit. PGP9.5-immunoreactive cells appeared within myenteric ganglia Figures -1, GFAP-The pattern of c-Ret expression observed with optical and confocal microscopy was similar Figures -2.α (GDNFRα-1) transduces GDNF mediated signal, which seems to be one of the neurotrophic factors related to ALS [The tyrosine kinase receptor c-Ret in association with another receptor GDNFR-d to ALS –12. The d to ALS , 13, is d to ALS .c-Ret is expressed during the embryonic period as a signal for the development of neurons in the enteric nervous system , 14–19. An immunohistochemical study published was the only one to show the existence of c-Ret positive neurons in the myenteric plexus of adult rats. No positive neurons were described in the submucosal plexus .Although some myenteric neurons in humans have been characterized based on morphology, projections, and neurochemical staining –41, the α1/c-Ret receptor complex) as described for cerebellar Purkinje cells cannot be ruled out [Given the additional fact that intestinal smooth muscle cells might even be a major source of GDNF, modulation and/or promotion of their own smooth muscle innervation in postnatal period might involve c-Ret receptors in adult enteric neurons . Howeveruled out .Sharkey and Parr (1996) revised the literature on this issue addressing the question about the enteric nature of these neurons, taking into account that these cells are postmitotic. These authors suggested that, since cell division in adult neurons was unlikely, the possible explanation for neuron addition in adulthood was a potential precursor nature of these cells in myenteric ganglia and their capability of late proliferation into adult neurons . AlthougOn the other hand, Stemple and Anderson (1992) isolated postmigratory neural crest cells from fetal rat gut usingc-Ret and examined the developmental and proliferative capacities of these cells using a clonal culture system . Many ofTherefore, it has been demonstrated that during the tissue repair process, possible recapitulation of embrionary development takes place. Geuna et al. have suggested that NCCs, besides driving the development of many different tissue and organs during development, can play a role also in the postdevelopmental period of animal life because some of the migratory elements persist as a peripherical reserve pool of multipotent stem cells that can undergo late differentiation into mature cells .We report immunohistochemical evidence for the presence of a group of adult enteric neurons stained with neural crest marker, c-Ret, in the normal adult duodenum. c-Ret was located in the myenteric and submucosal ganglia of the adult human duodenum and is expressed in some enteric neurons since it colocalized with pan-neuronal marker PGP9.5. Indeed, colocalization was more frequent in oval enteric neurons located peripherally in the ganglion, which suggested that c-Ret function is required not only during early foetal stages but also in the mature human ENS of the gut. The positivity to c-Ret may be indicative of different stages of neuronal maturation and, therefore, may be involved in adult enteric neurogenesis. We also suggest, like other authors, that multipotent NCCs coexist with differentiated cell types. Further studies are needed to study in depth the nature of these cells and their potential role in neurodegenerative diseases, such as ALS, could shed light on new molecular pathways possibly affected under neurodegenerative progression.