Smoking cessation is a key clinical intervention for reducing progressive lung destruction and lung function deterioration in patients with Chronic Obstructive Pulmonary Disease (COPD). Specialised Tobacco Cessation Guidelines for High-risk Groups (TOB-G) were developed and published in 2017 that present evidence-based recommendations to support smoking cessation in COPD patients. The purpose of this pilot study was to examine the real world effectiveness of the TOB-G guideline recommendations among a sample of COPD patients.A pilot study was conducted among a sample of COPD patients who smoke and were interested in quitting. Participants were recruited from inpatient and outpatient hospital admissions between October and December 2016 in Iasi, Romania. The intervention program was designed based on the recommendations of the TOB-G guidelines for COPD patients. Patients received a total of four contacts: at baseline, 1, 2, and 6 months. The primary outcome measure was biochemically validated point prevalence smoking abstinence measured at 6 months.Fifty patients  with diagnosed COPD took part in the pilot study. Self-reported rates of point prevalence smoking abstinence were 30.6%, 44.9% and 64.6% at the 1-, 2-, and 6-month follow-up, respectively. Carbon monoxide testing was completed with 51.6% of the sample at 6 months. The biochemically verified abstinent rate was 33.3% at the 6-month follow-up.This pilot testing of the TOB-G Clinical Practice Guidelines for COPD patients was associated with high rates of patient smoking abstinence, which are of clinical importance. Further research is needed to evaluate the guidelines large-scale effectiveness in clinical practice. Smoking cessation has been shown to improve both the prognosis9 and slow progression of the disease10, as well as reduce exacerbations of COPD and improve survival12. Respiratory symptoms  and immune response have been shown to improve within 3-9 months of cessation, and lung function may increase by 10%14. Improved efficacy of therapies, including oxygen therapy and COPD inhalator medication such as bronchodilators2 or inhaled corticosteroids15 are expected after quitting.Smoking cessation is the single most effective treatment for reducing the rate of COPD progression and for improving disease outcomes18. In particular, tobacco users with COPD have a history of smoking more cigarettes per day and are more highly addicted to nicotine compared to the general population of tobacco users18. COPD disease pattern that often includes the existence of co-morbidities  is associated with a lack of motivation and self-confidence for quitting, thus reducing the odds of quitting smoking20. Understanding these differences and tailoring cessation interventions may assist with increasing effectiveness of smoking cessation treatments among smokers with COPD21.There is emerging evidence that smokers with COPD have characteristics that may make it more difficult to quit smoking relative to the general population of tobacco users23. The TOB-G guidelines include a special chapter on tobacco treatment among patients with COPD with 16 evidence-based clinical practice recommendations  were developed with the aim to develop and implement an innovative and cost effective approach to prevent chronic diseases related to tobacco dependence by developing specialized tobacco cessation guidelines for populations at high riskndations . The guiUnderstanding the real world effectiveness of guidelines is key in supporting their dissemination. As such, we conducted a pilot study to examine the effectiveness of a cessation intervention based on the TOB-G guideline recommendations on cessation rates among a sample of patients with COPD.A single group non-randomized pilot study was conducted among a sample of COPD patients who smoke. Outcome measurement occurred at baseline, 1, 2, and 6 months. This study was approved by the Ethics Board of the Clinical Hospital of Pulmonary Diseases, Iasi, Romania (Protocol number 664292).The study sample was recruited from inpatient and outpatient visits to the Clinical Hospital of Pulmonary Diseases in Iasi Romania between October and December 2016. All COPD patients had their smoking status assessed and received brief advice to quit smoking and were invited to enrol in the pilot study. Eligible patients were: daily tobacco users (>5 cigarettes per day) with a clinical diagnosis of COPD, who were interested in making a quit attempt and had access to a telephone for follow-up. The TOB-G guidelines address both inpatient and outpatient populations and as such the study design included for recruitment of both patient populations. All eligible patients provided written informed consent and were scheduled for an appointment in the outpatient pulmonology clinic of the hospital. Fifty eligible COPD patients participated in the pilot study. The recruitment flow diagram is presented as 22.The intervention program was designed based on the evidence-based recommendations for tobacco treatment in the TOB-G clinical practice guidelines for COPD patients . The int1 measurement was taken and ‘lung age’ calculated and presented to patients. Each participant worked with a pulmonologist, trained in the 2017 evidence-based TOB-G smoking cessation guidelines, to develop a quit plan based on their individual smoking and personal history. A quit date was set in the next 7-14 days with all patients. The clinician provided patients with counselling  and reviewed personal triggers for tobacco use with the patients. The study intervention was counselling-based and while participants had the option to utilize first line pharmacotherapy  it was not provided by the study. Three follow-up sessions were delivered via telephone at 1, 2 and 6 months. For those patients who agreed to attend the in-person visit at the hospital, a CO evaluation was performed at 6 months. At each of the follow-up sessions, smoking status was assessed and the severity of cravings, withdrawal and risk of relapse were assessed. Patients were provided with brief counselling and asked to identify any risk of smoking relapse and to discuss strategies for addressing these high-risk situations.A standardized consult form was used to guide smoking cessation consultation and to document the consultations. At the initial smoking cessation consultation, the demographic characteristics, plus smoking related and medical history were documented. A carbon monoxide (CO) sample and FEV36. Patient smoking status was assessed at baseline, at 1 (±2 weeks), 2 (±2 weeks) and 6 months (±2 weeks). Point prevalence abstinence was defined as not having smoked in the previous 24 hours at each of the time points assessed. Continuous abstinence (weeks 1-26), defined as not having smoked at the 1-, 2- and 6-month follow-up was also reported. At the 6-month follow-up patients reporting they have quit smoking were asked to provide validation of smoking status using exhaled CO testing (cut-off level 10 ppm).The primary outcome measure was self-reported point prevalence smoking abstinence assessed using the Russell Standard26. The FTND consists of 6 questions scored on a scale of 1-10. A score 0-3 indicates no or low tobacco dependence, a score of 4-6 indicates moderate tobacco dependence, and a score of 7-10 is categorized as high tobacco dependence.Demographic characteristics  and medical history of patients were collected at baseline. Smoking history assessed pack-years (years of tobacco use x number of cigarettes per day), actual number of cigarettes per day, and years of smoking. The Fagerström Test for Nicotine Addiction (FTND) was used to assess level of nicotine addiction27. Deceased patients (n=2) were removed from the analysis. SPSS version 24.0 was used to analyse all data.Descriptive analysis was used to summarize the demographic characteristics of the sample. The proportion of participants who quit smoking was calculated for each follow-up time point. For any patients with missing data we assumed they had returned to active smoking as per Russell Standard1 30-50%), nicotine dependence as assessed by the FTND was consistently less than 7 (i.e. mild to moderate nicotine addiction). When we evaluated this parameter in this subgroup of Stage III patients, it was revealed that all these patients had the desire to smoke the first cigarette after awaking, much earlier than they could effectively manage to smoke. Patients reported that difficulty in breathing as their disease progressed and other severe COPD morning symptoms resulted in smoking fewer cigarettes per day and smoking their first cigarette later in the day than when they would have otherwise done so.Self-reported rates of smoking abstinence were 30.6%, 44.9% and 64,6% at the 1-, 2-, and 6-month follow-up, respectively . Continu25. Disease severity and COPD progression risk resulting from previous or continued smoking should be used as principal arguments to motivate cessation in COPD patients who smoke, as motivation and willingness to quit are important parameters of smoking cessation28.Within the context of this pilot study we found the implementation of the 2017 TOB-G tobacco treatment guideline recommendations within European clinical practice was associated with clinically important rates of point prevalence and continuous smoking abstinence among COPD patients sampled. Guideline recommendations were for the most part feasible to implement in clinical practice and acceptable to patients. We achieved high rates (100%) of follow-up contact with patients, providing evidence of the feasibility of the intervention. The pilot intervention integrated spirometry and ‘lung age’ as adjunct treatment strategies, perceived by clinicians involved in the study as a more feasible and tailored way to recommend smoking cessation among COPD, consistent with previous reports30. The TOB-G guidelines were developed to serve as a summary of the latest evidence-based practices for addressing tobacco use22. The guideline recommendations were informed by the available literature, clinical experience and vetted by a committee of experts to ensure their applicability to real world clinical settings.Clinical practice guidelines can have an important role in influencing professional practice and guiding the clinical treatment. These guidelines however are infrequently tested in terms of their feasibility, utility and efficacy in ‘real world’ clinical practice settings. Available guidelines for the management of COPD do not offer sufficient attention to evidence-based practices for addressing tobacco use in this important population of tobacco users32. The quit rates documented in this pilot study are somewhat higher than those reported by other investigations of COPD patients that have ranged from 4-34% at 6 months or at a longer follow-up33. A recent Cochrane Review examining smoking cessation for people with COPD found evidence that that a combination of behavioural treatment and pharmacotherapy is effective in helping smokers with COPD to quit smoking, but no evidence for any particular form of behavioural counselling or pharmacotherapy33. Authors did report some evidence that high-intensity behavioral treatment increased smoking abstinence compared with usual care  or low-intensity behavioral treatment 33. The present pilot study was primarily counseling based and included a total of 4 contacts. Higher quit rates may have been documented in the present study if pharmacotherapy was provided to participants. Despite this fact, overall quit rates documented are relatively high and offer preliminary evidence to support the ability of interventions tailored to COPD patients that are based on the TOB-G clinical practice recommendations to support cessation in this high-risk population of tobacco users. Additional research would be required to validate the findings of this initial pilot study.Others have also reported on the value of evidence-based guidelines in influencing tobacco treatment outcomes in clinical settings. Two evaluations of the United States Guidelines for Treating Tobacco Use and Dependence have been published and reported favourable outcomes in terms of efficacy in increasing cessation outcomes among the general population of tobacco users in primary care settings in both a pre-post evaluation and randomized controlled trial designOur study also found that patients with severe COPD were deterred from smoking in the morning and smoked fewer cigarettes per day as a result of their COPD-related symptoms. Given that time of first cigarette in the morning and number of cigarettes smoked per day are two key questions used by the FTND for assessing nicotine dependence, these suggest that this group of patients may have a much higher dependence to nicotine than the group we registered with the FTND. We hypothesize that the FTND’s reliability may not be as useful in assessing nicotine dependence for this category of patients, and more tailored smoking cessation approaches may be needed to accurately assess patients with severe COPD. This important practical learning from the TOB-G COPD pilot study warrants further examination.34. A significant proportion of the sample did not complete biochemical validation of smoking abstinence and were classified as smokers, so it is possible that these participants had quit but were unable or not interested in traveling to the hospital to provide validation. Other authors have reported on the challenges of compliance with biochemical validation in cessation studies27. Given that this was a single centre pilot study, future studies should examine the generalizability of the study findings to other settings and for larger samples of COPD patients. Finally, our study focused on the feasibility and effectiveness of the guidelines on COPD patients who are ready to quit smoking. We did not examine interventions to increase motivation to quit among patients not ready to quit, and we acknowledge that this is an important target for addressing tobacco use among the population of COPD patients35.Limitations of our study include the use of a single group non-randomized design. The lack of a control group means that we are unable to confirm a causal relationship between the intervention and changes in smoking outcomes, and it is possible that some participants would have quit smoking without the support of the intervention program tested. Available literature suggests that the natural quit rate among COPD patients involved in clinical studies ranges between 2-9%, indicating that the intervention program was associated with significantly higher rates of smoking abstinence than expected for an unaided quit attemptThe TOB-G tobacco treatment guidelines for COPD patients were feasible to implement in a ‘real world’ clinical practice setting in Europe and implementation was associated with high rates of patient smoking abstinence compared to that reported in the literature for COPD patients who smoke. The pilot study results will be used to further refine the guidelines to assist with maximizing uptake in clinical settings.


This study cross-sectionally examines in the elderly population: (a) the association of type 2 diabetes with executive function (EF); (b) the effect of BMI on both type 2 diabetes and EF; (c) the association between glycaemia control and EF in type 2 diabetes. 6823 older individuals with overweight/obesity and metabolic syndrome participating in the PREDIMED-PLUS study, were assessed with a battery of cognitive tests and a medical interview. ANOVA showed a significantly worse performance on EF in type 2 diabetes vs. non-diabetic individuals. Two complementary models were displayed: (1) in the whole sample, the presence of type 2 diabetes, depressive symptoms and BMI had a direct negative effect on EF, while apnoea had an indirect negative effect; (2) in the diabetes subsample, higher illness duration was associated with worse performance in EF. Participants with type 2 diabetes and HbA1c<53 mmol/mol displayed better cognitive performance when compared to those with HbA1c≥53 mmol/mol. Our results provide a controlled comprehensive model that integrates relevant neuropsychological and physical variables in type 2 diabetes. The model suggests that, to improve treatment adherence and quality of life once diabetes has been diagnosed, cognitive decline prevention strategies need to be implemented while monitoring depressive symptoms, BMI and glycaemia control. This is one of the most frequent diseases among the elderly individuals, with studies reporting a prevalence ranging from 19% to 33% in general population3. With global population ageing at significant pace and given the high prevalence of type 2 diabetes among the elderly, the negative effects of this chronic metabolic disease on health and cognitive functioning are of notable clinical interest.Type 2 diabetes is caused by a combination of resistance to insulin action and an inadequate compensatory insulin secretory response5. Similarly, when exploring other cohorts of elderly population (>65) with normal cognition, it was confirmed that type 2 diabetes participants obtained lower scores than non-diabetic patients in some cognitive domains such as attention, psychomotor function, information-processing speed and executive function, but they did not differ in memory7.With this regards, the main cognitive domains that have been related to type 2 diabetes are the following: attention, memory, processing speed and executive function , as well as global cognitive functioning. When examining cognitive impairment in older individuals (>60 years), results confirmed that patients with type 2 diabetes in comparison with those without type 2 diabetes displayed higher impairment in global cognitive function, working memory and cognitive flexibility8. Similarly, other longitudinal studies concluded that older women (>65) with type 2 diabetes experienced an accelerated cognitive decline in general functioning and other specific domains, including visual and working memory as well as verbal fluency10. In addition, research in old population exploring the duration of type 2 diabetes point towards an association between the duration of this metabolic disease and a poorer performance in verbal memory and concept formation11 as well as in general cognition10. A longer duration of type 2 diabetes together with an earlier onset, has also been reported to increase the risk of mild cognitive impairment development12.Different prospective studies have analyzed the association between the presence of type 2 diabetes and the risk of cognitive dysfunction. In older adults (42–89 years), results showed that women with type 2 diabetes had an increased risk of major cognitive decline in verbal fluency after 4 years when compared with non-diabetic women13. Some findings show that both high BMI and obesity are associated with a worsened performance in measures associated with EF, working memory, short-term memory and verbal fluency16.The increasing number of individuals suffering from type 2 diabetes among older people during the last few decades might be due to inappropriate dietary habits and a sedentary lifestyle, which lead to overweight and obesity. An elevated BMI may cause insulin resistance and it is common in adults with type 2 diabetes17. Nevertheless, other aspects seem to be relevant when examining the negative effects of type 2 diabetes on cognitive performance. In this line, different studies report that gender8, low glycaemia control19 as well as the presence of depressive symptoms20 are associated with worse cognitive performance in type 2 diabetes individuals. It has also to be note that sleep disorders can have an impact on cognition, especially in older age population at higher risk of cognitive decline21.Finally, type 2 diabetes has been associated with cognitive dysfunction regardless of ethnicity and cultural factorsIn light of previous studies and in order to specify the association between the presence of type 2 diabetes and cognitive decline in older individuals (above 55 years of age) with overweight/obesity and metabolic syndrome, the objectives of this study are: (a) to explore the associations of type 2 diabetes with cognitive performance (i.e.: EF); (b) to evaluate the effect of BMI on type 2 diabetes and cognitive performance when controlling for other confounding variables ; (c) to explore the association between glycaemia control and cognitive function performance in individuals with type 2 diabetes.http://predimedplus.com/wp-content/uploads/2016/07/Protocolo_PREDIMED_PLUS_eng_23112016_adl.pdf. This study was registered at the International Standard Randomized Controlled Trial  with number 89898870. All participants were recruited and randomized from October 2013 to December 2017 across 23 centres from different universities, hospitals and research institutes of Spain. Each of these centres recruited participants from several Primary Care Health Facilities belonging to the National Health System. The eligible participants were community-dwelling adults  with overweight/obesity (BMI between 27 and 40 kg/m2), who met at least three components of the metabolic syndrome according to the updated harmonized criteria of the International Diabetes Federation and the American Heart Association and National Heart, Lung and Blood Institute22. Specific PREDIMED-plus exclusion criteria can be found in the website http://predimedplus.com/en. Participants included in the current analysis provided data on cognitive performance and depressive symptoms, as well as detailed specification of BMI, physical conditions and sociodemographic data. Additionally, glucose and HbA1c concentrations were measured at the same time point of the mood and cognitive assessment. All participants provided written informed consent, and the study protocol and procedures were approved according to the ethical standards of the Declaration of Helsinki by the Research Ethics Committees from all the participating institutions (Supplementary Info 1).This is a cross-sectional analysis on baseline data within the frame of the PREDIMED-Plus study, a 6-year multicenter, randomized, parallel-group, primary prevention clinical trial conducted in Spain to assess the effect of an intensive weight loss intervention program based on an energy-restricted traditional Mediterranean diet, physical activity promotion and behavioural support (intervention group), compared to a usual care intervention and energy-unrestricted Mediterranean diet recommendations (control group). A more detailed description of the PREDIMED-plus study and the study protocol is available at Exclusion criteria for the present analysis were: (1) reported dementia (2) head trauma, learning disability or intellectual disabilities; (3) presence of depression or a lifetime mental disorder according to DSM-5. Finally, all the included participants presented glucose levels within the normal range during the neuropsychological assessment.All participants completed a self-report questionnaire for exploring depressive symptoms as well as a set of neuropsychological tests for assessing different cognitive domains that included attention, short term memory, verbal fluency, cognitive flexibility, visuospatial skills and working memory. Sociodemographic information was also collected by means of a self-reported questionnaire and a hetero-administered clinical interview assessing medical conditions .23 is a 21-item self-report measure for assessing the severity of depressive symptoms in adults and adolescents (ages from 13 to 80 years). The BDI-II reflects the diagnostic criteria for Major Depressive Disorder listed in the Diagnostic and Statistical Manual of Mental Disorders (4th ed.)24. Scores for each item range from 0 to 3; the total score is the sum of all responses. The Cronbach’s alpha in our sample shows a good internal consistency (α = 0.883).-The Beck Depression Inventory–II (BDI-II)25 consists of 25 circles spread out over two sheets of paper (parts A and B). In the part A, participants are requested to connect consecutive numbers (1–2–3–4-…) in the correct order by drawing a line. In the part B, they have to connect consecutive numbers and letters in an alternating numeric and alphabetic sequence . Each part is scored according to the time spent to complete the task (lower score mean better performance). TMT is frequently used to assess executive function. More specifically, TMT-A is used for measuring attention, psychomotor speed and visuospatial skills; while, TMT-B is used for measuring divided attention, cognitive flexibility and interference.The Trail Making Test (TMT)26 assess verbal ability and executive control. Participants need to retrieve words of their language by accessing their mental lexicon, but also focus on the task and avoid repetition that involves executive control processes. In the phonemic fluency task, participants are requested to produce as many words as possible that start with the letter P during 60 seconds. Participants were asked to avoid producing names of people or places and repetitions of the same word with a different suffix. Secondly, in the semantic fluency task, participants have to name as many different animals as they can without repeating them during 60 seconds. The total raw score for each of the tasks corresponds to the number of words the participant achieves to say.The Verbal Fluency Test27 is compound of two different subtests: DS Forward, considered as a test of attention and short-term memory capacity; and DS Backward recall, considered as a test of working memory capacity. The DS Forward recall requires participants to repeat orally a series of random single digits in the same order they heard them; the sequence of digits varies in length from three to nine. Contrary, in the DS Backward recall participants are asked to repeat a series of random single digits in reverse order; the sequence varies from two to eight. The performance on the DS can be reported by a direct score of the backward performance (ranging from 1 to 16) and the forward performance (ranging from 1 to 14) and, by the span of immediate recall (backward span range from 1 to 8 and forward span range from 1 to 7).The Digit Span (DS) test of the Wechsler Adult Intelligence Scale-III (Spanish version)1.Weight and height were measured with light clothing and no shoes with calibrated scales and a wall-mounted stadiometer, respectively. BMI was calculated as weight in kilograms divided by the square of height in meters. Serum glucose levels were measured using standard enzymatic methods. HbA1c was measured by standard routine methods and glycaemia control was specified (HbA1c<53 mmol/mol [7%])d coefficients measured the effect size for the mean comparisons 28. The Finner’s method, a procedure included into the Familywise error rate stepwise procedures which offers a more powerful test than the classical Bonferroni correction29, was used to control Type-I error due to multiple comparisons. The same method was used to explore the association between glycaemia control (HbA1c<53 mmol/mol [7%]) and cognitive function performance in the subsample of participants who presented type 2 diabetes.Analyses were carried out with Stata15 for Windows. We used the study baseline database generated in August 2017. The comparisons of the cognitive measures between participants with and without type 2 diabetes was done with ANOVA procedures adjusted for the individuals’ sex, age, years of school attendance and presence of sleep apnoea syndrome. Cohen’s-30. The Path analysis used in this study was implemented through Structural Equation Modelling (SEM) with exploratory aims, with the advantage  to allow the inclusion of multiple relationships among a set of variables, including mediational associations. The maximum-likelihood estimation method of parameter estimation was used and goodness-of-fit was evaluated using standard statistical measures31: the root mean square error of approximation (RMSEA), Bentler’s Comparative Fit Index (CFI), the Tucker-Lewis Index (TLI), and the standardized root mean square residual (SRMR). Adequate model fit was considered if the following criteria were met: RMSEA< 0.08, TLI> 0.9, CFI> 0.9 and SRMR< 0.1; the global predictive capacity of the model was measured by the coefficient of determination. Separate models were obtained for the whole sample  and for the type 2 diabetes sub-sample .The underlying mechanisms between BMI, type 2 diabetes and cognitive measures were examined with path-analysis modelling, a straightforward extension of multiple regression techniques that can be used for both confirmatory and exploratory modelling. The aim of this procedure is to estimate the magnitude and significance of hypothesized connections/relationships into a set of variables, and so they allow to theory testing and theory developmentThe initial sample was constituted of 6865 participants. According to the exclusion criteria of this study, for the present analyses forty nine participants were excluded for presenting hypo or hyperglycaemia during the cognitive assessment . The final sample for the present analysis consisted of 6823 participants  distributed as follow: 4964 with no type 2 diabetes and 1859 with reported type 2 diabetes. At the moment of the assessment most of the patients with type 2 diabetes had presented this affection for more than 5 years (55.4%), some of them during 1 to 5 years (32.2%) and a 10.4% of them were only diagnosed during the last year  and for the subsample with type 2 diabetes . Both models included a latent variable (labelled cognitive) which was defined by the cognitive scores analyzed in this study . Goodness-of-fit was obtained for both models, as well as very good global predictive capacity. Supplementary Info S2 and S3 contain the complete results of the path-analyses, including the parameters estimating the direct, indirect and total effects. contain the complete results of the path-analyses, including the parameters estimating the direct, indirect and total effects.Figure In the whole sample, the presence of type 2 diabetes was predictive of worse cognitive functioning (direct negative effect); while the BMI directly contributed on the cognitive performance (as high the BMI as poor the cognitive performance). In the subsample with type 2 diabetes, the higher duration of this metabolic condition was predictive of a lower cognitive functioning (direct effect), while BMI did not contributed to (with direct or indirect effects) on the cognitive levels.Regarding the remaining variables included in the SEM, for both models better cognitive functioning was directly related to male sex, younger age and higher education. The apnoea achieved indirect effects on the cognitive performance only in the whole sample, through one path-way: the presence of apnoea was directly related to type 2 diabetes, which was also a predictor of poor cognitive execution. No direct effect between the presence of apnoea and cognitive performance emerged in any of the SEM.As a synthesis of the results of the study, Figure The present study examined the effect of type 2 diabetes on cognitive performance specifically with EF tasks, in a large cohort of 6823 patients above 55 years of age. Additionally, the role that BMI, apnoea, age, sex, depressive symptoms and education play in these effects was also explored or controlled when appropriate. Mainly, results display worse cognitive functioning when participants present type 2 diabetes, with a larger effect on those who have a longer duration of this metabolic condition. Additionally, some other variables are also found to directly or indirectly worsen cognitive function among this population, namely BMI, sex, depressive symptoms and education.33; taken together, our results add some extra evidence to previous findings and point towards the need to target these neurocognitive domains once type 2 diabetes has been diagnosed and its treatment protocol implemented. In an attempt to further explore the underlying mechanisms of the reported association between type 2 diabetes and cognitive performance, the present study sought to step further, by exploring the association of glycated haemoglobin with the neuropsychological measures in the subsample with type 2 diabetes. Previous studies have found that individuals with a weighted mean HbA1c less than 53 mmol/mol (7%) performed significantly better on tests of motor speed and psychomotor efficiency than those subjects whose time weighted mean HbAlc was greater34. In our study, individuals with type 2 diabetes and HbA1c<53 mmol/mol (7%) displayed better cognitive function when compared to those with HbA1c≥53 mmol/mol (7%), specifically on verbal fluency and working memory which highlights the importance of maintain good glycaemia control in this population which is already at risk of cognitive decline.Within the first analysis when comparing patients presenting type 2 diabetes with individuals without type 2 diabetes and controlling for other co-related variables that could interfere with the cognitive assessment , results show a significantly worse cognitive performance on EF when type 2 diabetes is present. Thus, the presence of type 2 diabetes in patients above 55 years of age seems to have a clear impact on cognitive function which would not be explained by the education, age, sex or the presence of apnoea. In the same line, previous studies have also reported that type 2 diabetes has a negative impact on cognitive function and physical health35. Our findings with the whole sample model give extra evidence on the negative impact of higher BMI on cognitive performance with this older age sample population. Additionally, higher BMI also negatively affects the participants’ mood in our sample. In turn, the lower mood in our sample is linked to poorer cognitive performance, which has also been reported in another study conducted with clinically depressed individuals36. Thus, health programs should give special emphasis in promoting weight loss within these individuals as well as strategies to monitor and improve mood when needed. On the contrary, in our older age sample with overweight/obesity, BMI differences are not significant enough to be robustly linked to the presence or duration of type 2 diabetes. However, BMI is described as a risk factor for the occurrence of type 2 diabetes in older age population37. Thus, the results observed regarding the influence of BMI on type 2 diabetes may most certainly only be circumscribed to our sample.Additionally, we sought to assess the associations of the presence of type 2 diabetes and its duration, cognitive performance and BMI in a model that also took into account other variables reported to be associated with cognitive performance . As a result of this analysis, two complementary different models were obtained. These two models provide relevant information about the interaction and contribution of the described variables to the cognitive performance of population suffering from type 2 diabetes, which can help improving the current understanding and treatment of this affection. Firstly, it is important to note that, as expected, type 2 diabetes and a higher duration of this metabolic condition was associated with worse cognitive performance. Consequently, it seems highly important to implement cognitive stimulation protocols within the treatment of type 2 diabetes that could help to reduce the progression of the cognitive decline once type 2 diabetes has been diagnosed. Secondly, a high BMI has previously been linked to specific cognitive impairments in different sample populations, especially in extreme weight conditions21, in our sample sleep apnoea (which unsurprisingly presents an association with higher BMI) is only linked to cognitive decline through its role in type 2 diabetes. Finally, it is also relevant that as reported in older adult populations a better cognitive function is directly related to males, younger age and higher education39.With regards to the presence of sleep disturbances/disorders and cognitive declineThe present study should be considered under some limitations. Firstly, the cross-sectional design does not imply causality. Also, given that our study participants are senior adults with a metabolic syndrome the present findings cannot be extrapolated to other population groups. However, it is one of the strengths of this study that it is conducted with a large sample of men and women and analyses are adjusted for potential confounding factors. Thus, although the results cannot be generalizable to other populations, they are representative of individuals over 55 years of age who suffer from a metabolic syndrome. Future studies should further explore the associations reported regarding the presence of type 2 diabetes, cognitive decline and BMI in different sample populations. Cognitive dysfunction is of particular importance because of its impact on quality of life. Future studies should test if an early intervention for improving cognitive deficits as well as depressive symptoms would help to reduce de cognitive decline and to improve the type 2 diabetes treatment adherences as well as self-care and quality of life.To conclude, in this cross-sectional assessment type 2 diabetes (including illness duration), high BMI and lower mood was linked to poorer cognitive function in older individuals with overweight/obesity and metabolic syndrome. A good glycaemia control in type 2 diabetes was associated with a better cognitive performance. Our results provide a controlled comprehensive model that integrates different neuropsychological and physical variables that are relevant in type 2 diabetes. The model also suggests that, to improve treatment adherence and quality of life once type 2 diabetes has been diagnosed, cognitive decline prevention strategies need to be implemented while closely monitoring depressive symptoms, BMI and glycaemia control. However, the etiopathogenic factors associated with the reported associations are still unclear.Supplementary Information


During development cells become restricted in their differentiation potential by repressing alternative cell fates, and the Polycomb complex plays a crucial role in this process. However, how alternative fate genes are lineage-specifically silenced is unclear. We studied Ultrabithorax (Ubx), a multi-lineage transcription factor of the Hox class, in two tissue lineages using sorted nuclei and interfered with Ubx in mesodermal cells. We find that depletion of Ubx leads to the de-repression of genes normally expressed in other lineages. Ubx silences expression of alternative fate genes by retaining the Polycomb Group protein Pleiohomeotic at Ubx targeted genomic regions, thereby stabilizing repressive chromatin marks in a lineage-dependent manner. Our study demonstrates that Ubx stabilizes lineage choice by suppressing the multipotency encoded in the genome via its interaction with Pho. This mechanism may explain why the Hox code is maintained throughout the lifecycle, since it could set a block to transdifferentiation in adult cells. Multicellular animals owe their complexity to their capacity to produce and maintain different lineages composed of multiple cell types that share virtually the same genomic DNA. Such complexity requires a tight regulation of gene expression to unambiguously specify and constrain the developmental path taken by cells within and between different lineages. Cell fates are controlled by networks of transcription factors (TFs) that act in the context of the genomic chromatin state to activate transcriptional programs realizing the distinct properties of cells of a given lineage . The forHox genes that are selectively expressed in adult VM-MSCs  axis of bilaterian animals. Importantly, the famous and well-known identity switch of whole body parts, the homeotic transformation, which is induced by altered Hox expression , shows t VM-MSCs . HoweverOne major bottleneck in this direction is that genome-wide Hox chromatin binding studies have been performed so far mainly in cell culture systems , specialDrosophila embryos and by interfering with Ubx function specifically in the mesoderm lineage that is specified and fully committed to the mesodermal fate. To this end, we generated a GFP-Ubx gene fusion at the endogenous locus using CRISPR-Cas9 and homologous recombination. We show that Ubx is a key regulator of lineage development and diversification, as it controls the mesodermal and neuronal transcriptional programs with high specificity despite interacting with many genes in both lineages. Intriguingly, our study demonstrates that Ubx controls lineage specification and differentiation not primarily by activating lineage-specific gene programs but to a large extent by restricting cellular plasticity within each lineage. In the mesoderm Ubx executes this function by silencing alternative fate genes, and we show that this repression requires the interaction of Ubx with the Polycomb Group (PcG) protein Pleiohomeotic (Pho) on co-bound chromatin sites. We furthermore find that Ubx stabilizes Pho binding to Ubx targeted genomic regions, and that this interaction is critical for gene repression by controlling the balance of H3K27me3 as well as H3K27ac at these sites. Taken together, our study demonstrates that Hox TFs control not only the segmental identity but also the developmental programs intrinsic to each lineage with high precision, and that one of the prominent functions of this multi-lineage TF family is the repression of alternative lineage programs, which restricts the cellular potential in a lineage-specific manner.Here, we probe the Hox TF Ubx in the mesodermal and neuronal lineages using sorted nuclei of Drosophila embryogenesis. To this end, we used the isolation of nuclei tagged in specific cell types (INTACT) method (E. coli Biotin ligase (BirA) in the mesoderm via the pan-mesodermal twist (twi)  almost exclusively in the mesodermal collection (eagle (eg) and elav were found highly enriched in the neuronal fraction only  method  by inducst (twi)  or the pon (elav) regulatoelopment , resulteelopment . Mesoderllection , while Eion only .10-13; r = 0.53 for stages14-17)  (10-13 + stages14-17)  using INTACT-sorted mesodermal or neuronal nuclei obtained from stage 10 to 13 (4–9 hr after egg lay (AEL)) and stage 14 to 17 embryos (10–18 hr AEL). Robust and reproducible data were obtained for all samples in biological duplicates. In total, transcripts (containing >5 RPKM) corresponding to 5652 coding genes were identified for the early mesodermal, 5716 coding genes for late mesodermal, 6752 coding genes for the early neuronal and 6182 coding genes for the late neuronal nuclei populations, which included genes typical for the lineage type and developmental stage . Pearsones14-17) . This waes14-17) . Importa<5.3e-5) . In addies14-17) . Tissue-es14-17) . PCA anaes14-17) , which wes14-17) .6 INTACT-sorted mesodermal and neuronal nuclei and an Ubx specific antibody generated and verified in the lab . The sequencing was performed in biological duplicates and Pearson correlation coefficient analysis revealed a high similarity of the samples (r = 0.94–0.96). The data were benchmarked by the identification of Ubx binding to known target loci, including the well-characterized interaction of Ubx with the decapentaplegic (dpp) (β-Tubulin at 60D (βTub60D)  and βTub60D in early and tin (tin), α-Actinin (Actn) and Tropomyosin 1 (Tm1) in late mesodermal nuclei, while in early neuronal nuclei deadpan (dpn), huckebein (hkb) and Neurotrophin 1 (NT1) in late neuronal nuclei Neuroglian (Nrg), target of PoxN (tap) and castor (cas) was among the Ubx bound active genes. In contrast, a large portion of the Ubx chromatin interactions (64% to 73%) were close to inactive genes in the two lineages   using 1 × 10ic (dpp)    and β-TuβTub60D)    enhancerβTub60D) , showingβTub60D)  and whicβTub60D) , targeteβTub60D) . To resoβTub60D) , with moβTub60D) . This inlineages  While itlineages , the hig.004e-9) , while U.004e-9) –2D. For Drosophila genome (dm3). This resulted as expected in the identification of substantially fewer binding events  lineages. In addition, our analysis revealed that a substantial fraction of Ubx interactions were found in the vicinity of inactive genes, which encoded many mesoderm-unrelated functions. Thus, we hypothesized that one important function of Ubx in tissue development could be the repression of alternative transcriptional programs, which instruct the development of other lineages.Ubx gene using the Clustered Regularly Interspaced Short Palindromic Repeats-associated Cas9 (CRISPR/Cas9)-mediated genome engineering -GAL4 driver, which resulted in a strong reduction of GFP-Ubx protein levels , ventral veins lacking (vvl), Neurotrophin 1 (NT1) and myospheroid (mys), while 1452 genes exhibited reduced expression, including βTub60D, Ankyrin 2 (Ank2) and Notchless (Nle) (14-17 UbxDegrad) or presence (mesoderm14-17) of Ubx were substantially different  . PCA anaifferent . In totaifferent , while uifferent . To haveifferent . For exaifferent , supportifferent . Consistifferent .Intriguingly, we found differentiation processes to be enriched among the genes up-regulated in the absence of Ubx . AnalysiTaken together, these results showed that Ubx controlled a large number of genes encoding diverse functions. The strong correlation between Ubx binding with specifically repressed or activated classes of genes suggested that Ubx indeed exerts a dominant influence over the mesodermal transcriptome at the two stages analysed. In addition, this analysis highlighted that Ubx repressed many genes controlling the establishment of non-mesodermal, alternative lineages, indicating that Ubx could have a pivotal role in restricting the developmental potential of cells and tissues, thereby ensuring that lineages adopt a unique identity.One intriguing of this study finding was the high number of genes repressed by Ubx, which encoded primarily functions related to other lineages . As thisIn a next step, we asked how the epigenetic landscape changed in the absence of Ubx, in particular at genomic regions with overlapping Ubx and repressive H3K27me3 marks. To this end, we performed ChIP-seq experiments in biological triplicates, the two most similar duplicates were used for further analysis. In total, 1216 H3K27me3 peaks and 1480 H3K27ac peaks located in gene regulatory regions, which corresponded to 2768 genes, changed their chromatin state in the absence of Ubx, as they experienced either a reduction/loss or an increase/gain in tri-methylation/acetylation at lysine 27 of histone 3 . ImportaOne result that puzzled us was that the majority of genes primed for activation (894 of 1058) remained silent on the RNA level when Ubx was lineage-specifically degraded . In ordeIn sum, these results showed that Ubx mediated the repression of genes encoding non-mesodermal functions by controlling the epigenetic status, in particular H3K27me3, at Ubx bound chromatin sites located in enhancers.in cellulo using Drosophila S2R + cells transfected with tagged versions of Ubx and Pho as well as in vivo using GFP-Ubx embryos , a TALE class homeobox TF functioning as Hox cofactor in invertebrates and vertebrates , and Tri embryos . This re embryos . We foun embryos  and 6A,  embryos , 66% , a NK2 class TF encoding gene critical for patterning of the neuroectoderm as well as the formation and specification of ventral neuroblasts , a cytokine encoding a gene that regulates motor neuron survival and axon guidance, hamlet (ham), a gene encoding a PRDM class TF that regulates neuron fate selection in the peripheral nervous system , Olig family (Oli), bendless (ben), myospheroid (mys) and medial glomeruli (meigo). Strikingly, all five Ubx/Pho/H3K27me3 loci, which were co-bound by Ubx and Pho in the presence of Ubx  of the genes up-regulated in the absence of Ubx, which were highly enriched for non-mesodermal and stem cell related functions, to be located in the vicinity of Ubx/Pho binding regions . In controblasts , HGTX, a muscles , Neurotrs system , and Ptx muscles . In addie of Ubx , experiee of Ubx . In conte of Ubx . As Pho e of Ubx , we conc embryos  to be si embryos  as well  embryos . It has  embryos , and conl nuclei . Importa3 region . In conte of Ubx . These rIn a next step, we asked whether stabilization of Pho binding and maintenance of H3K27me3 and H3K27ac levels are generally dependent on Ubx. To this end, we performed ChIP-seq experiments in biological triplicates using a Pho antibody  and INTAIn sum, these results demonstrated that Ubx stabilized Pho binding to chromatin regions at alternative fate genes in the mesodermal lineage, which controlled the proper levels of H3K27me3 and H3K27ac marks, thereby ensuring repression of these genes.Drosophila development using a comparative genomic approach and an experimental system to deplete Ubx protein exclusively in the embryonic mesodermal lineage. This approach allowed us to dissect the cell-autonomous function of Ubx in a single lineage that was located in an otherwise normal cellular environment at the transcriptome and chromatin level. Using this experimental set-up, we found that Ubx comprehensively orchestrates the transcriptional programs of the mesodermal as well as of the neuronal lineage, as it bound and regulated a substantially fraction of genes specifically expressed in these tissue lineages. Strikingly, this analysis revealed that the majority of Ubx chromatin interactions were located in the vicinity of inactive genes, and lineage-specific interference with Ubx in the mesoderm demonstrated that about 20% of these interactions were important for repressing the close-by genes. Intriguingly, these genes were highly enriched for alternative cell fates, demonstrating that Ubx had indeed a pivotal role in restricting the developmental potential of cell and tissue types in a context-dependent manner.Cell and tissue types get different during development and their identity needs to be maintained also in adulthood to guarantee the survival of organisms. Here, we provide evidence that multi-lineage TFs of the Hox class stabilize the different lineage choices by restricting cellular plasticity in a lineage specific manner. To this end, we studied the broadly expressed Hox TF Ubx in the mesodermal and neuronal lineages during Hox gene Antennapedia (Antp), which is normally expressed anterior to Ubx, is ectopically activated when Ubx function is absent , mediates together with the p300/CREB-binding protein (CBP) the deposition of H3K27ac marks  enriched among the sites, which are bound by Ubx and marked by H3K27me3 in the mesodermal lineage, while in the neuronal lineage the Ventral nervous system defective (Vnd) motif is frequently found in Ubx/H3K27me3 sites. Motifs for tissue-specific TFs are also detected at active sites, as Ubx/H3K27ac regions in the mesoderm have the Slouch (Slou) motif enriched, while in the nervous system it is the motif for Intermediate neuroblasts defective (Ind). Thus, we assume these factors to be critical in determining the lineage-specific output . In futuHox genes can effectively induce cell type specific transcriptional programs in multi-potent cells thereby unambiguously inducing their differentiation. This is in line with our findings showing that Hox TFs activate lineage-specific transcriptional profiles with high precision. However, so far it is unclear why the conversion of somatic cells of one type into another one is so difficult. Our data now suggest that one reason for this hurdle could be the Hox encoded restriction of cellular plasticity via the repression of alternative gene programs using Polycomb based epigenetic mechanisms.One peculiar feature of Hox TFs is that they are not only active during the development but their input is continuously required throughout the lifetime of an organism to assess the positional values of cells and to maintain their proper identities and functions . Our fintwi-INTACT and UAS-INTACT (gift from the Henikoff lab). The UAS-INTACT was crossed in the elav-GAL4 background to generate elav-GAL4;;UAS-INTACT stable lines. For the degrade GFP experiments (arm-GAL4 (BL1561) Mef2-GAL4 (BL50742), UAS-TEV-P14 . UAS-Nslmb-vhhGFP4 (BL38419), the 3.005GFP-Ubx line was recombined with the UAS-Nslmb-vhhGFP4 and Mef2-GAL4 lines and crossed in the twi-INTACT background to generate 3.005twi-INTACT;Mef2-GAL4,GFP-Ubx and 3.005twi-INTACT; UAS-Nslmb-vhhGFP4,GFP-Ubx. Embryos from Ubx deGradFP experiments were collected at 10–18 hr AEL to ensure the knockdown of the GFP-Ubx protein. The 81A;pho1pho-like douple mutant was generously provided by Jürg Müller  as injection line and the resulting progenies (F0) were crossed with TM3/TM6 balancers and resulting F1 was used for single crosses against TM3/TM6 balancers to generate independent stocks. The F1 generation was screened by PCR for the presence of the GFP and the GFP containing stocks of the F2 generation were visually screened for the Ubx patterned GFP expression in vivo.The 9 system . For the9 system  containiHI and XhoI restriction site. The protein was purified according to the protocol (GElifesciences) and eluted by using the PreScission Protease site. The immunisation and antibody handling were performed by the Charles Rivers company.The Ubx antibody was generated using the pGEX-purification system (GElifesciences). The open reading of Ubx-RA was cloned in the pGEX-6P-2 vector using Bam2-382 (generously provided by J. Müller) and the homemade guanine pig Ubx antibody (gp-Ubx). Total RNA was isolated with TRIZol (Invitrogen) and DNA digest was performed with the TURBO DNA-free Kit (Ambion). The material was analysis or validated by qPCR (Invitrogen Syber-Green-Mix) (The nuclei were purified as described in een-Mix) , 3. Genobowtie2 --very-sensitive -x Bowtie2Index/genome sequence.txt -S output.sam  1) The authors used 10 RPKM as their threshold to distinguish active from inactive genes. This is a rather high threshold, and so one is left to wonder what the consequence would be on the downstream analyses/results if the more standard threshold of 1 RPKM would be applied?We thank the reviewer for this comment. The usage of such a stringent threshold was based on previous studies, which had used similar RPKMs, in particular when studying tissue-specific gene expression . In addition, other studies using the INTACT procedure and thus profiling nuclear and not whole cell RNAs used a similar stringent threshold to reduce the false positive recovery rate . We do understand the reviewer’s concerns, thus we have re-analyzed the data using a threshold of >5 RPKM, which is frequently used . As the reviewer will appreciate, this does not change the results and the main take home message of the manuscript.2) The authors show that Ubx binds to a very large number of genes  can be considered to be Ubx targets). This raises a couple of questions:a) In Figure 2B, the number of Ubx-bound genes in neuronal tissue at stages 14-17 is much lower than for the other three conditions. What could be the underlying reason?b) In Figure 2G, the authors show that there is a high correlation in higher order GO terms between the lineage specific transcriptional and Ubx binding profiles. As a control, the authors included the TF Tinman, which revealed a more distinct GO term enrichment profile. However, since Ubx binds to such a large number of genes, is it not entirely expected that the majority of active genes will also be Ubx targets, and thus yield the same GO term enrichment profile? The same holds true for the inactive genes which also fall mostly within the "Ubx target space" and would thus again yield the same profile. That Tin does not yield the same profile is also not surprising given that it binds much fewer genes so the overlap between (in)active genes and Tin gene targets will be much smaller. A better control (if available) would in this regard be a TF that, similar to Ubx, binds to a large number (10 K) of genes. In its current configuration however, this analysis is not very informative. Similar concerns apply to the statements "Strikingly, 85% (1227/1452) of the genes with reduced and 90% (1299/1393) of the genes with increased expression were bound by Ubx in mesodermal nuclei in wild-type embryos, implying that most of the expression changes were a direct consequence of altered Ubx chromatin interactions". Again, since >70% of Drosophila genes are Ubx targets, 85-90% is in fact not very striking and could just be a statistical aberration. This needs to be addressed.We thank the reviewer again for this valuable comment. Concerning the first point: We cannot explain why Ubx binds to less chromatin regions in the late neuronal time point, in particular as the quality of the reads is similar to the other stages. However, we assume that this is due to the different biology of the two lineages. In particular, as we already mention in the text, the neuronal lineage is a bit ahead of the mesodermal lineage with regards to developmental processes and initiates differentiation earlier than the mesoderm. This might result in a reduction of Ubx binding in the neuronal lineage.Drosophila genome), we then identified matching replicated via person correlation and combined the data sets for further analysis. This already changed the number of genes to which Ubx binds. We obtain now 6417 Ubx bound genes at stages 10-13 and 8256 Ubx bound genes at stages 14-17 in the mesoderm, with 80% of them corresponding to coding genes and 20% to non-coding genes. Thus, in total Ubx binds to 37-46% of the genes in the Drosophila genome  using these settings. Using the WEADE tool, we still see the same result, that Ubx binding reflects genome activity, suggesting that Ubx indeed comprehensively controls lineage development.The surprising outcome was that Ubx is indeed binding to a lot of sites. This is of course dependent on the TF type, on the way data are analysed, which thresholds are set. As requested by this reviewer before, we reanalyzed the Ubx data sets using a threshold of >5 RPKM  available for Tin  were done using this version. Alignment and peak calling against dm3 results in regions that are named “U” and “Uextra”, which are normally removed as described in the supplementary of Erceg et al., 2017. This led to a removal of nearly half of the mapped reads. Using dm6 as a reference genome, which we did for the Ubx data, maintains these peaks and results in more binding events. In order to make the Ubx and Tin datasets more comparable, we mapped the Ubx peaks against the dm3 genome, which resulted in the identification of only 1914 genes to which Ubx binds in the early mesoderm. This is now comparable to the binding events of Tin in the mesoderm. We then redid the WEADE analysis and still get a very similar result . In addition, we did the analysis also with another TF, Mef2, which generally controls mesoderm development and not like Tin a sub-aspect (cardiac development). Interestingly, we see that Ubx and Medf2 binding profiles are similar, while Tin binding preferences are more different. This makes in our opinion the main message even stronger, as Ubx binding is more similar to a general factor controlling mesoderm development. Thus, we are convinced that this finding reflects true differences between Ubx, Mef2 and Tin.We have studied the comparison of Ubx and Tin binding behavior in more detail, as we do agree with the reviewer that the number of bound genes will affect the outcome. For Tin, we had to rely on available mapping data onto the We do agree with the reviewer, that in the previous version the use of “striking” was not adequate, and we removed it from the text. Using the reanalyzed data, we now see that about 70% of the genes that were up- or down-regulated were bound by Ubx, and thus are very likely direct targets of Ubx. We would however highlight that binding does not necessarily mean a regulatory function, it is possible that Hox proteins bind without any functional consequences, and this could be a substantial fraction of the Hox binding events .3) The proposed model, while informative, is too simplistic. This is because, in its current form, the model lacks information on what the factors / determinants are that mediate either tissue-specific activation or repression. The identification of these factors is likely beyond the scope of the current study, but perhaps some first layer data analysis on motifs (beyond the listed generic ones) could be performed. In addition, these additional layers need to be incorporated in the model because, as an example, the Ubx-Pho complex is shown to repress neuronal genes in the mesoderm given the enrichment of the Pho motif in these repressed genes. But why then, given that the same motif will obviously be present in the DNA of a neuronal cell, does Pho-Ubx not repress these same genes in a neuronal cell ? What makes that the Pho motif in a neuronal cell is read differently in a mesodermal cell and vice versa? The same reasoning can be followed for active genes in the respective cell types. At a minimum, such uncertainties should be represented in the model to clearly indicate what type of regulatory information is still missing.We do agree with the reviewer that the factors that mediate the tissue-specific activation and repression are very interesting, but are indeed beyond the scope of this paper. We have however performed some preliminary analysis (motif search) and included this in an extended version of the model shown in Figure 8A and B. We have also now dedicated a section to the Discussion part where we explain more in depth how we envision that specificity in tissue-specific target gene regulation by Hox TFs is achieved.Reviewer #3:I have mixed responses to the paper by Domsch et al. On the one hand, the experimental design, to analyze Hox regulatory mechanisms in distinct embryonic tissues, seems at first glance important and exciting, I was less impressed with the analysis than I expected to be. My concerns fall in two categories:1) Data generation and analysis.For one, although replicates were analyzed for the RNA-seq experiments, it seems that all of the ChIP-seq datasets were only done once, without replicates. Given how noisy these type of data can be  I think it is imperative to compare at least two experimental replicates and use peak calling algorithms that require two independent datasets to assess statistical relevance. The Venn diagrams in Figure 2—figure supplement 1 for example suggest that there are ~14,000 (!) Ubx peaks in the mesoderm and that more than 7000 of these are mesoderm-specific. These numbers are quite striking but as it stands it is not possible to know how solid they are without replicate datasets and rigorous statistical tests.Mef2>UAS-Nslmb,GFP-Ubx). For the analysis of differential Expression  and differential Binding  the two most similar replicates were identified and analyzed. For the identification of active genes or Ubx bound regions, we identified matching Ubx ChIP-seq replicates via person correlation, combined the data sets for further analysis and have (as requested by reviewer #2) used a less stringent threshold of >5 RPKM threshold for the tissue specific transcriptome data sets (dm6 Drosophila genome). This changed the number of genes to which Ubx binds. We obtain now 6417 Ubx bound genes at stages 10-13 and 8256 Ubx bound genes at stages 14-17 in the mesoderm, with 80% of them corresponding to coding genes and 20% to non-coding genes. Thus, in total Ubx binds to 37-46% of the genes in the Drosophila genome  using these settings. Although this number is now reduced, the overall take home message is the same: Ubx binds a lot of sites, Ubx controls lineage development comprehensively (see comments to reviewer #2), and Ubx controls lineage development by repressing alternative fate genes and by activating lineage specific genes.We apologize if this was written in an unclear manner, but we had done all experiments of course at least in duplicates. To clarify: for the general mapping of tissue specific binding sites of Ubx we performed ChIP and HiSeq on two biological replicated. The experiments of the Ubx degradation (UbxDegrad) were performed in three biological replicates, the control  as well as the experiment . We would however highlight that the subtraction of the Input from the IP is a common method to show the binding peaks and is used by many researcher groups, as it is normalizing the ChIP-seq reads .There is little validation of the ChIP peaks. The only known enhancer that was highlighted was one from Dpp.βTub60D enhancer, this gene is significantly down-regulated upon Ubx degradation.As requested by the reviewer, we now show another example in Figure 4—figure supplement 1A, the The Pho co-IP signal is surprisingly weak given the mechanism of co-recruitment that the authors are proposing.w1118 fly line, and we found an interaction of Ubx with Pho in an in vivo context, which to our knowledge has so far not been shown for a Pho-TF interaction. And actually, we do not propose a direct recruitment or co-recruitment but we state that Ubx stabilizes Pho binding to repressed regions. The weak signal suggests that the interaction of Pho with Ubx is rather weak but essential for the maintenance of the repression. We have now stated this even clearer in the text.We have performed whole embryonic Co-IP using the GFP-Ubx line, as a control we used the GAGA-like motifs are often observed by motif discovery algorithms and I am not convinced they are meaningful here given the lack of solid follow up evidence.We assume a lot of genomic regions to contain the GAGA or GAGA-like motif and we agree that motif search does not that these regions are functional or bound by GAGA. However, we do think that some Ubx bound regions will have GAGA bound, as we could show an interaction between Ubx and GAGA at least in cell culture .One potential strength of the paper is the claim that Pho binding and K27me3 depends on Ubx binding. However, this conclusion is based on a small handful of loci analyzed by qPCR and the differences are small. Further, given that the Pc system can have very specific targets, I think that calculating the ratio of K27me3/K27ac can be misleading. Better to treat these marks independently, since when one is lower it isn't necessarily the case that the other will be higher. It is also the case that Pho binding is not equivalent to K27me3; the latter could simply be a consequence of whether the gene is repressed, which may or may not occur in a Pho-dependent manner.We agree with the reviewer and we have redone the analysis using the Pho ChIP-seq dataset we have generated (in control and Ubx degradation backgrounds) to show the global relevance of our finding. In addition, we show K27me3 and K27ac marks independently. This analysis now reveals that a subset of Ubx-Pho bound sites that lose Pho binding in the absence of Ubx have their K27me3 marks significantly reduced, while K27ac marks are increased, demonstrating that at least at a subset of sites Ubx is required for Pho binding and H3K27me3 marks. We have now emphasized this point also stronger in the paper.Many genes lose expression in the Ubx knockdown experiments, suggesting that Ubx also acts as an activator in the mesoderm, but this is not adequately discussed or studied in the paper. It is not clear why the authors choose to focus on the repressed genes.We agree with the reviewer that it has not been explained well enough why we chose to focus on repressed genes, which we have now done in the revised version. We have now dedicated also a paragraph in the Discussion on the activation of lineage-specific genes by Ubx to highlight that Ubx controls lineage development not only by repression.2) General comments.Ubx, and Hox genes in general, are well studied transcription factors that are known to be important in multiple cell types including mesoderm, the nervous system, and epithelium. So, seeing lots of genes change their expression when a broadly used transcription factor like Ubx is knocked down. It is also not surprising that the genes differ depending on the tissue . What would take the analyses beyond what is known already is to understand how Ubx carries out differential functions in these two (and other) tissues. Presumably this happens in conjunction with tissue specific factors (e.g. twist for the mesoderm). The role of lineage specific factors, however, is not addressed and only marginally mentioned in the paper.We would like to highlight that although it might seem trivial and expected to see a lot of genes changing their expression when Ubx is lost, this is the first time that a Hox TFs, which is active in many different cell types, is studied in individual lineages in vivo. Reviewer #1 and #2 appreciated this aspect a lot, as they thought this is an elegant study. It was in particular this set-up that allowed us to uncover the function of Ubx in repressing alternative fates. We do agree with the reviewer that the next interesting and important question to solve is how Ubx performs this function lineage-specifically, very likely via the interaction with lineage-specific factors. We work on that aspect, but this is beyond the scope of this paper, as this manuscript is already quite complex (stated by reviewer #1).Terms such as "Cell plasticity" and "lineage restriction" are used rather loosely. The authors show that many up-regulated genes after Ubx knockdown are expressed in other lineages, but this result would have to be the case when a TF that represses genes is removed. There is no hard-evidence of lineage conversion , nor is it clear why neuronal  genes are the focus here. It seems that the analysis is artificially restricted to repressed neuronal genes, while a more unbiased analysis of the changes that occur would be more valuable.We went critically through the manuscript and used the terms “cell plasticity” and “lineage restriction” in a more stringent manner. Concerning the second point, that there is no hard evidence for lineage conversion, we would like to highlight that we never state that the mesodermal lineage changes identity in the absence of Ubx. Actually, we dedicated a whole paragraph in the Discussion why we believe that there is no lineage conversion, one reason being that other Hox TFs (like Antp) are ectopically expressed when Ubx is removed, which are very likely involved in maintaining the expression of mesodermal master-regulators. Thus, it will be important to study lineage development in a Hox free environment. However, this is beyond the scope of the paper. We now state more clearly why we focused on neuronal genes in our GO term analysis. However, we would like to highlight that we show the global significance of the de-repression of different types of alternative fate genes in Figure 3O.


Magnaporthe oryzae is a destructive disease of rice and responsible for causing extensive damage to the crop. Pi54, a dominant blast resistance gene cloned from rice line Tetep, imparts a broad spectrum resistance against various M. oryzae isolates. Many of its alleles have been explored from wild Oryza species and landraces whose sequences are available in the public domain. Its cognate effector gene AvrPi54 has also been cloned from M. oryzae. Complying with the Flor’s gene-for-gene system, Pi54 protein interacts with AvrPi54 protein following fungal invasion leading to the resistance responses in rice cell that prevents the disease development. In the present study Pi54 alleles from 72 rice lines were used to understand the interaction of Pi54 (R) proteins with AvrPi54 (Avr) protein. The physiochemical properties of these proteins varied due to the nucleotide level polymorphism. The ab initio tertiary structures of these R- and Avr- proteins were generated and subjected to the in silico interaction. In this interaction, the residues in the LRR region of R- proteins were shown to interact with the Avr protein. These R proteins were found to have variable strengths of binding due to the differential spatial arrangements of their amino acid residues. Additionally, molecular dynamic simulations were performed for the protein pairs that showed stronger interaction than Pi54tetep  protein. We found these proteins were forming h-bond during simulation which indicated an effective binding. The root mean square deviation values and potential energy values were stable during simulation which validated the docking results. From the interaction studies and the molecular dynamics simulations, we concluded that the AvrPi54 protein interacts directly with the resistant Pi54 proteins through the LRR region of Pi54 proteins. Some of the Pi54 proteins from the landraces namely Casebatta, Tadukan, Varun dhan, Govind, Acharmita, HPR-2083, Budda, Jatto, MTU-4870, Dobeja-1, CN-1789, Indira sona, Kulanji pille and Motebangarkaddi cultivars show stronger binding with the AvrPi54 protein, thus these alleles can be effectively used for the rice blast resistance breeding program in future.Rice blast, caused by the ascomycete fungus  Magnaporthe oryzae, causes a big loss to the net production of rice Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The papers presents the results of the nucleotide polymoephism in the Pi54 alleles which affect the Pi54 and AvrPi54 interaction. The title of the papers should be more specific since many of studies have been done on the R-Avr protein interaction including in the author papers published in 2014,which revealed that the Pi54of interacted with AVR-Pi54 through its non-LRR region, but Pi54 and Pi54rh interacted through CC-non-LRR-LRR regions and non-LRR-LRR regions, and the susceptible Pi54tp protein did not show interaction with AVR-Pi54, respectively. So this papers judged from the title only provides a study is similar to the former’.The major problem of the papers is that the conclusions in the papers should be verified by experiment, the molecular simulation is not real happened after all. Furthermoer, there are many problems with sentence structure and clause constrution.So try to set the problem discussed above in more detail!Reviewer #2: The authors analysed the Pi54 alleles from 72 rice lines by using bioinformatics methods to understand the interaction of Pi54 (R) proteins with AvrPi54 (Avr) protein. And they found that the physiochemical properties of these proteins varied due to the nucleotide level polymorphism. Generally, the authors present a well conducted study. The manuscript is concise and the results are mainly convincing.Some minor points:1. Figure 1, it can be hard to distinguish the 13 different amino acids. It may be useful to draw some lines.2. Figure 2, add the letters to indicate which one is 2A or 2B.3. It could be better for this manuscript if the authors could do some Y2H to check the interactions between avrpi54 and Pi54 alleles from resistant and susceptible lines. These experimental results could provide stronger evidence to explain the different phenotype**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  9 Sep 2019Reviewer 1 comments The papers presents the results of the nucleotide polymorphisms in the Pi54 alleles which affect the Pi54 and AvrPi54 interaction. The title of the papers should be more specific since many of studies have been done on the R-Avr protein interaction including in the author papers published in 2014, which revealed that the Pi54of interacted with AVR-Pi54 through its non-LRR region, but Pi54 and Pi54rh interacted through CC-non-LRR-LRR regions and non-LRR-LRR regions, and the susceptible Pi54tp protein did not show interaction with AVR-Pi54, respectively. So this papers judged from the title only provides a study is similar to the former’.The major problem of the papers is that the conclusions in the papers should be verified by experiment, the molecular simulation is not real happened after all. Furthermore, there are many problems with sentence structure and clause construction.So try to set the problem discussed above in more detail!)Our reply Title of the manuscript is changed as per the suggestion of the reviewer that is “Variation in the LRR region of Pi54 protein alters its interaction with the AvrPi54 protein revealed by in silico analysis”.The experimental proof for the interaction between Avr-Pi54 and Pi54 proteins was already done by Ray et al, 2016 . Here, the main objectives of this manuscript was to analyse sequence variation at the LRR regions of those alleles of Pi54 gene isolated from both phenotypically resistant and susceptible rice lines and analysed sequence variation at the LRR region and look at their interaction with AvrPi54 gene. The hypothesis was that the proteins of Pi54 alleles isolated from resistant rice lines should show in silico interaction with the AvrPi54 proteins and the susceptible one should not show any interaction which is akin to the Flor`s gene-for-gene hypothesis (Flor 1972). The interaction analysis revealed that Pi54 protein from resistance lines, showed significant in silico interactions with AvrPi54 protein, while Pi54 from susceptible lines did not interact with its counterpart. Hence, it proves our hypothesis.We made corrections in the conclusion section of the manuscript in lines 527 and 529. We also corrected many sentences throughout the manuscript, e.g. in lines 56, 71, 77, 83, etc. These changes can be seen in the track change version of the manuscript.In addition in the revised version of the MS, molecular dynamics simulation analysis was carried out to see whether the interactions between Avr-Pi54 and Pi54 proteins are stable at the thermodynamics level or not, and found that the interaction was stable at the different thermodynamic parameters.Reviewer 2 comments: The authors analysed the Pi54 alleles from 72 rice lines by using bioinformatics methods to understand the interaction of Pi54 (R) proteins with AvrPi54 (Avr) protein. And they found that the physiochemical properties of these proteins varied due to the nucleotide level polymorphism. Generally, the authors present a well conducted study. The manuscript is concise and the results are mainly convincing.Some minor points:1. Figure 1, it can be hard to distinguish the 13 different amino acids. It may be useful to draw some lines.Our replyThe figure 1 has been removed from the main text and instead of this figure a comparative composition of amino acids of the resistant and susceptible proteins with Pi54tetep protein is given, but annexed to supplementary data as figure S1 titled as “Composition of different amino acids in Pi54 proteins of the alleles cloned from resistant and susceptible rice line (A) compared with wild type Pi54tetep protein”.2. Figure 2, add the letters to indicate which one is 2A or 2B.Our replyAs suggested the changes have been made. 3. It could be better for this manuscript if the authors could do some Y2H to check the interactions between avrpi54 and Pi54 alleles from resistant and susceptible lines. These experimental results could provide stronger evidence to explain the different phenotype.Our replyThe Y2H assay has been already performed between the AvrPi54 and Pi54tetep protein to confirm their interaction by Ray et al, 2016, “Analysis of Magnaporthe oryzae genome reveals a fungal effector, which is able to induce resistance response in transgenic rice line containing resistance gene, Pi54” published in Frontiers in Plant Science journal and this publication has been cited in this manuscript. In this article, authors were identified the AvrPi54 gene and validated the gene using the in vitro as well as the in planta experiments. The significant interaction between the candidates of AvrPi54 and the Pi54 protein was initially begun with the bioinformatics approaches and led the analysis to pinpoint the AvrPi54 gene. Thus we hypothesized that to check whether the phenotypic results of rice blast disease screening experiment, on Pi54 resistant rice line and susceptible rice line would be matched with the results of in silico interaction between AvrPi54 and variants of Pi54 proteins. For that we conducted this study and disrupted interactions were found when variants of Pi54 derived from susceptible rice lines interacted with the AvrPi54 protein, while a strong interaction with AvrPi54 was found in case of Pi54 variants obtained from resistant rice lines. Thus, any of the Pi54 allele could be initially determined for its function through the in silico interaction analysis without doing phenotyping experiment. That is the main objective of this study. The interaction between Pi54-AvrPi54 proteins has been taken as a basis to extend our knowledge to discover the strength of interaction between various allelic Pi54 proteins with the AvrPi54 protein.AttachmentReply to reviewers comments.docxSubmitted filename: Click here for additional data file. 7 Oct 2019Variation in the LRR region of Pi54 protein alters its interaction with the AvrPi54 protein revealed by in silico analysisPONE-D-19-16056R1Dear Dr. Sharma,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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