Seven districts in Andhra Pradesh, South IndiaTo a) determine treatment outcomes of patients who fail first line anti-TB treatment and are not placed on an multi-drug resistant TB (MDR-TB) regimen, and b) relate the treatment outcomes to culture and drug susceptibility patterns (C&DST).Mycobacterium TB Culture C&DST between July 2008 and December 2009.Retrospective cohort study using routine programme data and There were 202 individuals given a re-treatment regimen and included in the study. Overall treatment outcomes were: 68 (34%) with treatment success, 84 (42%) failed, 36 (18%) died, 13 (6.5%) defaulted and 1 transferred out. Treatment success for category I and II failures was low at 37%. In those with positive cultures, 81 had pan-sensitive strains with 31 (38%) showing treatment success, while 61 had drug-resistance strains with 9 (15%) showing treatment success. In 58 patients with negative cultures, 28 (48%) showed treatment success.Treatment outcomes of patients who fail a first-line anti-TB treatment and who are not placed on an MDR-TB regimen are unacceptably poor. The worst outcomes are seen among category II failures and those with negative cultures or drug-resistance. There are important programmatic implications which need to be addressed. In India, out of the 1.2 million ‘new’ cases of tuberculosis (TB) notified in 2009, 14,991(1.8%) were reported to have failed the first line ‘anti-TB treatment drug regimen. Similarly among 289,756 re-treatment TB cases , 11,265 (4%) failed the first-line re-treatment drug regimen. Multi-drug resistant TB, MDR-TB  is one of the important causes for failure on TB treatment. In order to identify such patients early and manage them with an appropriate drug regimen, sputum specimens of all ‘new’ TB patients who are sputum smear positive at 5 months or more after the initiation of treatment (defined as having failed their first-line anti-TB treatment regimen)-, should be sent for sputum Culture and Drug Susceptibility Testing (C&DST) in an Revised National Tuberculosis Programme (RTNCP) accredited laboratory and patients should be placed on a re-treatment regimen while waiting for their C&DST results. Similarly, for ‘Re-treatment TB’ cases, sputum samples of patients who are smear positive at 4 months or more after the initiation of treatment (defined as having failed their first line anti-TB treatment) should be sent for C&DST and patients should be continued on a re-treatment regimen while awaiting the laboratory results.As per RNTCP guidelines, patients with DST results showing resistance to rifampicin are considered eligible for MDR-TB treatment, irrespective of resistance to isoniazid, streptomycin, or ethambutol. In the absence of rifampicin resistance, patients simply continue their re-treatment regimen to completion Of the 340 isolates tested by C&DST approximately 60% of the isolates were found to be susceptible to rifampicin and were not eligible for MDR-TB treatment, and hence were continued on the ‘re-treatment’ regimen. There is very little published information in India on how such patient's fare, as all of them are already showing signs of poor response to conventional first-line anti-TB treatment, and some of them have considerable non-rifampicin mono or poly-drug resistance. Such information is essential to guide the choice of continuing (or not) the current re-treatment regimen for such patients.We thus conducted a retrospective cohort study in Andhra Pradesh, South India, to a) determine the treatment outcomes of patients who fail a first line anti-TB treatment regimen and are not placed on an MDR-TB regimen and b) relate their treatment outcomes to culture and drug susceptibility patterns.The study was conducted in seven districts (with a combined population of 18.4 million) in the state of Andhra Pradesh, South India, which are implementing RNTCP MDR-TB treatment services. The MDR-TB treatment services have been implemented in four of these seven districts since mid 2007 and the other 3 districts since early 2008.We selected all patients from these 7 districts who as per the programme guidelines were assessed as having drug resistance by C&DST at the two RNTCP accredited laboratories and were found not eligible for treatment with an MDR-TB treatment regimen. Patients registered during the period July 2008 to December 2009 were included in the study.Management of Tuberculosis patients who are not responding to TB treatment RNTCP uses World Health Organization (WHO) recommended disease classification and treatment management guidelines. Patient management is guided by type of disease, sputum smear status and history of previous TB treatment as recommended by WHO. The Intermediate reference laboratory at Hyderabad (Government sector) and Blue Peter research centre laboratory, Hyderabad were the culture and drug susceptibility testing (DST) laboratories that performed the DST. All the samples were collected by trained staff and transported to the laboratories in 1% cetylpyridium chloride (CPC) solution. These laboratories maintain a register to document the receipt of sputum specimens and the results of the C&DST.Mycobacterium tuberculosis strains is based on growth rate, morphology, and susceptibility to para-nitrobenzoic acid. For drug susceptibility, the proportion method on Lowenstein-Jensen (LJ) is used for standard anti-tuberculosis drugs  and tested using standard procedures. For quality assurance, the standard Mycobacterium tuberculosis H37Rv strain was used. Both the laboratories had passed proficiency testing which involves re-testing and panel testing. The performance of both the laboratories was satisfactory as determined by standard concordance of >95% for Isoniazid and rifampicin and >90% for streptomycin and ethambutol. The laboratories also participated in the periodic proficiency testing programmes being conducted by a supra national laboratory .These laboratories were accredited by RNTCP. Accreditation involves a pre-accreditation visit by a team of experts from the Tuberculosis Research Center (TRC), Chennai  which looks at the adherence of the laboratory to standard operating procedures laid down by the programme. The identification of We reviewed a) the laboratory registers at the C&DST laboratories which are the reference laboratories, b) the culture & DST register at district level which indicates the referrals made, and c) and the TB treatment cards and TB registers at sub-district level in which are documented the treatment outcomes. A line- list of all patients who had their C&DST done during the study period and whose DST results showed no resistance to rifampicin was prepared after ensuring the correctness of the records. Patients with negative C&DST results or no-growth on C&DST were also included in this data base. We collected baseline demographic data, the C&DST results and treatment outcomes of these line listed patients. The treatment outcomes that are reported for all cases in this study are a result of treatment with ‘retreatment regimen’. The data were cross verified by two investigators and compared for consistency; once it was found to be correct the data were entered in the Microsoft excel 2003. All variables were described by proportions and differences between independent groups were compared using Chi-square test and Fisher exact test as applicable by Epi-info software. Treatment outcomes were grouped as successful and adverse  and patients in each of these groups were compared. A ‘p value’ less than 0.05 were taken as statistically significant.The study was approved by the ethics committee of the Public Health Foundation of India and the Ethics Advisory Group of The International Union Against Tuberculosis and Lung Disease, Paris, France. The study was determined to be a retrospective audit of the programme surveillance data in its records and reports, and permission was obtained from the programme managers at the state and national levels to access these data. Individual patient consent was deemed un-necessary by both the ethics committees. Electronic databases created for this analysis were stripped of personal health identifiers and maintained securely.There were 204 patients who fulfilled the study eligibility criteria of whom 2 patients could not be identified in the tuberculosis register and were subsequently excluded from the analysis. Of the remaining 202 patients, 58 failed a Category I treatment regimen, 139 patients failed a Category II treatment regimen and 5 patients failed a Category III treatment regimen. The median age of the patients was 35 years (range 9–70 years) and there were 143 (70%) males. All patients were treated with the recommended RNTCP re-treatment regimen.Treatment outcomes as a result of initiating/continuing all patients on a re-treatment regimen are shown in Results of treatment outcomes in relation to C&DST are shown in Of the 5 patients who failed a Category III treatment regimen, 2 had a pan-sensitive organism, 1 had a strain with streptomycin mono-resistance, 1 a strain with isoniazid mono-resistance and 1 had negative C&DST pattern  using rapid diagnostic technology to ascertain if they have developed rifampicin resistance or not. If they do develop rifampicin resistance, they could then be changed to the MDR-TB treatment regimen. However, this proposed strategy has serious feasibility challenges. The recent introduction of GeneXpert into the market which allows detection of Rifampicin drug resistance within two hours is promising but is unlikely to be available at wide level for some time to come Finally, the entire approach of using the category II re-treatment regimen, in our opinion, merits urgent review. According to current practice, the retreatment regimen involves adding one new drug (streptomycin) to an already failing regimen. This contradicts the basic principle that at least four drugs to which the TB bacilli are sensitive are needed to ensure effective cure and prevent the development of drug resistance The strengths of this study are as follows: large numbers of district records were carefully reviewed and outcomes verified using patient cards; the findings come from the programme setting and are thus likely to reflect the operational reality on the ground; C&DST results were quality controlled; and we adhered to STROBE guidelines on reporting In conclusion, overall treatment success rates in failure patients placed on a re-treatment drug regimen are poor and these results need to be urgently reviewed. One of the immediate logical options particularly for patients who fail re-treatment category II regimens would be to start on an empiric MDR-TB regimen until C&DST results become available.


This study aimed to evaluate the effectiveness of systematic assessment of asthma-like symptoms and environmental tobacco smoke (ETS) exposure during regular preventive well-child visits between age 1 and 4 years by well-child professionals.Sixteen well-child centres in Rotterdam, the Netherlands, were randomised into 8 centres where the brief assessment form regarding asthma-like symptoms and ETS exposure was used and 8 centres that applied usual care. 3596 and 4179 children (born between April 2002 and January 2006) and their parents visited the intervention and control centres, respectively. At child’s age 6 years, physician-diagnosed asthma ever, wheezing, fractional exhaled nitric oxide (FeNO), airway resistance (Rint), health-related quality of life (HRQOL) and ETS exposure at home ever were measured. Linear mixed models were applied.No differences in asthma, wheezing, FeNO, Rint or HRQOL measurements between intervention and control group were found using multilevel regression in an intention-to-treat analysis (p>0.05). Children of whom the parents were interviewed by using the brief assessment form at the intervention well-child centres had a decreased risk on ETS exposure at home ever, compared to children who visited the control well-child centres, in an explorative per-protocol analysis .Systematic assessment and counselling of asthma-like symptoms and ETS exposure in early childhood by well-child care professionals using a brief assessment form was not effective in reducing the prevalence of physician-diagnosed asthma ever and wheezing, and did not improve FeNO, Rint or HRQOL at age 6 years. Our results hold some promise for interviewing parents and using information leaflets at well-child centres to reduce ETS exposure at home in preschool children.ISRCTN15790308.Controlled-Trials.com  Asthma is a highly prevalent chronic condition associated with considerable morbidity, reduced health-related quality of life (HRQOL) and significant costs for public health In the Netherlands, growth, development and health of all children (0–19 years) is monitored in a nationwide program with regular visits at set ages by well-child care physicians and nurses This study aimed to evaluate the effectiveness of systematic assessment of asthma-like symptoms and ETS exposure between age 1 and 4 years by well-child professionals. We hypothesised that systematic assessment of asthma-like symptoms and ETS exposure to parents of preschool children  reduces the prevalence of physician-diagnosed asthma ever and wheezing frequency, and improves fractional exhaled nitric oxide , airway resistance (Rint) and HRQOL measurements at age 6 years. In addition to the study protocol This study is embedded in the Generation R Study, a prospective population-based cohort Details of our study design were published previously see  [12]. ThWhen parent and child attended the well-child centre allocated to the intervention group, the professionals used a brief assessment form regarding asthma-like symptoms and ETS exposure during the regular visits at age 14, 24, 36 and 45 months. Details of this form were published previously When parents reported that their child had at least 3 episodes of any asthma-like symptoms during the past 12 months and at least 1 episode of asthma-like symptoms in the past 4 weeks, the well-child professionals could provide them with a leaflet with information about asthma. If the child had been free of asthma-like symptoms during the past 4 weeks, the well-child professionals could advise a visit to the general practitioner should the child’s asthma-like symptoms return. When parents reported that their child had at least 3 episodes of asthma-like symptoms during the past 12 months, of which at least 1 in the past 4 weeks, and the child had not yet been treated by the general practitioner or paediatrician in the past 4 weeks, the well-child professionals could refer to the asthma nurse and/or general practitioner. If the child had already been treated by the general practitioner or paediatrician in the past 4 weeks, the well-child professionals could refer to the asthma nurse.If the child had been exposed to ETS , the well-child professional could discuss health risks of ETS exposure to preschool children , and discuss whether parents could be motivated and prepared to stop ETS exposure to their child (house rules) and provide them with an information leaflet about preventing their child from exposure to ETS. The well-child professionals from the intervention centres were informed during a two-hour session about the intervention.The control centres applied current routine practice, addressing the presence of general health symptoms during the regular well-child visits and ETS exposure (at least at age 18 months) nd, 3rd, 4th and 6th year of life. Response rates for these questionnaires were 71%, 76%, 72%, 73% and 68%, respectively. The primary outcome measure was physician-diagnosed asthma ever, obtained by a parent-reported questionnaire at age 6 years.Data from parents were collected in the Generation R Study by postal questionnaires at enrolment, and at the first, 2Secondary outcomes were current wheezing frequency (as reported by parents), FeNO, Rint and HRQOL as reported by parents. Reducing ETS exposure to preschool children was one of the objectives of counselling following systematic assessment of ETS. Therefore, in addition to the proposed outcomes Wheezing frequency  in the past 12 months was assessed using a parent-reported question from the International Study of Asthma and Allergies in Childhood (ISAAC) elog transformation.FeNO was measured according to American Thoracic Society guidelines At age 6 years, Rint  was measured at the research centre during tidal breathing, with occlusion of the airway at tidal peak expiratory flow. Median values for at least 5 acceptable Rint measurements were calculated and used to calculate Z-scores, additionally adjusted for median variation of the study period : Physical functioning, Role functioning: emotional, Role functioning: physical, Bodily pain, General behaviour, Mental health, Self-esteem, General health perceptions. These multi-item scales were summarised into a Physical summary measure and a Psychosocial summary measure. Furthermore we used the Change in health item. The impact of the child’s health on the caregiver’s and family’s HRQOL was measured across the remaining four multi-item scales: Parental impact: emotional, Parental impact: time, Family cohesion and Family activities. All scale measures were transformed to scores ranging from 0 to 100. Lower scores correspond to lower HRQOL. Summary measures were standardised with a mean of 50 and standard deviation of 10 to reflect general US population norms for children The CHQ-PF28 in the parent-reported questionnaire was used to measure HRQOL of the child at age 6 years The outcome ‘ETS exposure at home ever ’ at age 6 years was defined and based on parent-reported questionnaires at age 2, 3 and 6 years, using the question: ‘Do people smoke occasionally at home? ’. ‘ETS exposure at home ever’ at age 6 years was scored ‘yes’ if there was ETS exposure at home at age 2 or 3 or 6 years.st year of life; respiratory tract infections  and wheezing  at the 1st year of life.We used information collected in the Generation R Study on maternal characteristics  for the intervention and control group. Information about the highest attained maternal educational level , maternal ethnicity  and single motherhood  and maternal history of asthma or atopy  were obtained at enrolment by questionnaires. Maternal educational level and maternal ethnicity were defined according to the classification of Statistics Netherlands Baseline data for the intervention and control group were described using descriptive statistics, which were tested for differences using multinomial regression adjusted for randomisation stratum (cluster). All participants were analysed according to the “intention-to-treat” principle.P values for differences in the prevalence of ‘ETS exposure at home’ between intervention and control group were calculated by means of the Chi-square test. Although not according to the study protocol, several children participating in the control group also visited the intervention centres and assessment of asthma-like symptoms and ETS exposure by a brief form was applied to a part of the parents of these children. Contamination of intervention and control condition may possibly also have occurred by moving to another neighbourhood in the city and visiting another well-child centre. Because this contamination may have reduced the differences in results between intervention and control group, we amended the study protocol The prevalence of ETS exposure at home before , during (at age 14–45 months) and after (at age 6 years) the study period was described. elog transformation.To prevent bias associated with attrition, missing data at baseline and missing outcomes were multiple imputed (10 imputed datasets) on the basis of the correlation between each variable with missing values and other parental and child characteristics Potential effect modification of socio-demographic characteristics and baseline values of the outcomes on the association between the research condition  and the outcomes was explored. First, we fit a multinomial regression model with randomisation stratum and baseline values of the outcome. Second, we added socio-demographic characteristics  and baseline values of the outcomes as an interaction separately Random treatment allocation ensures that intervention status will not be confounded with either measured or unmeasured baseline characteristics It should be considered that given multiple comparisons, there is an 1-in-20 chance of a false association for each comparison (Type I error at p = 0.05) In addition, a process evaluation of the intervention was performed. The study is reported according to the CONSORT standards for reporting RCTs There were 8 intervention and 8 control well-child centres, involving 3596 and 4179 children (and their parents) visiting these well-child centres, respectively. The different rates of participation of the children in the different elements of the study are shown in the flow diagram .At age 6 years, multilevel regression analysis indicated no differences in asthma, wheezing frequency, FeNO or Rint measurements between the intervention and control group (p>0.05)  and 3.The response rate regarding the CHQ-PF28 scales at age 6 years was different for each scale and varied between 57–59% (n = 4410–4590). Baseline measurements were available for 8 out of 13 CHQ-PF28 scales. At age 6 years, no differences in HRQOL were found between the intervention and control group, after adjustment for baseline HRQOL and randomisation stratum (p>0.05)  and 3.No differences in ETS exposure at home at age 2 and 3 years were found between intervention and control group after adjustment for baseline ETS exposure at home  using multinomial regression in an intention-to-treat analysis, . However, in the per-protocol analysis (n = 1560), multinomial regression analysis indicated a decreased risk on ETS exposure at home in the intervention group at age 2 and 3 years .At age 6 years, no differences between intervention and control group were found on the outcome ‘ETS exposure at home ever’ using multilevel regression in an intention-to-treat analysis including adjustment for baseline ETS exposure at home   . HoweverNo interaction effects on the outcomes were found of the research condition (intervention or control group) with socio-demographic characteristics or baseline values of the outcomes (p>0.10) (data not shown). We found no effect of the frequency of the intervention on outcomes.In total, professionals at well-child centres completed 6826 forms to assess asthma-like symptoms and ETS exposure for 2718 children (75.6% of the 3596 children) participating in the intervention group; and 1566 forms were completed for 682 children (16.3% of the 4179 children) participating in the control group (see discussion). In half of the children participating in the intervention group, the brief assessment form was applied at age 14 months  was already treated by general practitioner or paediatrician. Of the children with ≥3 episodes of asthma-like symptoms in the past year and asthma-like symptoms during the past month, 86% (119/139) was already treated by general practitioner or paediatrician.Using the assessment forms, well-child professionals in the intervention group reported a decreasing prevalence of ETS exposure to children participating in the intervention group with increasing child’s age: 19% (276/1447) at the age of 14 months, 16% (266/1627) at age 24 months, 17% (301/1767) at age 36 months and 13% (225/1760) at age 45 months. At age 14 months, 89% (245/276) of the children with ETS exposure received the information leaflet regarding the prevention of ETS exposure. However, after the first year, the information leaflet regarding prevention of ETS exposure was less often provided to the parents of children who were exposed to ETS: 61% (163/266) at age 24 months, 64% (192/301) at age 36 months and 53% (119/225) at age 45 months.Systematic assessment of asthma-like symptoms and ETS exposure by professionals at well-child centres, followed by counselling  did not lead to a lower prevalence of physician-diagnosed asthma ever, reduction in parent-reported wheezing symptoms and did not improve FeNO, Rint or parent-reported HRQOL at age 6 years. A decreased risk on ETS exposure at home in the intervention group was found at age 2 and 3 years, but at age 6 years no difference between intervention and control group was found. Process evaluation results showed that most children with wheezing were already treated by their general practitioner or by a paediatrician. Further, half of the parents of children with ETS exposure participating in the intervention group did not receive the information leaflets on ETS exposure at the intervention centres at age 45 months.This is a community health worker-delivered intervention study using physician-diagnosed asthma ever, wheezing frequency, FeNO, Rint, HRQOL and (in addition) ETS exposure at home ever at age 6 years as outcomes. In contrast to the positive outcomes associated with community health worker-delivered interventions (including decreased asthma-like symptoms) reported by Postma et al In addition to the review by Priest et al The strengths of this study include the integration in current practice with a brief assessment form regarding asthma-like symptoms and ETS exposure, the large number of parents participating, the longitudinal design (with follow-up until child age 6 years) and large number of FeNO and Rint measurements. Limitations include shortcomings in the application of the brief assessment forms and counselling. Possible reasons are falling attendance of parents to the well-child centre; lack of time or priority is given to other health questions during the well-child visit or professionals who are not familiar with the intervention, that is still not routine practice. In this study, the professionals were provided with a two-hour specific training on how to apply and use the brief assessment form regarding asthma-like symptoms and ETS exposure. This level of instruction may not be optimal as we did not organize refreshment sessions nor provided feedback on performance or assessed its effect The study faced some difficulties. In contrast to what was described in our study protocol In addition to the proposed outcomes, we evaluated whether the intervention had reduced ETS exposure at home. Children participating in the control group also visited the intervention well-child centres and systematic assessment and (when indicated) counselling of asthma-like symptoms and ETS exposure was applied to the parents of these children. Contamination of intervention and control condition may possibly have occurred by moving to another neighbourhood in the city and visiting another well-child centre. Because this contamination may have reduced the differences in results between intervention and control group, we amended the study protocol and in addition to the intention-to-treat analysis we performed a per-protocol analysis.The following limitations would be a possible explanation for the negative study results: the study included a relatively low-intensity counselling intervention. However, the systematic assessment of the presence of asthma-like symptoms in early childhood by well-child professionals was prioritized and was considered feasible and essential in the Dutch youth healthcare system We consider selection bias unlikely because a multiple imputed analysis including all eligible children did not change the results. Information bias should be considered for different measurements. Although the validity of assessing ETS exposure by questionnaires in epidemiological studies has been shown, misclassification may occur due to underreporting The decreased risk on ‘ETS exposure at home ever’ in the intervention group remained statistically significant even after correction for multiple testing.This study raises questions about whether it is feasible to prevent the development of asthma by using systematic assessment and counselling of asthma-like symptoms and ETS exposure by using brief forms at well-child centres. We recommend further studies to evaluate whether professionals at well-child centres can contribute to optimal asthma management in other ways, and efforts are needed to optimize the protocols that can be implemented in this setting.We also recommend further studies to improve the current intervention to optimise asthma management at well-child care. Based on previous results, it is recommended that professionals at well-child centres encourage breastfeeding and advise parents of children at high-risk of developing asthma to avoid ETS and indoor allergens exposure to their children to reduce the prevalence of asthma Our study was embedded within the Dutch system of preventive health care provided by well-child centres in Rotterdam, the Netherlands. This may have consequences for the generalisability of our results in other areas and countries and therefore evaluation of our study in other, varied populations is recommended.A systematic assessment of asthma-like symptoms and ETS exposure by using brief assessment forms at well-child centres was not effective in reducing the prevalence of physician-diagnosed asthma ever and wheezing, and did not improve FeNO, Rint or HRQOL at age 6 years. Our results hold promise for interviewing parents and using information leaflets at well-child centres to reduce ETS exposure at home in preschool children.Table S1Age at enrolment in intervention group (N = 3596).(DOCX)Click here for additional data file.Table S2Frequency of applied intervention to preschool children participating in the intervention group (N = 3596).(DOCX)Click here for additional data file.Checklist S1CONSORT Checklist.(DOCX)Click here for additional data file.File S1Study design paper .(PDF)Click here for additional data file.Protocol S1Original Study Protocol.(PDF)Click here for additional data file.


A 14-year-old girl presenting with visual loss in both eyes was diagnosed to have healed toxoplasma retinochoroiditis in the right eye with active choroidal neovascularization (CNV) secondary to toxoplasmosis in the left. She underwent combination photodynamic therapy (PDT) and intravitreal bevacizumab as primary treatment. PDT was performed as per the ‘Treatment of Age-related Macular Degeneration by Photodynamic therapy’ study protocol and was followed by intravitreal bevacizumab after 2 days. CNV regressed at 8 weeks of follow-up and remained stable at 8 months of follow-up. The initial visual acuity improved from 20/120 to 20/30. Combination therapy with PDT and intravitreal bevacizumab appears to be effective in the treatment of CNV secondary to toxoplasma retinochoroiditis. Ocular toxoplasmosis is an important cause of choroidal neovascularization (CNV) in the pediatric age group. The deveA 14-year-old girl presented with a 1-year history of deterioration of vision in both eyes. There were no significant ocular problems in the past. Medical and family history and systemic examination were unremarkable. On ocular examination, her best corrected visual acuity (BCVA) at presentation was CF 1 mt, N36 in the right eye and 20/120, N18 in the left eye. Biomicroscopic examination did not show any evidence of intra-ocular inflammation and the anterior segment was unremarkable in both eyes. Fundus evaluation of the right eye showed the presence of a punched-out pigmented lesion with well-demarcated borders at the macula that was suggestive of a healed toxoplasma lesion . ClinicaThe clinical findings were confirmed on fundus fluorescein angiography (FFA) and optical coherence tomography (OCT). The FFA of the left eye revealed an active subfoveal classic choroidal neovascular membrane with profuse leakage. Blocked choroidal fluorescence due to the overlying hemorrhage was also noted . OCT revAt 8 weeks follow-up, the CNV showed regression with no evidence of subretinal fluid or hemorrhage on clinical examination or OCT. The BCVA improved to 20/30, N6 and the fundus remained stable up to the eighth month of follow-up . No adveet al.[The natural history of CNV secondary to toxoplasmosis reveals a poor visual prognosis.–5 The CNet al. assessedet al. However,et al.[et al.[The use of verteporfin–PDT, although initially used for the treatment of age-related macular degeneration and myopic CNV, has now been extended beyond these indications to inflammatory and infectious etiologies. Mauget Faysse et al. have foul.[et al. recentlyThere is however no report of any patient with CNV secondary to toxoplasmosis who has been managed with combination PDT and intravitreal bevacizumab as a primary treatment. The combined regime is postulated to have a beneficial synergistic effect that could reduce the need for repeated injections. Combination therapy using PDT and bevacizumab as the first-line management in such cases could be a viable option. Larger s


This study presents efficient vision-based finger detection, tracking, and event identification techniques and a low-cost hardware framework for multi-touch sensing and display applications. The proposed approach uses a fast bright-blob segmentation process based on automatic multilevel histogram thresholding to extract the pixels of touch blobs obtained from scattered infrared lights captured by a video camera. The advantage of this automatic multilevel thresholding approach is its robustness and adaptability when dealing with various ambient lighting conditions and spurious infrared noises. To extract the connected components of these touch blobs, a connected-component analysis procedure is applied to the bright pixels acquired by the previous stage. After extracting the touch blobs from each of the captured image frames, a blob tracking and event recognition process analyzes the spatial and temporal information of these touch blobs from consecutive frames to determine the possible touch events and actions performed by users. This process also refines the detection results and corrects for errors and occlusions caused by noise and errors during the blob extraction process. The proposed blob tracking and touch event recognition process includes two phases. First, the phase of blob tracking associates the motion correspondence of blobs in succeeding frames by analyzing their spatial and temporal features. The touch event recognition process can identify meaningful touch events based on the motion information of touch blobs, such as finger moving, rotating, pressing, hovering, and clicking actions. Experimental results demonstrate that the proposed vision-based finger detection, tracking, and event identification system is feasible and effective for multi-touch sensing applications in various operational environments and conditions. The firSome consumer systems, such as the touch screens on electronic check-out machines, use resistive surfaces for touch sensing. These resistance-based systems are cheap to implement, but cannot provide sufficient sensing accuracy and clarity to support more sophisticated user operations and commands. Systems based on capacitive technologies can provide more accurate sensing results using multiple touch contacts on the screen –9. Howevetc… . The segmentation results of homogeneous objects can be optimized by maximizing the 𝒮𝒡 value. Observation of the terms comprising WCv (T) indicate that if the pixels in each class are broadly spread, i.e., the contribution of the class variance nσ2 is large, then the corresponding 𝒮𝒡 measure becomes small. Hence, when 𝒮𝒡 approaches 1.0, all classes of grayscale values decomposed from the original image I are ideally and completely separated. Previous research presents a detailed derivation of this property [The aforementioned statistical measures can be used as a measure of separability, among all existing pixel classes, decomposed from the original image property .I until the brightest objects of interest are clearly obtained, regardless of the number of existing objects and various illuminated conditions. This segmentation process is conducted by recursively thresholding the grayscale values of the image I until the 𝒮𝒡 measure is large enough  to indicate that the appropriate discrepancy among the resulting classes of grayscale intensities is achieved. Thus, the bright objects of touch blobs are clearly segmented into a separate thresholded sensing image. Through the above mentioned properties, this objective can be reached by minimizing the total within-class variance WCv (T). This can be achieved by a recursive partition strategy that selects the pixel class with the maximal within-class variance contribution (nσnw2), denoted by pC. The bi-class partition procedure, as described in [pC into two more classes in each recursion. This partition process is recursively performed until the separability among all pixel classes becomes satisfactory, i.e., the 𝒮𝒡 measure approximates a sufficiently large value:Using the above-mentioned statistical discriminant measure, the bright object segmentation process can be implemented by recursively segmenting homogeneous objects from the multi-touch sensing image ribed in , is perfSTh as 0.92 based on the experimental analysis in [i.e., the obtained brightest pixel class kC, extracted by this recursive thresholding strategy are ensured to achieve maximum separation. This approach achieves satisfactory segmentation results for these objects using the fewest recursions. Previous research provides detailed descriptions of this multilevel thresholding technique [This study sets the value of the separability measure threshold lysis in  to yieldechnique .To obtain blobs of potential touching fingers from the extracted bright object plane, a connected-component extraction process  is perfo3.2.The proposed touching blob extraction method effectively obtains the blobs of the touching fingers in each image frame captured from the proposed sensing interface. In multi-touch applications, users perform various gestures by moving their fingers to issue their desired control commands. These gestures are mostly formed by the movements and actions of one, two, or more blobs of touching fingers. Since the complete features of touching gestures may not be immediately identified from single image frames, a tracking procedure for the blobs of touching fingers must be applied to analyze the information of moving blobs to recognize touching gestures from consecutive image frames. During the tracking process, the spatial-temporal information of moving blobs can be used to refine the detection results and correct the errors due to occlusions caused by noise and interference during the blob object segmentation process. The tracking information of blobs can be utilized to determine and recognize meaningful touch events, such as finger moving, rotating, pressing, hovering, and clicking actions. The proposed blob tracking and touch event recognition process includes two phases. The blob tracking phase first associates the motion of blobs in succeeding frames by analyzing their spatial and temporal features. The touch event recognition phase then identifies possible touch events based on the tracked motion information of blobs.3.2.1.The spatial-temporal features of the blob regions of touching fingers make it possible to progressively refine and correct the detection results of the moving blobs by associating them in sequential frames. Therefore, this study presents a tracking process for analyzing the motion features of the detected touch blobs to address the above-mentioned problems. Moreover, the tracking information of the moving blobs can be applied to the following touch event recognition process to determine possible touch events from the tracked motion information of the blobs.thi detected touch blob appearing in tht frame captured by the sensing systemThe position of the blob t, and is defined as:The tracker tth frame captured by the vision system, which is:φ is the path coherence function of t − 1th frame to the tth frame.Let φ can then be determined by computing the deviation formula of the motion vectors φ consists of two deviation terms. The first term reveals the directional deviation formed by The path coherence function iP corresponding to its tracker Hence, the path deviation of a given blob m blobs are detected within a time slot of image frames, the overall tracker deviation function D of the tracker sets of these m blobs can be obtained by:Accordingly, when When the vision system initially detects the blob of a given finger, it creates a tracker to associate this blob with those from the same finger in subsequent frames based on their contiguous spatial-temporal features. The features used in the tracking process are described and defined as follows:D associated with these detected blobs. Accordingly, in each recursion of the tracking process for a incoming frame t, the newly detected touch blobs appearing in this incoming frame, denoted by t − 1 and t − 2, denoted by TPt can be updated by minimizing the overall tracker deviation function D associated with the sets of finger blobs in Pt, TPt−1, and TPt−2.Based on the above-mentioned definitions utilized in the tracking process, the trackers formed by the detected touch blobs in the sequential images can be determined by minimizing the overall tracker deviation function 3.2.2.The proposed system can efficiently obtain trackers that reflect the spatial-temporal information of the detected finger blobs by applying the above-mentioned tracking process to the detected finger blobs. By analyzing the spatial-temporal information of the tracked finger blobs, the changing processes of the amount of finger blobs and the variation of the finger blob trajectories can reflect possible touching events of commands, or some occlusions of blob trackers caused by the blob extraction and tracking process. Such touch events may be: (1) When users press down or lift up their fingers, they change the number of finger blobs; (2) When users conduct a single-click or double-click action using their fingers, the corresponding finger blobs will quickly disappear and re-appear; (3) When users activate a zooming action by moving two fingers apart or close together along a straight line, the motion trajectories of a pair of finger blobs will form a straight line with increasing or decreasing distances; (4) Users may move two or more fingers close to each other for a while then split their fingers, or vice versa. In this case, the corresponding finger blobs might be occluded for a while during the blob extraction and tracking process. Using the trackers of the finger blobs t and t’, is defined as:To perform the touch event analysis and occlusion resolution process on the tracked finger blobs, the following spatial-temporal overlapping score of two finger blobs Update: When the number of tracked finger blobs TPt for the next recursion of the blob tracking process.Appearing / Finger Pressing-down: If the newly appearing finger blobs TPt in the following frames.Disappearing/Finger Lifting-up: Some existing trackers of finger blobs i.e., k = 0,1,…,5) quickly re-appear, they may successfully match Clicking Action: If a tracked finger blob mτ should have reasonable spatial-temporal coherences for mτ = 0.25 to obtain satisfactory click event identification results.To identify clicking events accurately, the threshold matching condition [TPt will be updated.Merging: If a detected finger blob TPj+nt−1 , this inondition  of the doS with TPt.Splitting: If one tracked finger blob nt frame , then thnt frame  between merging event, then a zooming out event is identified. When a touched blob of two fingers starts with a splitting event into a pair of two blobs zooming in event is identifiedZooming Action: When a pair of two tracked finger blobs While the finger blobs are being tracked, the touch event analysis and occlusion resolution process will determine the associated possible event states based on their spatial-temporal features. In the proposed system, the tracked finger blobs might be in one of seven possible event states, and the touch event analysis and occlusion resolution process will activate different touch events or operations according to the given event states of the tracked finger blobs in each given period. The proposed system uses the following tracking event states and associated actions for the tracked finger blobs:After identifying the touch events based on the moving blobs in the video sequences captured from the proposed touch-sensing interface, the touch events are then be transformed into interactive commands for the corresponding computer applications, such as selecting, moving, clicking, or zooming some interactive objects on the display screen.4.This section presents several representative experiments for evaluating the performance of the proposed vision-based multi-touch finger detection, tracking, and event identification techniques using a prototype system of the proposed multi-touch sensing and display system .The proposed vision-based multi-touch sensing and display techniques were implemented on a 2.4 GHz Pentium-IV personal computer platform. To provide better portability and maintainability for the proposed system, the software modules of the proposed vision-based techniques were implemented using C++ programming language with the Visual C++ 2008 development environment. By releasing the source code project associated with the software modules, the proposed vision-based techniques can be conveniently distributed and migrated onto different hardware platforms (such as ARM-based platforms) with various operating systems . Thus, the application developers can easily implement and design many customized multi-touch interactive application systems under various hardware platforms and software environments. The frame rate of the vision system is approximately 30 frames per second, and the resolution of each frame of captured image sequences is 640 pixels by 480 pixels per frame. An experimental set of several multi-touch sensing videos captured in various illumination conditions and application environments was adopted to evaluate the system’s finger detection and touch event identification. This experimental set consists of five experimental video sequences with a total of 16,841 captured frames and 1,113 desired interactive touch events in various conditions.To evaluate the performance of the proposed vision-based multi-touch detection and tracking technique, The experimental samples in pT (true positives) represents the number of correctly detected touch fingers, pF  represents the number of falsely detected touch fingers, and nF  is the number of missed touch fingers. The Jaccard coefficient J for the touch finger detection results of each frame of the touch-sensing image sequences were determined by manually counting the number of correctly detected touch fingers, falsely detected touch fingers, and missed detections of touch fingers in each frame. The average value of the Jaccard coefficients J was then obtained from all frames of the captured video sequences using:N is the total number of video frames. The ground-truth of detected touch fingers was acquired by manual counting. This study adopts the Jaccard coefficient  as a detThis table clearly shows that the proposed approach provides high detection accuracy on touch fingers, and can thus provide efficient multi-touch sensing results that accurately reflect the user’s interactive commands.In addition to evaluating the detection accuracy of the touch fingers of the proposed vision-based technique on single frames, this study evaluates the identification performance of touch events based on the spatial-temporal features of the finger blobs detected and tracked in consecutive image frames. Manually analyzing and discriminating the touch events appearing in the five test sequences shows that there are 1,113 discernible touch events of possible interactive gesture commands, including finger moving, clicking, and zooming action events . These 1The identified finger touch events can further be transformed into interactive commands for various applications. The computation time required to process one captured frame depends on the complexity of the touch command in the frame. Most of the computation time is spent on the connected-component analysis and touch blob tracking process. For an input video sequence with 640 × 480 pixels per frame, the proposed vision-based multi-touch sensing system requires an average of 12.2 milliseconds processing time per frame. This frugal computation cost ensures that the proposed system can effectively meet the requirements of real-time processing at over 80 frames per second. The above-mentioned experimental results of our numerous real tests on many different application scenarios and conditions on multi-touch sensing confirm that the proposed system can provide fast, real-time, and effective finger touch detection, tracking, and event identification for human-computer interactive applications. In our experimental analysis of the prototype system, the software modules of the proposed vision-based multi-touch sensing and display techniques only costs less than 5.0% of the CPU computational usage on average, and about 8,640 Kbytes of memory resource. These economic costs of computation and memory resources can efficiently provide the interactive multi-touch interfaces for many simultaneously executed software applications  by the analyzing spatial-temporal features of touch blobs in successive frames. The experiments in this study implement the proposed vision-based multi-touch finger detection, tracking, and event identification techniques on a low-cost hardware framework, forming a practical multi-touch sensing and display system. Experimental results in various application scenarios and operational conditions demonstrate that the proposed vision-based multi-touch sensing and display framework achieves fast, real-time, and effective finger touch detection, tracking, and event identification for many practical human-computer interactive applications and environments.Regarding to the further researches on the vision-based multi-touch sensing and display systems, an extension to a large-scale and high-resolution multi-touch sensing and display system is desirable for extensive interactive applications. However, this extension will require much more computational costs and resources for achieving real-time operation performance on exploiting and processing large volumes of sensing video data, as well as the demand on guaranteeing high precision capabilities of the multi-touch finger detection, tracking, and event identification functions. In this aspect, further improvements and developments of the proposed system on the computational efficiency can be achieved by taking advantage of the computational power of the graphic processing units (GPU) ,41. Thus


To avoid artefacts introduced by culturing cells for extended periods of time, it is crucial to use low-passage patient-derived tumour cells. The ability to enrich, isolate and assay sub-populations of cells that behave as cancer stem cells (CSCs) from these primary cell lines is essential before performing characterizations such as gene-expression profiling. We have isolated cells from glioblastomas which show characteristics of CSCs. Although glioblastomas contain only a relatively small amount of putative CSCs, these cells express many genes which seem to be worthy targets for future therapies. The tumourigenic cell population was identified based on its cell surface phenotype. This population could initiate tumours in immuno-compromised mice with as few as 200 cells, while as many as 500,000 or more of the remaining cells in the tumour did not initiate new tumours in mice [Al-Hajj  in mice .et al have shown that upon culturing of metastatic melanoma cell suspensions under appropriate conditions, a subset of cells could be propagated as non-adherent spheres, which could then be induced to differentiate in vitro and to generate tumours in vivo [Fang  in vivo . The abiGlioblastoma (GBM) tissues were obtained intra-surgically. A trained pathologist evaluated stage and grading of the removed tumour tissue. For our experiments, we only cultivated cells from tissues containing at least 90% tumour cells as determined by H&E staining . In a moTissues that met our criteria were used for low-passage cell culture. We enriched CSCs using embryonic stem (ES) cell medium supplemented with FGF2 . Cells cTo show the de-differentiation towards a more primitive neural phenotype, we performed immunofluorescence microscopy of cells from the normal medium and ES medium with NESTIN.To investigate if the treated cells were enriched for putative cancer stem cells, we employed magnetic sorting with a bead-coupled anti-CD133 antibody. Enrichment of CSCs by selective cell culture methods resulted in higher yields of CD133+ cells . This enTo determine the quality of enrichment, we kept the cells from the positive and negative fractions in culture for four days and stained them with a fluorescence coupled anti-CD133 antibody .To identify the mechanisms and pathways underlying CSCs, we conducted RNA-expression analysis using the Illumina platform. For expression analysis, we used CD133+, CD133– and bulk cells from the GBM1207 cell line and compared this data to the expression profile of hES lines H1/H9 . ExpressWe validated the differential expression of eight selected marker genes from the expression analysis by real-time PCR . All marStill only little is understood about the origin and nature of CSCs . From ou


Saccharomyces cerevisiae is a multi-step developmental path on which dormant spores re-enter the mitotic cell cycle and resume vegetative growth. Upon addition of a fermentable carbon source and nutrients, the outer layers of the protective spore wall are locally degraded, the tightly packed spore gains volume and an elongated shape, and eventually the germinating spore re-enters the cell cycle. The regulatory pathways driving this process are still largely unknown. Here we characterize the global gene expression profiles of germinating spores and identify potential transcriptional regulators of this process with the aim to increase our understanding of the mechanisms that control the transition from cellular dormancy to proliferation.Spore germination of the yeast Employing detailed gene expression time course data we have analysed the reprogramming of dormant spores during the transition to proliferation stimulated by a rich growth medium or pure glucose. Exit from dormancy results in rapid and global changes consisting of different sequential gene expression subprograms. The regulated genes reflect the transition towards glucose metabolism, the resumption of growth and the release of stress, similar to cells exiting a stationary growth phase. High resolution time course analysis during the onset of germination allowed us to identify a transient up-regulation of genes involved in protein folding and transport. We also identified a network of transcription factors that may be regulating the global response. While the expression outputs following stimulation by rich glucose medium or by glucose alone are qualitatively similar, the response to rich medium is stronger. Moreover, spores sense and react to amino acid starvation within the first 30 min after germination initiation, and this response can be linked to specific transcription factors.Resumption of growth in germinating spores is characterized by a highly synchronized temporal organisation of up- and down-regulated genes which reflects the metabolic reshaping of the quickening spores. A greater understanding of the control mechanisms that establish, maintain and end quiescence is important for basal, medical and biotechnological research.Cellular adaptation to environmental changes ensures fitness and survival of cells and organisms. Adaptation programs may strengthen cell walls in response to stress, switch the metabolic machinery upon starvation or trigger morphological differentiation during multicellular development and sexual reproduction. Entering and exiting GSaccharomyces cerevisiae responds to carbon and nutrient limitation either by entering stationary phase (haploids), or sporulation (diploids) [a and α, enclosed in an ascus sack (reviewed in [Budding yeast iploids) ,2. Sporuiewed in ). The spiewed in -7. Upon iewed in ,9. Duriniewed in ,11. If tiewed in . The matiewed in -15. The In this study, we analysed the transcriptional reprogramming of germinating spores and show that exit from dormancy results in rapid and global changes consisting of sequential gene expression subprograms. We identified a complex candidate network of transcription factors (TFs) regulating this response, thereby extending our understanding of the global regulatory program during germination . By compWe performed a time course microarray analysis to investigate the global response during germination Figure A. The anWe further aimed at identifying regulators of the global transcriptional germination program and its subprograms. The on-line-tool T-profiler  was usedThe main trigger for spores to exit dormancy is a fermentable carbon source, which suffices to induce early germination events such as spore wall degradation and swelling ,11. To i-214; chi-square test). We conclude that the vast majority of genes changed expression in the same direction, even though expression change for some genes passed the two-fold criterion in YPD but not glucose treatment. There is one noteworthy exception; 45 genes showed a two-fold or more up-regulation in glucose but not in YPD . The coD Figure D. AlmostD Figure E. This sThe similarity in gene expression pattern between germination in YPD and glucose is reflected in similar TF activity profiles. T-profiler analysis of the global germination program in glucose predicted eight additional TFs and the combined TF set of germination in YPD and glucose encompass thirty three TFs Figure . The majTo relate the temporal organisation to the candidate TF network structure, we extracted the TF sub-network corresponding to the thirty three TFs identified above from a global TF network . Figure We proceeded to compare our results to a previous transcriptome analysis of germination of SK1 spores . Two dimSeveral lines of evidence suggest that a large portion of the germination program reflects the metabolic evolution of the quickening spores rather than germination specific functions. To pursue this connection in more detail, we compared the germination program to the transcriptional changes during stationary phase exit . We starFurther, a T-profiler analysis of stationary phase exit and comparison with YPD-induced germination revealed that the rapid and transient upregulation of many genes seen during germination was also prominent during stationary phase exit Figure B, C. WheThe ability to exit and re-enter the cell division cycle in response to altered nutrient conditions is of paramount importance for cellular viability and cooperation. When starved, diploid yeast cells enter the sexual cycle by sporulation, while haploid cells arrest in a stationary phase. Cells in these two quiescent stages share many properties; they do not proliferate, their metabolic, transcriptional and translational rates are low, and they are surrounded by a thickened cell wall that provides resistance to harsh environments . ConsistFurther, we identified a common early and transient peak of transcription, including genes encoding transporters and transcriptional regulators that presumably are required for setting up proliferation after spore dormancy . We alsoThere are also discrepancies between dormancy exit of spores and stationary cells. In our hands, germination is associated with down-regulation of Gcr1/Gcr2-regulated glycolytic mRNAs, which does not occur during quickening from stationary phase. This is counterintuitive as resumption of growth in glucose containing medium requires glycolysis. We can only speculate about the possible purpose of this. To germinate quickly is crucial in the competition with other micro-organisms for nutrients and colonization of the micro environment; perhaps the spores contain high levels of these glycolytic mRNAs for translation immediately upon germination signals as mRNAs already present in the dormant spores most likely is the first choice for translation upon germination initiation . It is aWe further characterise the spore quickening by examining the effect of re-entry of media on the gene expression program. Rich growth medium with glucose has proved to be a more powerful germinant than glucose alone when it comes to acquisition of zymolyase sensitivity and trehalose breakdown ,28. ThisFinally, we resolve the gene expression profile in temporally distinct subprograms that are orchestrated by interconnected TFs. The data set we present here has a higher initial time resolution than previous studies, more stringent evaluation criteria, less noise and hence more coherent clusters, and benefit from the increased resolution gained by two-dimensional clustering. This allows us to analyse the temporal and compositional organisation of the germination gene expression program. As shown in Figure In conclusion, we have determined the gene expression response of dormant spores to YPD and pure glucose, identified distinct expression subprograms, and linked them to distinct TFs. We found strong similarities between quickening of spores on YPD and glucose, and to quickening from stationary phase. Resumption of active metabolism coincides with a transcriptional response with clearly distinct subprograms that reflect each nutritional condition. Interestingly, the program does not encompass any clear germination specific genes, and the process can be explained by a combination of metabolic programs including trehalose mobilisation, glucose repression, amino acid and protein biosynthesis, and mating. Future challenges in the field of yeast germination include deciphering the translational and metabolic responses of germinating spores, and to identify proteins and pathways essential for the process of exiting dormancy.HO gal3 MALI SUCI) strain background [Diploid cells of Y55  and labelled in a reverse transcription reaction with Cy3-dUTP or Cy5-dUTP (Amersham Pharmacia Biotech) in a total volume of 30 μl, following standard protocols (http://.cmgm.stanford.edu/pbrown) [http://microarrays.ca) was prehybridized with 1% BSA in DIGeasy hybridization buffer at 42°C for 1 h. Hybridization was performed at 42°C for 12–18 h. The slides were scanned on a GenePix 4000B scanner and quantized using the software GenePix Pro 4.0 resulting in data for 13056 spots, two spots represented one gene. RNA from dormant spores was used as reference RNA for all microarrays.Samples for RNA isolation were taken in three biological replicates at nine different time points  after addition of YPD or glucose 2%. For RNA extractions, 10 ml sample volumes were taken at each time point and transferred to 40 ml ice-cold 0,5% TritonX-100 in 50 ml falcon tubes, left on ice for a few minutes and then centrifuged briefly to collect the cells . The pellet was immediately frozen in dry-ice. Total RNA was extracted as described previously [/pbrown) . LabelleFor microarray data analysis, normalization was performed by a correction for dye-bias and intensity dependent trends. The normalization method was chosen by fitting a robust line (loess line) on the M-values and then corrected each value according to the value of the line. Statistical data analysis was carried out to obtain significantly different gene expression by using a moderated t-test analysis in R and Limma package . EmpiricTranscription profiles of germination and stationary phase comparative datasets were collected from databases NCBIs Gene Expression Omnibus (Series accession number GSE7393), and The ArrayExpress Archive (Experiment E-UMCU-12), respectively. Normalized log2 values were subjected to transformation so that all genes were 0 at 0 min time points.http://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE29960.MIAME-compliant microarray data has been deposited in the microarray database GEO (http://.rana.lbl.gov/EisenSoftware.htm) [http://www.yeastgenome.org).The gene expression profiles were filtered to select genes with a reliable expression change by applying the criterion of a two-fold change (in the same direction) in expression level in two or more consecutive time points. K-means and hierarchical clustering was performed and visualized using Cluster and TreeView (are.htm) . Two dimare.htm) . Brieflyhttp://www.t-profiler.org) compares the mean expression ratios of groups of genes, and all genes within each group contribute to the evaluation of statistical significance, not just those genes that are judged to be differentially expressed. Two statistical parameters are utilized; (i) a t-value measuring the up-regulation (t > 0) or down-regulation (t < 0) in units of the standard error of the difference and (ii) a Student t test derived E-value that reflects the degree of difference in the mean log2-transformed expression ratio of a pre-defined group of genes and the mean for the rest of the genome. An E-value of <0.05 is considered as a statistically significant difference in gene expression. Only TFs and motifs with T-values ≥3.5 or ≤ −3.5 (in one or more time points) in one or both conditions compared are included in the analyses. Most but not all of these TFs have statistically significant E-values. Where several T-values for a transcription factor are identified, the highest absolute T-value at each time point is used. For TF identification in k-means clusters, the time point with the largest change compared to previous time point for each cluster is analysed. Only those TFs identified also for the whole microarray dataset are listed , without consideration of statistical significance. For cluster 7 and 8 in Figure T-profiler  was usedFor calculations of the differences in TF T-values between two different conditions (X-Y), the following rules were set up: If the T-values of the TF has the same sign in both conditions, then purple (positive numbers) indicates a higher absolute value in YPD germination compared to glucose germination/stationary phase exit, and green vice versa (negative numbers). If the TF T-values are of different signs, the highest absolute value of the two conditions dictates the direction of the comparison.For Cytoscape visualization, the data on transcription factor promoter binding was collected elsewhere , and theThe authors declare that they have no competing interests.CG participated in the design of the study, analyzed and interpreted the data, made figures, participated in discussions of the work and has been highly involved in drafting the manuscript. IP designed and carried out the microarray analysis and performed initial data analysis. WV and AE performed the statistical analysis and processing of data and helped with initial data analysis and interpretation. JN participated in design and coordination of the study. MK assisted and advised CG on analysis and interpretation of the data and has been highly involved in drafting the manuscript. SH conceived of the study, and participated in its design and coordination and has been involved in drafting the manuscript. All authors read and approved the final manuscript.K-means clustering of the complete gene expression dataset of YPD induced germination is largely consistent with clusters in Figure1. Genes were subjected to K-means clustering and grouped in eight clusters. The graph displays the average expression profiles of the eight clusters, named and coloured according to the clusters in Figure Click here for fileT-profiler analysis of the global K-means clusters identified in Additional file. TFs identified both in the global analysis in Figure Click here for fileTwo-dimensional visualisation highlights the correlation between the transcriptional responses of spores to rich growth medium and pure glucose. Two dimensional hierarchical clustering of the expression response to YPD and glucose. The x-axis represents the YPD cluster trees whereas the y-axis holds the glucose cluster trees. Three plots are separated by a grey line; genes whose expression changed two-fold in both data sets to the upper left, genes whose expression changed two-fold after glucose addition but not after YPD addition to the upper right, genes whose expression changed two-fold after YPD addition but not after glucose addition to the lower left. Each cluster is subdivided into groups of genes with correlation factor >0.75 by thin lines. The colour panels along each axis indicate diminished (blue) or stimulated gene expression (red) over time, compared to dormant spores. Each gene occurs once on each axis and the intersection is marked with “■”.Click here for fileTwo-dimensional comparison of the YPD data set with previous study on yeast germination. Two dimensional hierarchical clustering of the transcriptional response of spores to YPD, comparing the datasets from this study and a previous study (Joseph-Strauss) [Strauss) . The x-aClick here for file


Gene duplication is central to genome evolution. In plants, genes can be duplicated through small-scale events and large-scale duplications often involving polyploidy. The apple belongs to the subtribe Pyrinae (Rosaceae), a diverse lineage that originated via allopolyploidization. Both small-scale duplications and polyploidy may have been important mechanisms shaping the genome of this species.This study evaluates the gene duplication and polyploidy history of the apple by characterizing duplicated genes in this species using EST data. Overall, 68% of the apple genes were clustered into families with a mean copy-number of 4.6. Analysis of the age distribution of gene duplications supported a continuous mode of small-scale duplications, plus two episodes of large-scale duplicates of vastly different ages. The youngest was consistent with the polyploid origin of the Pyrinae 37-48 MYBP, whereas the older may be related to γ-triplication; an ancient hexapolyploidization previously characterized in the four sequenced eurosid genomes and basal to the eurosid-asterid divergence. Duplicated genes were studied for functional diversification with an emphasis on young paralogs; those originated during or after the formation of the Pyrinae lineage. Unequal assignment of single-copy genes and gene families to Gene Ontology categories suggested functional bias in the pattern of gene retention of paralogs. Young paralogs related to signal transduction, metabolism, and energy pathways have been preferentially retained. Non-random retention of duplicated genes seems to have mediated the expansion of gene families, some of which may have substantially increased their members after the origin of the Pyrinae. The joint analysis of over-duplicated functional categories and phylogenies, allowed evaluation of the role of both polyploidy and small-scale duplications during this process. Finally, gene expression analysis indicated that 82% of duplicated genes, including 80% of young paralogs, showed uncorrelated expression profiles, suggesting extensive subfunctionalization and a role of gene duplication in the acquisition of novel patterns of gene expression.This study reports a genome-wide analysis of the mode of gene duplication in the apple, and provides evidence for its role in genome functional diversification by characterising three major processes: selective retention of paralogs, amplification of gene families, and changes in gene expression. Because it provides the raw material for gene diversification and hence the creation of genetic novelty, gene duplication is recognized as a major force in the evolution and adaptation of species ,2. The cArabidopsis and more than half of the poplar and rice genes are grouped into gene families [Although small-scale duplications or large segmental duplications seem to be a phenomena common to all eukaryotes, polyploidy appears to be more restricted to specific lineages and is particularly well recognized among flowering plants . It has families -13. Accufamilies -16.The prevailing theory predicts gene loss or functional diversification as the main fate for one of two copies of a pair of duplicated genes . Gene loWhen the two copies of a duplicated gene are retained in a functional state, they can diversify by partitioning the original function  or by evolving a new function . Of these two processes, subfunctionalization processes, in which each duplicate gene develops a distinct expression pattern, seems to be the most common phenomena and takes place early after gene duplication . On the Malus domestica Borkh.) is one of the most important fruit crops grown in temperate climates and is widely appreciated by consumers worldwide. Together with other fruit species such as pears, quince, and loquat, apples are grouped into the subtribe Pyrinae  within the family Rosaceae, and have a pome-type fruit and chromosome number x = 17 as the main distinctive characteristics. This high base chromosome number compared with other members of the Rosaceae family  was recognized early as the result of polyploidy. Sax [x = 8 and x = 9. Moreover, he suggested that these progenitors may have been related to extant members of the Rosaceae family belonging to the Amigdaloideae (x = 8) and Spiroideae (x = 9) lineages. A more recent molecular phylogenetic analysis by Evans and Campbell [Gillenia (x = 9), followed by loss of one pair of chromosomes [The apple (idy. Sax  proposedCampbell  supporteomosomes . Despitein silico methods for monitoring gene expression [Collections of expressed sequence tags (ESTs) are providing plant scientists with a valuable source of data for the large-scale characterization of duplicated genes in non-sequenced genomes ,19,25-27pression . Based opression  for the The present study reports the characterization of duplicated genes and their derived gene families in the apple using EST sequence collections. I dated the age of gene duplications using synonymous site substitutions, to identify the major events that contributed to the formation of paralogous genes in the species. This approach allowed characterization of a continuous mode of small-scale duplications and two putative episodes of polyploidy. The imprint left by this mode of gene duplication was studied and suggested that the selective retention of paralogs, the amplification of gene families, and the acquisition of novel patterns of gene expression by duplicated genes have been important mechanisms shaping the apple genome.Thirty-three publicly available EST libraries containing 119,177 sequences from the apple cultivar Royal Gala were used to construct a unigene dataset of tentative non-redundant sequences , were likely candidates for apple-specific genes, genes with uncharacterized open reading frames, transcripts from non-coding genes, or transcribed genomic regions corresponding to mobile elements or other repetitive DNA, and were discarded from further analysis. A further selection of this initial dataset of putative protein-coding genes was made by discarding transcripts with open reading frames shorter than 300 nucleotides. After removing those unigenes matching (retro)transposon-related proteins and the sequences putatively corresponding to the same transcript (see Methods), the resulting dataset consisted of 13,598 non-redundant protein-coding genes (Table Ks distance (synonymous substitutions/synonymous site) between pairs of paralog sequences. Figure Ks distance.Pairs of duplicated genes  were characterized based on protein sequence similarity. In the collection of 13,598 protein-coding genes, 9,339 (~68%) were found to have at least one paralog, whereas 4,259 (~32%) did not match with any other sequence in the dataset and were considered to be single-copy genes (singlets). The ages of the gene duplications were estimated by computing the Ks values of 0.185 and 1.650 respectively, which were attributable to two putative episodes of whole-genome duplication , fostering two conclusions. First, the overall estimated gene family sizes in the apple seem to remain proportionate with actual sizes. Second, using Arabidopsis as a reference, no major changes in gene-copy number seem to have occurred in the apple.The sizes of 111 apple gene families with more than five members  and three families encoding ribosomal proteins , which are all directly involved in protein biosynthesis. Two other families encoded proteasome subunits: the proteasome alpha/beta subunit and the 26S proteasome regulatory subunit, which are involved in controlled proteolytic activity. Of note, two important families encoded proteins involved in the degradation of starch: starch phosphorylase and β-amylase. On the other hand, other families seemed to exhibit a variety of different unrelated functions including diverse metabolic functions or photosynthesis (chlorophyll a/b-binding protein).Interestingly, some of the amplified families developed related functions, suggesting the amplification of interrelated gene families . For example, six families were involved in protein metabolism; these included elongation factor 1  categories that were over-represented or under-represented in gene families as compared with single-copy genes. Annotation of gene families and single-copy genes to the plant GOslim categorization system was performed using Blast2GO and showed that 70% 2998 of 4259) of single-copy genes and 72% (1472 of 2027) of gene families could be associated with at least one GO category. On average, singlets were associated with 3.5 GO terms, whereas gene families were associated with 4.0 GO terms, and the two groups were distributed equally across the three general sections provided by the GO annotation scheme: biological processes, molecular functions, and cellular components (data not shown). Several GO categories were over-represented or under-represented in gene families, which suggested functional bias in the process of gene retention in the apple  or carbohydrate metabolism . The Lhc family of chlorophyll a/b-binding proteins also constitutes an important photosynthetic gene family. Other families could not be related to any of the biological processes over-represented among young paralogs and may represent cases of selective gene retention only associated with specific families.To explore the extent to which this pattern of gene retention might reflect characteristics specific to the Pyrinae/apple genome, over-represented or under-represented GO terms were searched within the group of young paralog families Figure . Some teIt is now well supported that different modes of gene duplication result in the retention of different functional-types of genes . The balAccording to the preceding analysis, different mechanisms of gene duplication seemed to have operated in the apple genome, which agrees with the analysis of mixture densities in suggesting that both polyploidy and small-scale duplications may have had a prominent role in gene duplication; particularly after the origin of the Pyrinae lineage Figure . AlthougLhc gene family. Ten major types of highly abundant Lhc proteins have been identified: four associated with PSI (Lhca1 to 4) and six mainly associated with PSII (Lhcb1 to 6), although Lhcb1 and Lhcb2 can also be involved in PSI. Four additional forms  are encoded by genes expressed at very low levels, and their products are still poorly characterized [Arabidopsis and 23 genes in poplar encode Lhc family members [Light-harvesting complexes (LHC) of photosystem I (PSI) and II (PSII) contain a group of chlorophyll a/b binding proteins encoded by the cterized . Twenty- members .Lhc family suggested the existence of at least 27 members. The gene genealogy, including the genes from the tree species , suggested that the apple dataset contained more than one member for the 10 major types of Lhc proteins, whereas for the four Lhc genes expressed at low levels, only a single ortholog, that of Lhca6, was identified , which are contemporaries to the Pyrinae episode of polyploidy , thus favouring gene retention and ultimately creating the conditions for neofunctionalization ,61. By cr = 0.78) estimated by considering two genes as being significantly co-regulated (α = 0.05). This proportion of genes showing divergent patterns of gene expression seemed to be consistent with estimates made for other species with proportions usually ranging from 74% to 85% [Ks < 0.4) and their older counterparts  between paralogs  were all over-represented among young paralogs, a pattern of gene retention that seems to have had a role in the amplification of a number of gene families. The origin of the Pyrinae lineage through polyploidy may be somewhat related to this observation. Although the mechanisms by which polyploids develop phenotypic novelty are still largely unknown, it is well accepted that increased growth vigor is seen in polyploids, which is apparently a key aspect of success under natural conditions . A recen parents . InteresEriobotrya and Rhaphiolepis in the tropical forests of China, Indochina, and Malaysia illustrate this process within the subtribe Pyrinae [An alternative hypothesis may also relate the pattern of gene retention observed in the apple with the acclimatization to a seasonal habitat because extant temperate deciduous Pyrinae species likely descended from Tertiary tropical or subtropical ancestors. Fossil records date the origin of Pyrinae to the middle Eocene . At that Pyrinae . For the Pyrinae -72 and i Pyrinae  is well  Pyrinae . Further Pyrinae . Such an Pyrinae ,76.Another remarkable feature of gene duplication in the apple was the high proportion of paralogs showing divergent patterns of expression, in some cases by developing bias towards tissue-specific expression/suppression. This behaviour was found to be similar regardless of the age of paralog formation, which suggested evolution by subfunctionalization early after gene duplication. Extensive subfunctionalization may have set the basis for gene diversification and the development of novel gene functions. These might have been mechanisms that played a role in the acquisition of new traits specific to the apple or the Pyrinae lineage and thus merit further research. Recent experimental evidence has suggested that phenotypic novelty can evolve quickly through changes in the pattern of gene expression of duplicated genes ,77.With the advent of more information about the biology of model organisms, researchers are using comparative genomic approaches to make functional knowledge portable across species. Comparative genomics largely relies on the characterization of orthologs-those genes derived from a speciation event that are therefore likely candidates to develop conserved functions . The dynThis study provides for the first time a genome-wide characterization of the mode of gene duplication in the apple, a major fruit crop and representative of the subtribe Pyrinae—important taxonomic group of the Rosaceae family.EST sequences from 33 non-normalized libraries obtained from different tissues  between each pair of sequences was estimated using the maximum likelihood method of Goldman and Yang [Ks values >2 were discarded from further analysis as they may be associated with uncertainty due to saturation of substitutions [Ks values were computed five times, and the estimation showing the best likelihood score was retained for further analysis.For each pair of paralogs, their deduced protein sequences were aligned using ClustalW , and theand Yang  as impleand Yang  under thitutions . For eacKs = 0), one of the two sequences was discarded from the dataset as they were likely multiple entries of the same gene . Finally, redundant Ks values corresponding to the same duplication event within a gene family were omitted by computing mean values for each node of the gene family phylogenies. For this, gene families were constructed by using single-linkage clustering, and the gene topology was ordered based on Ks distances between paralogs.The dataset was cleaned according to the two criteria used by Blanc and Wolfe . First, Ks distribution were evaluated using the Kolmogorov-Smirnov test.Gene duplications in a genome can originate from a continuous mode of small-scale duplications and discrete episodes of large-scale duplications . As a consequence, the age distribution of gene duplications closely follows a mixture of an exponential distribution representing the constant birth and death process of small-scale duplications and normal distributions representing discrete bursts of duplicated genes derived from large-scale duplication events . To testGene families were constructed based on protein sequence similarity. For this, pairs of paralogs were grouped using a single-linkage clustering approach. Simple sequence counting data provided initial estimates of gene family sizes. These estimates were directly used to calculate the gene family size distribution. The unigene collection only partially represents the gene complement of the apple. To evaluate the extent to which gene family sizes could be biased due to differences in gene expression, I investigated any relationship between gene expression levels and gene copy-number  were selected from the whole dataset and were newly clustered into gene families following the same approach used when building the families from the whole dataset. Hierarchical reconstruction of gene families was used to estimate the proportion of gene duplications for each gene family in the whole dataset and in the group of young paralogs.To evaluate putative events of gene family amplification in the apple, the gene-copy numbers of families with more than five members were compared with the gene family sizes of A drawback associated with the analysis of ESTs is that, within a gene family, some unigenes may correspond to partial sequences, making it difficult to construct alignments of sufficient length for phylogenetic reconstruction. Thus, for phylogenetic analysis, unigene sequences were manually assembled de novo using all the apple EST resources available in dbEST. Deduced protein sequences were aligned using ClustalX. Protein sequence alignments were further inspected manually and were used as a reference for the alignment of their corresponding nucleotide sequences .A standard strategy for gene annotation was followed in this study as outlined by Götz et al. . Brieflyr) [r value below which expression divergence could be considered statistically significant was defined according to Blanc and Wolfe [r values calculated for 10,000 randomly selected gene pairs was constructed. The frequency distribution indicated that 95% of the randomly selected gene pairs had an r < 0.78. Thus, duplicated genes with r ≥ 0.78 were considered to be significantly co-regulated (α = 0.05). Conversely, paralogs with r < 0.78 were considered to have developed divergent patterns of gene expression.The expression profile for each unigene was obtained by evaluating its EST representation among 14 datasets derived from different tissues and/or developmental stages  , which gnd Wolfe . For thiTo evaluate tissue-specific expression, the 14 EST datasets were further classified according to the four tissue types represented . EST counting data for each unigene were extracted from the four datasets as described before, and Fisher's exact test was applied to a 2 × 2 contingency table where genes were assigned to rows and the tissue type to be tested and the bulk of all the other tissue types were assigned to columns.Custom Perl scripts and SQL database searches were used for dataset management and protocol automation.Unigene collection derived from assembling 119,177 apple ESTs cv. 'Royal Gala'.Click here for file'Royal Gala' apple EST libraries used in this study.Click here for fileApple gene families with more than five membersClick here for fileApple 20S proteasome gene family members.Click here for fileApple Chlorophyll a/b-binding protein gene family members.Click here for fileApple β-amilase gene family members.Click here for fileApple Eukariotic elongation factor 1 gene family members.Click here for fileApple Malate deshydrogenase gene family members.Click here for fileApple Glyceraldehyde 3-phosphate dehydrogenase gene family members.Click here for fileApple α-tubulin gene family members.Click here for fileApple S-adenosylmethionine synthetase gene family members.Click here for fileApple Vacuolar sorting receptor gene family members.Click here for fileApple 26S proteasome regulatory subunit (RPN8) gene family members.Click here for fileApple Starch phosphorylase gene family members.Click here for fileApple 40S ribosomal protein S5 gene family members.Click here for fileApple 40S ribosomal protein S4 gene family members.Click here for fileApple 60S ribosomal protein L12 gene family members.Click here for fileApple ADP-glucose pyrophosphorylase gene family members.Click here for fileApple Aconitate hydratase gene family members.Click here for fileApple GDP-mannose 3,5-epimerase gene family members.Click here for fileScatter-plot of Ks distance (synonymous substitution/synonymous sites) between pairs of duplicated genes, plotted against divergence in gene expression (Pearson's correlation coefficient).Click here for fileScatter-plot representing gene family size plotted against gene expression. (A) Individual values represent mean EST counts for each gene family. (B) Individual values represent raw EST counts for each unigene within each gene family.Click here for fileArabidopsis and poplar aligned nucleotide sequences, used for phylogenetic analysis of the Chlorophyll a/b-binding protein (Lhc) gene family.Apple, Click here for fileArabidopsis and poplar aligned nucleotide sequences, used for phylogenetic analysis of the α-tubulin gene family.Apple, Click here for fileArabidopsis and poplar aligned nucleotide sequences, used for phylogenetic analysis of the β-tubulin gene family.Apple, Click here for fileArabidopsis and poplar aligned nucleotide sequences, used for phylogenetic analysis of the 40S ribosomal protein S5 gene family.Apple, Click here for file