A single-blinded randomized controlled trial among patients requiring an upper third molar extraction was performed to evaluate the anxiety degree after receiving information or not about the functioning of The Wand system. Secondarily, perceived pain and the need of re-anesthesia were assessed.  Patients were randomly assigned to the experimental group (detailed explanation about The Wand) or control group (no specific information). Local anesthesia with The Wand consisted in a supraperiosteal infiltrative technique injection 1.6 mL at the buccal and 0.2 mL at the palatal side. Distinct questionnaires for assessing dental anxiety and 100-mm visual analog scales to assess pain were delivered. Demographic data, radiological parameters, operative time and type of intervention were also registered.A descriptive bivariate analysis by non-parametric tests to detect differences in anxiety, pain and re-anesthesia was performed by SPSS 22.0 . A total of 85 patients were assessed for eligibility but 17 participants were lost due to the cancellation of the visit for the surgical intervention. Finally, sixty-eight patients were included (34 participants in each group), 47 women (69.1%) and 21 men (30.9%), with an average age of 28.8 (± 9.3) years. Patients that received a detailed explanation of The Wand did not have a significant reduction of the anxiety degree and perceived pain during the anesthetic act compared to patients that received no information. The need of re-anesthesia was not related to the anxiety level but was significantly related to increasing operative time. Key words:Dental anxiety, pain, local anesthesia, third molar, computerized anesthesia. Anxiety and pain during dental procedures are well described in the literature -4. DespiThe research into this field aims to develop less invasive and painful systems compared to conventional anesthesia ,9,10. Thet al.  guidelines . All pat- Experimental designA single-blinded randomized controlled trial was performed among patients requiring an upper third molar extraction treated by postgraduate students of the Master of Oral Surgery and Orofacial Implantology at the dental hospital of the University of Barcelona.- ParticipantsInclusion criteria were patients from 18 to 45 years old, coming for an upper third molar extraction  in absence of symptomatology, willing to participate in the study and signed the informed consent for treatment.On the contrary, exclusion criteria were dental professionals or students because of the possible knowledge of the anesthetic system, patients with systemic diseases (≥ASA III), suffering from allergy or intolerance to the local anesthesia administered, patients who needed other tooth extraction in the same surgical act, surgical interventions lasting more than 60 minutes, active infection in maxillofacial area, concomitant treatment with systemic antibiotics and/or analgesics and abnormal hemodynamic parameters .- Study interventionAt the 1st visit, the patients answered the STAI-T (State-Trait Anxiety Inventory) questionnaire to identify their anxiety trait. Then, in a 2nd visit when the patients came to perform the surgical procedure, they were randomly assigned to the experimental group (detailed explanation about the functioning of The Wand system) or control group (no specific information about the system) by means of a computer generated random sequence through www.randomization.com web page.The explanation for experimental group was verbal and standardized: “The Wand is a local anesthesia system whose main objective is to reduce pain and anxiety during the anesthetic act. It has a pen-shaped design producing less visual impact, thus reducing patient anxiety. Moreover, this device releases anesthesia regardless of tissue resistance, at a low pressure and a constant flow, which produces less pain during puncture and injection. In your case, the anesthetized area will be the buccal gingiva and the palate associated to the third molar that is going to be extracted”. No explanation was given to participants belonging to the control group.- Registered variablesAnxiety degree measured by distinct questionnaires , Modified Dental Anxiety Scale (MDAS), Dental Fear Survey (DFS) and State-Trait Anxiety Inventory (STAI)) was considered the primary outcome variable. Perceived pain measured by 100 mm visual analog scales (VAS) in 4 different times  and need of re-anesthesia during the surgical procedure were considered secondary outcome variables. Demographic data (age and gender), radiological parameters (Pell & Gregory (13), Winter  and ParaThe ISAR questionnaire consists in drawing a horizontal stroke into a 90 mm vertical VAS to set the anxiety level. The MDAS is a modification of Corah’s Dental Anxiety Scale that contains 5 questions measuring anxiety in distinct stages of a dental treatment. The maximum punctuation is 25 and patients with a mark ≥ 19 should be considered as very anxious. DFS is specifically designed to measure dental anxiety. It has 20 questions and the total punctuation varies from 20 (no fear) to 100 (terrified). The cut-off point that differentiates patients with or without dental anxiety is considered to be at 63. The STAI questionnaire consists in 40 questions divided in two groups; the first evaluates anxiety as a transitory state (anxiety state) and the second, as a latent feature or trait (anxiety trait). Anxiety trait (STAI-T) identifies relatively sTable individuals who have a tendency to perceive some situations as a threat. Thus, patients answered this questionnaire at the 1st visit. On the other hand, state anxiety (STAI-S) is considered to be a transient emotional state related to subjective feelings, apprehension and hyperactivity originating from autonomous nervous system.- Surgical techniqueThree blinded and previously calibrated researchers  performed the local anesthesia with The Wand system by a supraperiosteal infiltrative technique injection 1.6 mL at the buccal and 0.2 mL at the palatal side, using a short dental needle of 25 mm and 30 G  and one cartridge of 4% articaine with 1:100.000 epinephrine . After concluding the anesthetic act, one investigator (ARL) delivered the questionnaires  and the four VAS scales were administered at the same time.The surgeries were performed by the Master fellows with similar experience and the same technique. The surgical technique employed for partially or totally impacted third molars consisted in raising a full-thickness flap to perform bone removal (if needed) with a hand piece at high speed (40.000 rpm) and a tungsten carbide round bur with constant sterile saline solution irrigation. The wound was closed with 3/0 silk interrupted suture.- Sample size calculation and statistical analysisA sample size calculation with an α – error of 0.05, a power of 0.9 with and a size effect d=0.8 was performed by G* Power software version 3.1.9.2 . Size effect (d) was calculated from a previous sample of patients anesthetized with The Wand® system. The total sample was 68 patients divided into two groups of 34 individuals  each one.p < 0.05.A descriptive and bivariate analysis was performed by SPSS 22.0 . Normality was assessed by means of Saphiro-Wilk test and non-parametric tests were used in case of absence of a normal distribution to detect differences for anxiety, pain and need of re-anesthesia between the experimental and control groups. The results of MDAS and DFS questionnaires were analyzed as dichotomic variables. The level of significance was set at A total of 85 patients were assessed for eligibility at the 1st visit although 17 participants were lost due to the cancellation of the visit for the surgical intervention. Fig. The results of the anxiety questionnaires for the overall sample and for each study group are highlighted in p=0.007), as the surgical interventions from patients with no need for additional anesthesia lasted for 15.5 (SD=8.8) minutes and the ones that had to be re-anesthetized lasted for 24 (SD=13.3) minutes. Fig. A total of 29 (42.6%) patients had to be re-anesthetized intraoperatively. There was a statistically significant relation with the increasing operative time (p=0.806). Moreover, no significant relationship was found neither between re-anesthesia and radiological variables  nor with the type of intervention (Parant: p=0.059).No significant differences were found for the need of re-anesthesia depending on the level of anxiety measured by the distinct questionnaires  or by prThe fear concerning the pain from the anesthetic injection is very frequent  and thesWe selected the upper third molar removal as a model to our study because it is a very reproducible model of surgical intervention. In particular, palate is one of the most painful areas  and thisThis study showed that previous explanation of the anesthetic system does not decrease the level of anxiety, as measured by distinct questionnaires. Therefore, no statistically significant differences between groups were noticed. However, the fact that individuals with a previous upper third molar extraction were also included could constitute a bias when answering the anxiety questionnaires. To date, there are no studies addressing the anxiety depending on the information delivered to the patient about the anesthetic system. Recent research  found thComputerized controlled anesthetic delivery systems are able to better control the flow and pressure of the liquid which can reduce pain during local anesthesia infiltration ,17,19. Aet al.  was correlated to increasing operative time but not to the anxiety level nor to the previous explanation. We can assume that it is a high rate for re-anesthesia given the mean operative time. However, the fact that surgical procedures were performed by fellows could overestimate the results as some cases could have been treated as an intraoperative painful sensation in order to detect patients feeling uncomforTable with the exerted pressure during the surgery.et al. (Interestingly, a randomized clinical trial from Patini et al.  that treet al. .Overall, the use of a computerized anesthesia system seems to be preferred as it can reduce disruptive behavior in children  and painTo conclude, patients that received a detailed explanation of The Wand system previous to an upper third molar extraction did not have a significant reduction of the anxiety degree and perceived pain during the anesthetic act compared to patients that received no information.The need of re-anesthesia was not related to the anxiety level but was significantly related to increasing operative time.


Was the 2013 expansion of US Medicare Part D prescription drug coverage to include benzodiazepines associated with increased rates of fall-related injuries or overdoses among older adults?This ecological study of more than 4.6 million Medicare Advantage beneficiaries found an increase in the rate of overdose after Part D coverage began to include benzodiazepines among adults aged 65 to 69 years and 80 years or older and an increase in the rate of fall-related injury in adults 80 years or older.Medicare’s expansion of benzodiazepine coverage to older adults may have been associated with an increased rate of overdose among those 69 years or younger and 80 years or older and of injury in those 80 years or older. Benzodiazepines, which are associated with safety-related harms for older adults, were not covered when the US Medicare Part D prescription drug benefit began. Coverage was extended to benzodiazepines in 2013.To examine whether the expansion of benzodiazepine coverage among Medicare Advantage (MA) beneficiaries was associated with increases in fall-related injuries or overdoses among older adults.This ecological study used interrupted time-series with comparison-series analyses of MA claims data from 4 635 312 age-eligible MA beneficiaries and 940 629 commercially insured individuals (comparison group) stratified by age  to separately compare trends in fall-related injury and overdose before  and after  coverage expansion for benzodiazepines. Data analysis was performed from September 1, 2018, to August 31, 2019.Expansion of benzodiazepine coverage in Medicare Part D in 2013.Monthly rate of fall-related injury and overdose.P = .04 for interaction). The overdose trend changed from decreasing to increasing among MA beneficiaries after coverage for all age groups, with a statistically significant increase compared with the comparison group among those aged 65 to 69 years  and among those 80 years or older . Results among MA beneficiaries were consistent when stratified by sex and when limited to those prescribed opioids.In 2012 (the year before the policy change), women constituted 57.5% of the MA group and 47.4% of the comparison group. A total of 25.8% of individuals in the MA group were aged 65 to 69 years, and 29.3% were 80 years or older ; 56.7% of individuals in the comparison group were aged 65 to 69 years, and 15.1% were 80 years or older . In the MA group, 4 635 312 individuals contributed 156 754 749 person-months from 2010 through 2015; in the comparison group, 940 629 individuals contributed 25 104 534 person-months. After coverage of benzodiazepines began, the rate  of fall-related injury among MA beneficiaries increased from before to after coverage among all age groups. Compared with the comparison group, the increase in rate was statistically significant for those 80 years or older (rate changes for the MA vs comparison groups: 0.12  vs −0.01 ; Medicare’s expansion of benzodiazepine coverage may have been associated with increases in the rates of overdose among adults ages 65 to 69 years and in the rates of overdose and fall-related injury among those 80 years or older. This ecological study examines whether the expansion of benzodiazepine coverage among Medicare Advantage beneficiaries was associated with increases in the rates of fall-related injury or overdose among older adults. Furthermore, no studies to our knowledge have examined the association of benzodiazepine coverage with overdose among older adults. In this analysis, we examined the association of fall-related injury and overdose with Medicare’s expansion of Part D coverage to include benzodiazepines. Given heightened public attention to opioid prescribing and the potential for harms when opioids and benzodiazepines are coprescribed,20 we also examined outcomes limited to older adults prescribed opioids. We hypothesized that this nationwide expansion of benzodiazepine coverage would be associated with an increase in the rates of fall-related injury and overdose both overall and among those prescribed opioids.In states that have implemented policies that limit benzodiazepine prescribing, fall-related adverse events among older adults have not decreased.STROBE) reporting guideline.21For this ecological study, we used administrative health claims from Optum’s deidentified Clinformatics Data Mart Database derived from a large national health insurance company in the US. We created monthly measures of the outcomes using a denominator of all age-eligible  Medicare Advantage (MA) beneficiaries who were enrolled during that month. All older adults in the population had Medicare Part D prescription drug coverage as part of their MA plan. There were no other exclusion criteria. As a comparison group, we used adults 65 years or older in the database with commercial  insurance; the prescription plans of these older adults would not have been subject to the benzodiazepine coverage change experienced by MA beneficiaries. The population-level numerators for the 2 analyses were the number of fall-related injury episodes that began in a given month and the number of overdoses. This study was considered not regulated by the Michigan Medicine Institutional Review Board, so informed consent was waived for this analysis of deidentified data. This study followed the Reporting of Studies Conducted Using Observational Routinely Collected Health Data (RECORD) extension of the Strengthening the Reporting of Observational Studies in Epidemiology  across the spectrum of severity from nonfracture injury to hip fracture. If a single injury episode crossed calendar months, it was attributed to the month when the episode began.Episodes of fall-related injury were determined using an algorithm developed and validated using MA claims data.International Classification of Diseases, Ninth Revision, Clinical Modification diagnoses from emergency department or acute inpatient encounters regardless of intent .Overdoses were captured using We obtained information on patient sex and age along with select additional demographic characteristics  from demographic data available in the Optum database. For descriptive purposes, we also determined the presence of select clinical conditions for which benzodiazepines may commonly be prescribed in clinical practice, including anxiety disorders, depression, dementia, and insomnia , and a term for the change in the linear time trend  between the precoverage and postcoverage periods. We did not include a term that tested for an immediate-level shift in outcomes  because we hypothesized that any change in benzodiazepine prescribing and associated injuries would accumulate over time and not change immediately on January 1, 2013. We constructed analogous models in the comparison group . To test for the association of the Medicare benzodiazepine coverage policy expansion , we fit a single model with interactions between group and each term in the interrupted time-series model, specifically reporting the significance of the interaction between groups (MA vs comparison) and the postcoverage rate shift.t tests to test the statistical significance (α = .05) of the association of benzodiazepine coverage expansion with injury . In addition, given safety concerns about opioid and benzodiazepine coprescribing,24 we completed additional analyses that limited the monthly MA population denominator to beneficiaries with 1 day or more of opioid supply in a given month to determine whether results limited to the population prescribed opioids were consistent with those for the overall MA population. Analysis was completed from September 1, 2018, to August 31, 2019.Descriptive analyses revealed secular trends in the age distribution of the study population. To guard against confounding on this basis, we stratified analyses by age . Results of the primary analyses for both outcomes were similar between men and women within each age group; thus, we present primary findings stratified only by age. Models also included monthly fixed effects to account for seasonality of the outcomes. We did not detect any other temporal autocorrelation in the model errors. We used 2-tailed In 2012 (the year before the policy change), women constituted 57.5% of the MA group and 47.4% of the comparison group. A total of 25.8% of individuals in the MA group were aged 65 to 69 years, and 29.3% were 80 years or older ; 56.7% of individuals in the comparison group were aged 65 to 69 years, and 15.1% were 80 years or older . There were 4 635 312 individual patients , who contributed 156 754 749 person-months from 2010 through 2015. In the comparison group, there were 940 629 individual patients, who contributed 25 104 534 person-months during the same period. Select population characteristics are given in P = .04 for interaction). The MA analyses stratified by sex yielded similar results for men and women by age group  of fall-related injury was 1.34 per 10 000 person-days  among those aged 65 to 69 years and increased for each age group; the rate was 3.20 per 10 000 person-days  among those 80 years or older . MonthlyAmong MA beneficiaries prescribed opioids, the fall rate was markedly higher than in the overall population; however, the precoverage and postcoverage changes were consistent (eTable 3 in the P < .001 for interaction) for the comparison group. For those 80 years or older, the postcoverage rate increased by 0.07 encounters per month  vs −0.20  for the comparison group. The MA analyses stratified by sex yielded similar results for men and women by age group  of overdose was 1.31 per 100 000 person-days  among those aged 65 to 69 years. The rate was highest among those aged 75 to 79 years  . The oveAs with the fall rate, the monthly rate of overdose was markedly higher among beneficiaries prescribed opioids than in the overall population  before Medicare Part D existed but then excluded benzodiazepines when Medicare Part D began, nearly 75% of MA beneficiaries continued use of benzodiazepines after exclusion by paying out of pocket.15 In addition, a recent analysis4 of benzodiazepine prescribing trends from 1996 to 2014 using the Medical Expenditure Panel Survey did not demonstrate an increase in prescription benzodiazepine use among respondents 65 years or older in 2013.4Although the increase in benzodiazepine claims among MA beneficiaries was increased by 5.5% or more than 10-fold, there were some benzodiazepine claims during the precoverage period . This finding likely reflects that some MA plans could provide more generous prescription coverage than required by Medicare that included benzodiazepines.19 of policies that restricted benzodiazepine access that did not demonstrate a decrease in injury. One such analysis19 that predated Medicare Part D examined hip fracture rates, comparing New York, which instituted a triplicate prescription policy that increased the administrative burden of benzodiazepine prescribing, with New Jersey, which had no such policy. Although there was an immediate 60% reduction in benzodiazepine prescribing in New York, the rate of hip fracture did not decrease compared with that in New Jersey. When Medicare Part D went into effect, some state Medicaid programs provided supplemental coverage of benzodiazepines, whereas other states did not. In an analysis18 of dually eligible nursing home residents, benzodiazepine prescribing varied with states’ supplemental coverage, but the risk of hip fracture was not lower in states that limited benzodiazepine coverage.18We hypothesized that the expansion of coverage would be associated with both types of injury. However, the minimal effect sizes observed align with prior studies26 new users may be particularly susceptible to experiencing an injury when first exposed. A study by Hernandez et al27 examining the risk of opioid-related overdose among concurrent opioid and benzodiazepine users suggests that the risk of overdose is highest in the first 90 days of concurrent use and then decreases with each successive time increment.27 In addition, some practitioners and patients may have interpreted Medicare’s benzodiazepine coverage expansion as an indication that safety risks had been overstated, further lowering the prescribing threshold and leading to prescriptions that practitioners previously avoided because of safety concerns.Given these prior analyses, what may account for the limited associations with increased injury in the present analysis? The expansion of Medicare Part D may have had a limited association with overall prevalence of benzodiazepine use among older adults but still would have been associated with a reduced threshold for new use among benzodiazepines-naive patients, particularly those with limited resources. Because those who regularly use benzodiazepines develop physiologic tolerance with repeated exposure,28 Their association with overdose other than when prescribed with opioids receives less attention, and unlike for fall-related injury, we are unaware of prior analyses examining the association of benzodiazepine coverage policies with overdose. This lack of attention may be partially because overdose is less common among adults 65 years or older than among younger adults29 and is therefore not perceived as a problem. However, a recent analysis9 found that the largest relative increases in opioid-related overdose from 2001 to 2016 were among adults aged 55 to 64 years followed by those 65 years or older.We did not anticipate that the change in the overdose rate associated with benzodiazepine coverage would be greater in relative terms than the change in fall-related injury. Safety warnings cautioning against benzodiazepine prescribing in older adults typically focus on the risk of falls.31 An 80-year-old individual may be more sensitive to these effects than a 65-year-old individual. The 65- to 69-year age group includes members of the baby boom cohort, who started turning 65 years of age in 2011. Compared with prior cohorts of older adults , baby boomers have higher rates of alcohol use, nonmedical prescription opioid use, and marijuana use,35 each of which might be associated with increased susceptibility to benzodiazepine-related injury from a new prescription. By occurring at the height of the opioid overdose crisis, the benzodiazepine coverage expansion potentially exacerbated the risks associated with prescription opioids.Why might the associations with increased injury have been limited to those aged 65 to 69 years and those 80 years or older in the study population? It is possible that these age groups were vulnerable to the effects of policy change for different reasons. Adverse effects related to benzodiazepines are generally attributed to the role of benzodiazepine as a central nervous system depressant, and older adults are particularly sensitive to these effects.29 Although favorable selection into MA plans has decreased,37 evidence remains that patients with the most complex needs may exit MA,39 and MA patients at the end of life are healthier, more educated, and have fewer functional limitations.40 The rate also decreased among the comparison group except among those 80 years or older. The population of adults 65 years or older who continue to maintain employment with employer-sponsored insurance coverage may be healthier and are likely not representative of the general older adult population.We did not expect the overdose rate to have decreased during the precoverage period; across the US overall, overdose-related deaths among those 65 years or older increased slightly during that time.This study has limitations. First, the study was limited to MA beneficiaries and not the overall population with Medicare Part D coverage; thus, our findings may not generalize to the entire population of older adults. Second, claims data have limited ability to detect and appropriately classify both of the primary outcomes, which thus may have been limited to events serious enough to require medical attention and to those appropriately coded by practitioners. Third, we cannot account for the true prevalence of benzodiazepine use in the population because patients were likely paying for prescriptions out of pocket or receiving coverage through other sources . Fourth, we did not adjust for patient-level characteristics that may have been changing during this time and potentially contributed to increased falls or overdoses, although our findings remained when restricted to those prescribed opioids.Compared with commercial coverage among similarly aged older adults, Medicare’s expansion of benzodiazepine coverage to older adults may have been associated with an increased rate of overdose among those 69 years or younger and 80 years or older and of injury in those 80 years or older. To our knowledge, this was the first analysis to consider the association of benzodiazepine coverage policy with the risk of overdose.


Type 2 diabetes is characterized by considerable heterogeneity in its etiopathogenesis and clinical presentation. We aimed to identify clusters of type 2 diabetes in Asian Indians and to look at the clinical implications and outcomes of this clustering.From a network of 50 diabetes centers across nine states of India, we selected 19 084 individuals with type 2 diabetes (aged 10–97 years) with diabetes duration of less than 5 years at the time of first clinic visit and performed k-means clustering using the following variables: age at diagnosis, body mass index, waist circumference, glycated hemoglobin, serum triglycerides, serum high-density lipoprotein cholesterol and C peptide (fasting and stimulated). This was then validated in a national epidemiological data set of representative individuals from 15 states across India.We identified four clusters of patients, differing in phenotypic characteristics as well as disease outcomes: cluster 1 , cluster 2 , cluster 3  and cluster 4 . While SIDD and MARD are similar to clusters reported in other populations, IROD and CIRDD are novel clusters. Cox proportional hazards showed that SIDD had the highest hazards for developing retinopathy, followed by CIRDD, while CIRDD had the highest hazards for kidney disease.Compared with previously reported clustering, we show two novel subgroups of type 2 diabetes in the Asian Indian population with important implications for prognosis and management. The coexistence of insulin deficiency and insulin resistance seems to be peculiar to the Asian Indian population and is associated with an increased risk of microvascular complications. Recently five distinct ‘clusters’ of individuals with diabetes with significantly different characteristics have been identified in a Scandinavian population.The unique Asian Indian phenotype predisposes them to young-onset type 2 diabetes (T2D).For the first time in India (and South Asia), clustering was done on 19 084 individuals with T2D using eight clinically relevant variables .Four replicable clusters were identified, two of which were unique to the Asian Indian population.The novel cluster ‘Combined Insulin Resistant and Deficient Diabetes’ is of particular importance as it is characterized by difficult-to-control hyperglycemia and increased hazards of kidney disease and retinopathy.Classifying Asian Indians with T2D into phenotypic clusters provides insights into the pathophysiological processes driving diabetes in this ethnic group, which could help in predicting the risk of complications and in focusing attention to individuals with the highest risk of morbidity and mortality.Global estimates suggest that 463 million individuals have diabetes as of 2019 and that this number will increase to 700 million by 2045.Recently, distinct ‘clusters’ or subgroups of individuals with type 2 diabetes have been identified in a Scandinavian population of 8980 individuals, based on five parameters representing the clinical presentation as well as the presence of insulin resistance and beta-cell dysfunction.Asian Indians (South Asians) represent an ethnic group with high predilection for developing type 2 diabetes; indeed, some of the largest increases in diabetes prevalence have been reported from the South Asian region. Type 2 diabetes in Asian Indians differs from that in white Caucasians in a number of significant ways. They tend to develop diabetes at a younger age and at lower levels of obesity than do white Caucasians. They also tend to progress faster from stages of ‘pre-diabetes’ to frank diabetes than members of other ethnic groups. The ‘Asian Indian phenotype’, characterized by high levels of abdominal fat and increased insulin resistance even at low levels of body mass index (BMI), has been postulated as a reason for this increased propensity to develop type 2 diabetes.It is therefore possible due to the above and the well-known younger age at diagnosis that clusters of type 2 diabetes identified in Asian Indians based on parameters used in the Western population might not behave exactly in the same manner with respect to treatment outcomes and risk of complications. In this paper, we attempt to identify distinct clusters of type 2 diabetes in Asian Indians and to look at the clinical implications and outcomes of this clustering. This study is part of the INdia-Scotland Partnership for pRecision mEdicine in Diabetes (INSPIRED) project.Data for this analysis were obtained from the electronic medical records of a tertiary care center for diabetes, which has 50 branches spread across nine states of India. Clinical data of more than 400 000 patients have been captured and stored in the common diabetes electronic medical records (DEMR) system of the center,The following examinations and investigations are performed for every patient during their clinic visits. Height, weight and waist circumference are measured using standardized techniques and the BMI calculated as weight (in kilograms) divided by the square of height (in meters). Blood pressure is recorded to the nearest 2 mm Hg from the right arm in a sitting position with a mercury sphygmomanometer .Blood samples are collected for the measurement of various parameters including fasting and postprandial plasma glucose, lipid profile, kidney and liver function tests, glycated hemoglobin (HbA1c), and C peptide (fasting and stimulated), while Glutamic Acid Decarboxylase (GAD) autoantibodies are measured in a selected subset of patients. Fasting plasma glucose (FPG), serum cholesterol, serum triglycerides and high-density lipoprotein (HDL) cholesterol are measured using Hitachi 912 Autoanalyzer . Fasting C peptide levels and stimulated (postbreakfast) C peptide levels are estimated by the electrochemiluminescence method on an Elecsys 2010 machine (Hitachi). To obtain the C peptide values, a fasting blood sample is obtained after an overnight fast of at least 8 hours and a postprandial sample after 90 min of a standard South Indian breakfast .Diabetes is diagnosed if the FPG level is ≥126 mg/dL (7.0 mmol/L) and/or 2-hour postload glucose level is ≥200 mg/dL (11.1 mmol/L) and/or if the patient has been prescribed pharmacotherapy for diabetes by a physician.26 mg/dL .0 mmol/LType 2 diabetes is diagnosed by absence of ketosis, good beta-cell reserve as shown by fasting C peptide assay >0.6 pmol/mL, absence of pancreatic calculi , and response to oral hypoglycemic agents for at least 2 years.8Assessment of complications is done as follows:A detailed retinal (fundus) examination is done by both direct and indirect ophthalmoscopy by a retinal specialist. Fundus photography is done using four-field stereo color retinal photography . An Early Treatment Diabetic Retinopathy Study grading system that has been modified and standardized in other population-based studies is used for the diagnosis of retinopathy.9 10Microalbuminuria is diagnosed if the albumin excretion was between 30 and 299 µg/mg and macroalbuminuria if albumin excretion is ≥300 µg/mg.2 as calculated by the CKD-Epi formula.Chronic kidney disease (CKD) is defined as an estimated glomerular filtration rate of less than 60 mL/min/1.73mDiabetic kidney disease is defined as the presence of CKD and/or albuminuria.Homeostasis Model Assessment of beta-cell function (HOMA-B) and insulin resistance (HOMA-IR) are assessed based on C peptide concentrations and plasma glucose using the HOMA calculator .12From the DEMR, we selected 373 000 individuals aged 10–97 years who had a diagnosis of type 2 diabetes. From these, we further selected 55 429 individuals who had baseline data available for the variables of interest, namely age at diagnosis, BMI, waist circumference, HbA1c, serum triglycerides, serum HDL cholesterol, and C peptide (fasting and stimulated). Of these, we selected 20 850 individuals with reported duration of diabetes less than 5 years at first clinic visit (mean 1.74±1.4 years). After merging the HOMA-B and HOMA-IR and removing individuals with HOMA-B >500 and HOMA-IR <20 (n=1512), removing outliers for the remaining variables by the 5SD method (n=188) and excluding individuals who had a positive test for GAD autoantibodies (n=66), we had a final sample size of 19 084.The k-means clustering method was done with k value of 4 using k-means function (max iteration=10 000) in R V.3.6.0. Cluster forming tendency of the data was validated by the Hopkins statistic value. The optimal number of clusters was determined based on silhouette width. Cluster-wise stability was computed by Jaccard bootstrap method through resampling of the data set 2000 times. A stable cluster generally yields Jaccard similarity index of greater than 0.75.Sensitivity analysis was done using three time periods for duration of diabetes <1 year, <3 years, and <5 years. Clustering tendency of the three different baseline diabetes duration data was found to have Hopkins statistic values of 0.19, 0.18, and 0.16, indicating that there were no significant differences in the cluster groups. Reclustering was done on men and women separately to validate the clustering and avoid the sex-dependent stratification effect on the phenotype variables. The minimum silhouette width was similar in both genders .The risk of development of diabetes complications was calculated using Cox regression models with covariate as a cluster label and adjusted for age at diagnosis and sex, after excluding individuals who already had complications at their first visit to the clinic. Cluster-wise time to reaching target goal was analyzed by Cox regression model. Cox proportional hazards assumptions were tested using R V.3.6.0.In order to validate the applicability of this clinic-based clustering to the general population, we attempted to replicate the clustering in the data set derived from the nationally representative Indian Council of Medical Research-India Diabetes (ICMR-INDIAB) study. ICMR-INDIAB is a nationwide population-based study on diabetes and related non-communicable diseases being carried out in all 29 states and 2 of the Union Territories of India based on a representative sample of each state. The detailed methodology of ICMR-INDIAB has been published elsewhere.15For purposes of this validation study, we selected 3851 individuals with type 2 diabetes from the INDIAB study population in 15 states of India and clustering was performed using the following six variables: age at diagnosis, BMI, waist circumference, HbA1c, serum triglycerides and serum HDL cholesterol. We excluded C peptide from the analysis as we did not have data on this variable in the population-based sample. After excluding 1521 individuals who did not have data on all our variables of interest as well as 88 outliers (5SD), we had a final sample size of 2204 individuals for the analysis. The k-means clustering was applied with k value as 4 (max iteration=10 000) in R V.3.6.0. The optimal cluster number was determined based on the silhouette width method.et al,Using the same five variables  reported by Ahlqvist We were unable to perform the Scandinavian clustering in the population-based INDIAB sample as we did not have information on variables such as HOMA-B and HOMA-IR in the epidemiological data set.Using cluster analysis based on eight clinically relevant variables , we were able to identify four replicable clusters of patients with type 2 diabetes in this Asian Indian population. The optimal number of clusters was based on silhouette width obtained from both the DEMR and the national ICMR-INDIAB data sets . Table 110.1136/bmjdrc-2020-001506.supp1Supplementary dataCluster 1, referred to as Severe Insulin Deficient Diabetes (SIDD), included 26.2% of clustered patients and was characterized by the lowest BMI and waist circumference, as well as the lowest C peptide (fasting and stimulated) levels. HOMA-B and HOMA-IR were both low in this cluster. These individuals had the highest HbA1c values and were more likely to be using insulin compared with the other clusters.Cluster 2 is a novel cluster which we refer to as Insulin Resistant Obese Diabetes (IROD). This comprised 25.9% of clustered patients. These individuals had the highest BMI and waist circumference and the highest C peptide levels. HOMA-B and HOMA-IR were also the highest for this cluster. Metabolic control was intermediate and individuals were more likely to be on metformin.Cluster 3, another novel group identified in this population, is referred to asCombined Insulin Resistant and Deficient Diabetes (CIRDD) and constitutes 12.1% of the study population. This group was characterized by the lowest age at onset. BMI and waist circumference were intermediate between SIDD and IROD. CIRDD had the highest triglyceride and lowest HDL cholesterol levels of all the four groups. C peptide levels were higher than SIDD, but lower than IROD. HOMA-B and HOMA-IR values were also intermediate between SIDD and IROD, indicating coexistence of insulin deficiency and insulin resistance. Metabolic control tended to be poor; however, only 15% were on insulin.Cluster 4, referred to as Mild Age-Related Diabetes (MARD), represented the most frequent cluster in this population (35.8%) and was characterized by later age at onset than other clusters. They were characterized by the highest HDL cholesterol, fairly preserved C peptide values and the best metabolic control of all the four groups. This group had the least use of insulin.The characteristics of the clusters did not differ when split by gender  and dura10.1136/bmjdrc-2020-001506.supp2Supplementary dataResults from the validation study with the INDIAB data show that the clusters identified in the clinic population are replicable in this nationally representative data set .In the INDIAB population, MARD was the most frequent cluster (34.8%), followed by IROD (30.3%), SIDD (24.7%) and CIRDD (7.6%). SIDD had the lowest BMI and waist circumference and highest HbA1c. IROD had the highest BMI and waist circumference and intermediate metabolic control. Individuals in the CIRDD cluster had BMI intermediate between SIDD and IROD, the highest triglycerides and lowest HDL, and high HbA1c and diastolic blood pressure. MARD had the highest age at diagnosis, the highest HDL levels, the lowest diastolic blood pressure and the best metabolic control. In all these respects, the clusters derived from the ICMR-INDIAB population behaved similar to those derived from the DEMR.et al.We then undertook the clustering based on the five variables used by Ahlqvist et alIn this analysis of around 20 000 individuals with type 2 diabetes from South India, we were able to identify four clusters of patients, differing in phenotypic characteristics as well as disease outcomes with respect to diabetes control and risk of complications. These findings replicated in a population-based study in India across 15 Indian states. Two of these subgroups (SIDD and MARD) correspond to the clusters identified by Ahlqvist One of these novel subgroups, which we have termed CIRDD, comprises a minority of patients with type 2 diabetes in our population, but represents a more aggressive phenotype in that the age of onset is lower and their metabolic control is almost as poor as those of the severe insulin deficient (SIDD) group. Also, they took almost as long as those in the SIDD cluster to reach treatment goals. It is likely that the presence of dual pathophysiology renders these individuals at high risk of developing diabetes at younger ages and predisposes them to poorer glycemic control. These individuals also had the highest serum triglyceride levels among all the clusters, possibly on account of accelerated lipolysis, secondary to insulin resistance. The insulin deficiency in these individuals could, in part, be attributed to beta-cell damage due to lipotoxicity. Patients with CIRDD also had the highest hazards of developing kidney disease and the second highest hazards for retinopathy. More aggressive therapy with a combination of agents targeting multiple pathophysiologies of type 2 diabetes may be indicated in these patients (perhaps as early as at the time of diagnosis) so as to help them develop a favorable ‘legacy effect’ and thus help prevent long-term complications. They also need to be screened more aggressively for complications, particularly nephropathy and retinopathy. All these of course have to be tested prospectively through well-planned randomized clinical trials.Individuals with the second novel subgroup, IROD, had better metabolic control than either SIDD or CIRDD, implying that they have sufficient beta-cell function to at least partially compensate for the obesity-related insulin resistance. However, they also had high risk of developing kidney disease. The higher risk of kidney disease in the two insulin resistant phenotypes (CIRDD and IROD) can be explained by the association of insulin resistance with increased salt sensitivity, glomerular hypertension and hyperfiltration.et alThe SIDD phenotype is similar to that described by Ahlqvist Individuals in the MARD subgroup formed the most frequent cluster in our population and behaved similar to the corresponding cluster in the Scandinavian population. They had the best metabolic control and the lowest risk of complications of all the four subgroups. However, these individuals had a significantly lower age of onset of diabetes (50.2 years) compared with those in the Scandinavian MARD cluster (67.3 years). This is likely explained by the lower age of onset of diabetes in the Asian Indian population in general. We cannot be certain whether our patients with MARD will continue to exhibit features of a mild phenotype of diabetes for the remainder of their lifespan; regular follow-up and monitoring is therefore essential even in this seemingly benign subgroup of type 2 diabetes.et al.Recent attempts to replicate the subclassification of type 2 diabetes in the US and Chinese population applying similar variables to those applied in the Scandinavian population have suggested that this European-oriented classification is generalizable to other ethnic groups.The strengths of our study include the use of a very large database on diabetes and identification of clusters based on phenotypic variables appropriate to the Asian Indian phenotype. However, the study does have a few limitations. Our institution being a private, pay-for-service clinic, data on all the variables of interest were not available for every patient due to financial constraints. Similarly, as our institution is also a tertiary referral center for diabetes, only the more severe or advanced cases of diabetes tend to visit the clinic, and this could have introduced an element of bias into our results. Our results, however, show that these clusters are replicable when applied to a nationally representative population derived from a large epidemiological study, suggesting that they are generalizable to the Asian Indian population with diabetes.In conclusion, we show that type 2 diabetes in the Asian Indian population can be classified into four phenotypic clusters with important implications for prognosis and management. While two of these clusters correspond to those reported in the white Caucasian population, the other two are novel and unique to the Asian Indian population. Of the four clusters, CIRDD is of particular importance as it represents a more aggressive phenotype of type 2 diabetes characterized by difficult-to-control hyperglycemia and markedly increased hazards of kidney disease and retinopathy. We acknowledge that categorizing patients into subtypes will have less power to predict complications than using the continuous data,


Caenorhabditis elegans, wounding the epidermis triggers an immune reaction and a repair response, but it is not clear how these are coordinated. Previous work implicated the microtubule cytoskeleton in the maintenance of epidermal integrity . Here, by establishing a simple wounding system, we show that wounding provokes a reorganisation of plasma membrane subdomains. This is followed by recruitment of the microtubule plus end-binding protein EB1/EBP-2 around the wound and actin ring formation, dependent on ARP2/3 branched actin polymerisation. We show that microtubule dynamics are required for the recruitment and closure of the actin ring, and for the trafficking of the key signalling protein SLC6/SNF-12 toward the injury site. Without SNF-12 recruitment, there is an abrogation of the immune response. Our results suggest that microtubule dynamics coordinate the cytoskeletal changes required for wound repair and the concomitant activation of innate immunity.The skin protects animals from infection and physical damage. In  Drechmeria coniospora infects Caenorhabditis elegans, by piercing the cuticle and growing through the underlying epidermis, it triggers an innate immune response, characterized by the induction of expression of a battery of antimicrobial peptide (AMP) genes  genes . These i cluster , which a cluster . Sterile cluster . In both cluster . The p38 cluster .C. elegans also triggers a Ca2+-dependent signalling cascade that promotes wound repair through actin ring closure  mutant, resulted in a delay of actomyosin assembly at the wound edge in multi-cellular wounds  cooperate with the actomyosin cytoskeleton during wound closure. In a single-cell wound-healing model using und edge . A studyr wounds . The dirC. elegans have been described to act in parallel to the innate immune response that accompanies wounding  domains, as well as a recruitment of EB1 and reorganisation of MTs around the wound. Rings of EB1 and actin close at the same pace, with EB1 at the leading edge. We demonstrate that specific inactivation in the adult epidermis of several alpha and beta tubulin isotypes leads to a decrease in MT dynamics, revealed by an absence of EB1 comets. This inactivation also blocks EB1 accumulation and decreases the recruitment of actin at the wound site. Further, it also abrogates the directed trafficking to the wound site of the key signalling protein SNF-12 and causes a block of the subsequent immune response. These results suggest that the recruitment of MT + end and EB1 to wounds coordinates the activation of the innate immune response and wound repair.Here, we develop the use of a 405 nm laser to wound the C. elegans is a single cell layer, covered on its apical surface by an impermeable cuticle that serves as an exoskeleton. The epidermal syncytium hyp7 can be divided into two distinct regions, one lateral, where the nuclei and most of the cytoplasm are, and the dorso-ventral part above the muscles where the apical and basolateral plasma membranes of hyp7 are juxtaposed and crossed by hemidesmosomes that anchor the muscles to the cuticle exoskeleton . Within 15 s after wounding, large heterogeneous membrane domains that are normally stable regrouped and extended towards the wound . Under resting condition in the young adult, in the lateral epidermis, we observed a disorganised pattern of foci linked with fine filaments   are another essential component of the cytoskeleton, but their role in wound healing has not previously been addressed directly in wing MTs , specifibrevity) . Under rbrevity)  similar brevity) . Upon wobrevity) . This wabrevity) . When acbrevity)  and 5. UC-2::GFP , we obseC-2::GFP .To address further the nature of the association of EB1 with actin, we quantified the respective speeds of their accumulation during ring closure. Kymograph analyses of the EB1 signal revealed two phases, the first one almost 10 times more rapid that the subsequent one . SimultaC. elegans tubulin proteins, TBB-2, was labelled with GFP . We observed that MTs were arranged predominantly in longitudinal but haphazard bundles in the lateral epidermis, in marked contrast to their ordered, parallel and circumferential organisation underneath the muscle quadrants in the dorsal and ventral epidermis as well as during molting  or tbb-2(RNAi) worms, EB1::GFP comets were no longer visible suggesting that MT dynamics had been severely compromised  conditions defined above. In resting conditions, short abrogation of tbb-2 from the L4 stage, altered the MT network in young adult worms, as visualised with TBB-2::GFP; it became less dense and less longitudinally structured in the lateral epidermis  and 6 β (TBB) genes. Some have been shown to act in specific cells or tissue, such as mec-7 (β) and mec-12 (α) in the mechanosensory cells  expression in the adult epidermis a few hours before infection did not affect AMP reporter gene induction nor the overall MTs pattern . Since tpidermis , howeverpidermis . Thus, i2 and the most apical RAB-11 recycling endosomes, and apical to LAAT-1-positive lysosomes  and fed with E. coli OP50, as described ]; frIs43 (rde-1(ne219) V; juIs346 III; frIs7 IV  II; zif-1(gk117) III  III  I (dnc-2[prt42(3xflag::GFP::DHC-2)] III (tba-1(pg77[TagRFP-T::TBA-1]) I (arx-2(cas607[GFP::ARX-2]) , RT343 pwIs82 ] ]; mcIs54 X  III , CZ147483p::RFP] , SA854 t-2]) III , SV1009 mCherry] , BOX188 -1.1]) I , GCP417 -2)] III , GN675 tBA-1]) I , GOU2047:ARX-2]) , MBA365 -119(+)] , XW10992::RAB-7] , ML1896 19(+)] X . All thefrSi9 is a single copy insertion on chromosome II at the location of the Nemagenetag Mos1 insertion (ttTi5605 of pNP151 (col-62p::Lifeact::mKate2_c-nmy3’utr). pNP151 was obtained by insertion of the col-62 promoter fused to Lifeact::mKate2 (myo-2p::mCherry) at 1.25 ng/µl, pCFJ104 (myo-3p::mCherry) at 5 ng/µl, pMA122 (hsp16.41p::PEEL-1) at 10 ng/µl, pCFJ601 (eft-3p::Mos1 transposase) at 20 ng/µl and pNP21 (unc-53pB::GFPnsertion  ttTi5605::mKate2  into the::mKate2 . It was frSi13 is a single copy insertion on chromosome II (ttTi5605 location) of pNP159 (dpy-7p::GFP::RAB-11) by CRISPR using a self-excising cassette (SEC) (dpy-7p::GFP::RAB-11 (kindly provided by Grégoire Michaux) into the pNP154 vector. pNP154 was made from a vector containing the SEC for single insertion on Chromosome II at the position of ttTi5605 . pNP159 was injected in N2 at 10 ng/µl together with pDD122 (eft-3p::Cas9) at 40 ng/µl, pCFJ90 (myo-2p::mCherry) at 2.5 ng/µl, pCFJ104 (myo-3p::mCherry) at 5 ng/µl, and #46168 (eef-1A.1p::CAS9-SV40_NLS::3’tbb-2te (SEC) . pNP159 frEx597 and IG1663 frEx577.Different transgenic strains containing several tagged version of SNF-12 were generated including IG1784 sta-1 was used as a negative control. To avoid developmental delay or lethality, RNAi experiments of tubulin or actin genes were either performed from the L4 stage or in a strain allowing gene silencing primarily in the epidermis from the L4 stage (IG1327 (rde-1(ne219) V; juIs346 III; frIs7 IV) rde-1 RNAi resistant mutant in the adult epidermis as previously described  before being exposed to fungal spores as previously described  as described in Analysis of Since in preliminary experiments we observed that immobilization using latex beads impacted vesicle and protein dynamics in the epidermis, young adult worms (containing less than five eggs) were immobilized on a slide using 0.01% levamisole. Time lapse and/or Z stack were acquired using two different spinning disk microscopes: an inverted Visitron Systems GmbH spinning disk with a Nikon 40X oil 1.3 NA objective and 1.5X lens, and a Roper inverted spinning disk with a Nikon 100X oil 1.4 NA objective, were used with their respective acquisition software (VisiView or Metamorph). For spatially resolved time-lapse imaging, a Zeiss LSM780 confocal microscope whose GaAsP detector was chosen for its high sensitivity and controlled through the Zen acquisition software.Laser wounding was performed by using a 405 nm laser at its maximum power using either Visitron, iLas FRAP or Zeiss FRAP systems. The respective laser power before objectives was: 7.3 mW, 1–2 mW and 3 mW. 1–3 s of laser pulses were applied on a circular region of interest (ROI) of 8 × 8 pixels with the Visitron (~2.13 µm in diameter), 21 × 21 pixels with the Nikon (~1.68 µm in diameter) or with the confocal (~1.8 µm in diameter) on the apical plane of the lateral epidermis either left or right of the seam cells. Each worm was wounded from 1 to 3 times.Time lapse acquisitions were 10 to 20 min long. Z stack acquisitions were performed on 4–6 planes spaced by 0.3 or 0.5 µm. High temporal resolution was obtained on one single plane with a time interval of 0.2 to 0.5 ms. Laser wound was performed between the first and the second Z stack. For colocalisation analysis, we initially controlled that there was no signal cross talk using single labelled strains. We then performed sequential acquisitions on the strains expressing two fluorescent proteins. Experiments requiring comparative analysis were performed on the same day.All image processings were done using Fiji software. A custom Image J macro was used to correct for Z-drift, while XY drift was corrected either with the Image Stabilizer or the Correct 3D drift plugin from ImageJ or the online available macro ‘NMSchneider/FixTranslation’.2 ROI in the lateral hyp7 or in an 80 µm2 ROI in the seam cell in five worms per RNAi condition. Similarly, we measured SNF-12 cluster density in the lateral hyp7, analysing six worms per RNAi condition. Quantification of EB1 recruitment at the wound site was performed by measuring the RawIntDen (sum of the pixel intensities) of a selected ROI of 256 µm2 for EB1 centred at the wound site. Since EB1 brightness differed before wounding between worms, for each single wound, only pixels with values above a defined brightness threshold were considered, the threshold being set to the [mean + 3 x standard deviation] in the ROI before wounding. Quantification of SNF-12 recruitment was performed by measuring the mean intensity of a selected ROI of 18 µm2 centred at the wound site at specific time points. Statistical significance was determined using a non-parametric Mann-Whitney test (Graphpad Prism).To analyse EB1 density before wounding, we manually counted fluorescent dots as described in To visualise the speed and directionality of EB1, RAB-11 and SNF-12, a temporal projection was done using a temporal-Color code (plugin from Kota Miura) on 120 frames over 2 min (one frame per second) before wounding for all tree makers and on 32 frames over 10 min after wounding for SNF-12.To analyse particle motility before and after wounding, we used the macro KymographClear described in 2 located on the centre of the wound was selected 15 min post-wounding. The area inside the actin ring was detected using the Huang automatic threshold method from Image J. To first compare the recruitment of EB1 versus actin, the radial profile (plugin from Paul Baggethun) was calculated, after determination of the centroid of the wound for each time point by manually selecting the wound area. For each radial profile, we extracted the radius corresponding to the maximum intensity and plotted this radius versus time. To further quantify, we build kymographs by considering masks that corresponds to stripes oriented in the anterior-posterior (AP) direction and including the ablation region. We obtained one-dimensional profiles by averaging the signal intensity over the circumferential direction; to account for variations in signal intensity during time, we normalised each profile by its maximal value. These colour-coded profiles were concatenated to produce a kymograph.To compare the actin ring closure upon different RNAi condition, a ROI of 80 pixelsTo analyse the microtubule orientation, we obtained maps of the local orientation of microtubules of an ROI of 20 × 20 µm of confocal images of MAPH-1::GFP reporter strain using the ImageJ plugin OrientationJ and custom Matlab scripts (Source file plotorientation), which provide the direction of the largest eigenvector of a local intensity structure tensor . We then In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen Didier Stainier as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: William Bement (Reviewer #2); Andrew D Chisholm (Reviewer #3).Thank you for submitting your article "Microtubule plus-end dynamics link wound repair to the innate immune response" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:C. elegans epidermis. The authors characterize the cellular responses to wound healing and note a remarkable accumulation of the microtubule plus end binding protein Eb1 at the wound site. They further suggest that this regulates both subsequent actin accumulation and the enrichment of a transcription factor that mediates the immune response to wounding. These interesting observations link a novel microtubule-dependent wound response to a local immune response.This manuscript establishes a new model for cellular wound healing, using laser induced cutting of the Essential revisions:1) Organization of microtubules. The data on accumulation of Eb1 puncta at the wound site is very clear. However, neither the organization of their paths, nor a clear image of microtubule organization is presented. Further investigation of this will allow increased clarity of the model for microtubule function in wound healing.2) Quantitation of data. This manuscript characterizes a new method for wounding and describes the dynamics of various cellular components during the healing process. To make it a more valuable resource for future research, increased quantitation of the data, showing both reproducibility over a number of cells, as well as showing the kinetics of various components relative to each other should be provided.There were a number of points that were not entirely clear in the manuscript, such as how microtubules control SNF-12 localization (and further discussed in detail below). Changes to the text to clarify many of these confusions, listed in the reviews below, would strengthen the manuscript.Reviewer #1:C. elegans lateral epidermis. Using this assay, the authors characterize the membrane and cytoskeletal responses to wounding, which are similar to previously studies Drosophila and Xenopus models. They also show a remarkable increase in the microtubule end-binding protein Eb1, at the wound site. Functionally, they find that disrupting microtubule dynamics inhibits actin accumulation and the normal immune response. There are several really interesting observations in this paper, however, the conclusions at present could be strengthened by increased rigor throughout as different interpretations of the data exist.This manuscript establishes a robust assay for wounding the 1) As they are establishing a novel wounding methodology and characterizing the response, increased quantitation of the dynamics and extent of responses in Figures 1-3 are required. For all of these, it would be ideal to have (in addition to the videos shown) quantitations over several videos to see the reproducibility of the responses. This will also be helpful in laying out the temporal order of changes occurring. I find this to be especially important as the authors suggest that the microtubule response occurs before the actin accumulation, though this is not clear from the video presented.2) Constriction of the F-actin at the wound. This is documented for a single wound in Figure 3H, but is not obvious in the videos provided. It is important to document whether this is a constriction or just a loss of F-actin over time. This is especially confusing as the Introduction states that healing hear is not driven by acto-myosin contraction, but rather by Arp2/3 mediate F-actin assembly.3) The accumulation of tubulin at wound sites was dramatically less than Eb1. This is perhaps surprising and should be discussed. Along these lines, it was not clear whether the authors could track Eb1 puncta within the wound site due to density of puncta. Are all puncta motile and analyzable?4) Epistasis of actin and microtubule accumulation. The authors state in the Discussion that Eb1 accumulates before actin. However, this is not clear from the videos provided. Functionally, there is a decrease in F-actin recruitment when tubulin genes are knocked down or microtubules disrupted. However, the timepoints here were late and don't address whether initial accumulation occurs but does not increase or whether it peaks and goes down, or never increases in the first place. An additional concern along these lines is that the F-actin as reported by LifeAct appears to be significantly disrupted in the unwounded tubulin mutant epidermis (S2B – to a similar extend as the Act2 mutant). This raises the caveat that it may not be a wound-induced effect, but a general effect on cytoskeletal organization.5) Connection to the immune response. I felt that I needed more discussion of how the SNF-12 localization connected to the phenotype. Is the localization of the SNF-12 transcription factor at the wound site necessary for its transcriptional activity? How do you imagine this occurs. Are microtubules required for MAPK kinase activity or DCAR-1 signaling in any way? Authors should rule out the possibility that microtubules affect this signaling axis and that they work through localization of SNF-12. Along these lines, in the induction of AMP's in response to cuticle defects similarly dependent on tubulin genes?6) SNF-12 clusters were significantly lower in unwounded epidermis of tubulin mutants compared to controls. One possibility is that microtubules are required for cluster formation and not localization of SNF-12.Reviewer #2:While not widely recognized, the response of single cells to damage is just as important as the response of tissues to damage. Not surprisingly then, a growing body of work suggests that cell damage elicits a conserved response that involves the mobilization of a variety of signaling, lipid, and cytoskeletal components. There are at least two biological consequences of this response: First, the hole in the cell is patched or otherwise closed, preventing the cell from simply lysing. Second, the damage material is cleaned up and removed, such that the wound site is eventually restored to its original state . To a first approximation, these biological imperatives parallel those of damaged tissue, with clotting being responsible for plugging the hole and the action of immune system being responsible for the clean-up.C. elegans embryo. To accomplish this, they develop a laser wounding system that permits them to make relatively precise wounds in the embryonic syncytial epiderms. They first show that repair of these wounds has several characteristics in common with the repair response in other damaged cell types. They then identify a striking accumulation of EB1, a protein that binds to the plus ends of growing microtubules, around wounds, leading them to conclude that wounding elicits local microtubule reorganization around wounds. They report that they can block this response by depleting microtubule subunit proteins  and that this manipulation not only impairs the immediate wound response, but also impairs the innate immune response as monitored by expression of an AMP reporter gene. They further link microtubules to recruitment of SNF-12-a protein involved in the innate immune response-to wound sites. Based on these results the authors conclude that the microtubule response is an important feature of both the basic  wound response and the innate immune response.In the current paper, Taffoni et al. ask whether the single cell wound response is somehow linked to and coordinated with the immune response in the Several features of this paper are very appealing: the question is of fundamental importance for those who seek to understand wound repair, the authors nicely make the link between the basics of cell damage repair as it occurs in their system and in other systems, and much of the imaging clearly illustrates the points the authors are making. Perhaps most importantly, the link between the microtubule response in a single cell  and the mobilization of the innate immune response is an important one.However, several points need to be addressed:1) It is very difficult to tell what, exactly, is happening with the microtubules at the wound site because the different approaches seem to be suggesting different things. That is, in some cell types microtubules accumulate around wounds in a radial array such that many microtubules are focussed toward the wounds . Such an arrangement would be consistent with the accumulation of the EB1-GFP fluorescence around wounds (but see below). However, it is not consistent with the movement of the EB1-GFP comets, which appear to move in all directions and in the absence of any obvious bias toward the wounds. The live imaging of the microtubules themselves  is also inconsistent with a radial array. As with the movement of the comets, it seems to indicate that the microtubules are randomly oriented with respect to the wound. Now, one might be tempted to imagine that the intense EB1-GFP signal that accumulates around the edge of the wound is sufficient evidence of some kind of wound-localized array of microtubules but there are two problems with this notion. First, that signal is not obviously in comet form, rather, it just appears to be a uniform slathering of GFP on the plasma membrane. Second, EB1 only associates with growing microtubules plus ends; it does not associate with stable microtubule plus ends . Thus, the apparently stable accumulation of EB1-GFP at the wound edge is rather mysterious. To settle this point the authors need to fix wounded embryos and stain for microtubules or find an alternative way to characterize the organization of microtubules around wounds.2) This may reflect my ignorance, but it is not at all clear to me what links the transport of SNF-12 to wounds and the triggering of the innate wound response. Does something special happen to the SNF-12 at wound edges? I was under the impression that SNF-12 exerts its effects on transcriptional control of the response after entering the nucleus as previously reported by the authors . While it is not incumbent on the authors to trace out all of the steps in the pathway that leads to an upregulation of the immune transcriptional response, it would be helpful to have a least a little bit of information on why recruitment to the wound is important.Reviewer #3:C. elegans. Microtubule dynamics have been implicated in several cell repair processes, and were implicated in C. elegans skin wound repair, but had not previously been examined directly. The authors use extensive live imaging of fluorescently tagged markers to show that MT dynamics are affected by wounding and link physical wound repair to innate immune response to damage via transport of the signalling molecule SNF-12. The authors provide extensive descriptions of the dynamics of various membrane and organelle markers after injury. Overall this is a thorough and careful study that advances our understanding of the cell biology of wound repair in vivo.Taffoni et al. examine the role of microtubule dynamics in epithelial wound responses in the epidermis of the nematode  Essential revisions:1) Organization of microtubules. The data on accumulation of Eb1 puncta at the wound site is very clear. However, neither the organization of their paths, nor a clear image of microtubule organization is presented. Further investigation of this will allow increased clarity of the model for microtubule function in wound healing.We thank the reviewers for their insightful comments that led us to define a more precise model. We have now better characterised EB1 and microtubule dynamics upon wounding. We have generated several new strains and combined imaging with advanced analysis techniques.Detailed analysis of EB1 revealed 2 regimes. The first was analysed by tracking EB1 comets after wounding. It matches the typical speed of microtubule + tips. This tracking also revealed that EB1 comets were not associated with any directional bias toward the wound, but seem to stop at the wound en-passant. The second regime of EB1 starts when it accumulates at the wound in the form of a ring. Kymograph analyses of both EB1 and actin signals reveal that the EB1 ring closes at the same speed as the actin ring, while also confirming our previous observation that EB1 precedes actin, EB1 being found at the leading edge of actin while the ring closes.C. elegans embryo , and MAPH-1, a MT associated protein MAP1, as well as more resolutive confocal analyses of the tubulin TBB-2 reporter. The observations with DNC-2 were fully consistent with what we had seen with EB1 and confirmed an accumulation of MT + tips at the wound at the same time as actin. Second, confocal analysis revealed MT growing and retracting at the wound just before and during actin accumulation. Third, quantification of MT orientation and tracking of EB1 comets, allow us to rule out a radial organisation of MT at the wound, consistent with the non-directional dynamics of EB1 comets. These data support a dynamic orthoradial organisation of the MT at the wound, and suggest that the microtubules reorganised to knit a web around the wound as the actin ring closes.We have complemented these observations with more detailed examination of MT by analysing several MT-binding protein reporters, like the dynactin protein DNC-2, known to bind MT + tips in the Reviewer #2 pointed out (see below), generally, “EB1 only associates with growing microtubules plus ends; it does not associate with stable microtubule plus ends ”. To reconcile this model with our observations that EB1 stops at the wound site and then migrates inward with the actin as the wound closes, we propose as a working model that MT-associated EB1 binds to actin at the wound and then dissociates from MT during closure. The actin and MT cytoskeleton have long been known to be intimately linked, for example during the directional migration of axonal growth cones. Indeed the main molecular linkers connecting actin and microtubules belong to the microtubule plus-end tracking proteins (+TIPs), including EB1 , and a direct association of EB1 with actin has been demonstrated in vitro . Recent studies also proposed a role of actin in the MT spindle formation during mitosis . Our results suggest a model where MT dynamics at the wound would permit the association of the +TIPs EB1 with actin that would then facilitate ring closure.2) Quantitation of data. This manuscript characterizes a new method for wounding and describes the dynamics of various cellular components during the healing process. To make it a more valuable resource for future research, increased quantitation of the data, showing both reproducibility over a number of cells, as well as showing the kinetics of various components relative to each other should be provided.The formation of an actin ring is a highly reproducible phenotype in our hands. It has become our general control  that we use to adjust the 405 laser settings to obtain a normal wound response in control worms. And it has allow us to implement a new wounding platform on a confocal microscope, still using a 405 laser from a FRAP module, in addition to our previous setting on two different spinning disk microscopes. We have now generated several new strains combining the actin reporter with MT binding proteins, and added quantification for actin and EB1 recruitment as mentioned above. In addition to new videos, we now provide kymograph analysis. We also have added the number of worm analysed in the different figures and a detailed source data file reporting for all quantification we have made . Finally, we have added a supplementary figure  with quantification of the onset of recruitment and a proposed timeline of events.There were a number of points that were not entirely clear in the manuscript, such as how microtubules control SNF-12 localization (and further discussed in detail below). Changes to the text to clarify many of these confusions, listed in the reviews below, would strengthen the manuscript.We have changed the text as explained in more detail below.Reviewer #1:C. elegans lateral epidermis. Using this assay, the authors characterize the membrane and cytoskeletal responses to wounding, which are similar to previously studies Drosophila and Xenopus models. They also show a remarkable increase in the microtubule end-binding protein Eb1, at the wound site. Functionally, they find that disrupting microtubule dynamics inhibits actin accumulation and the normal immune response. There are several really interesting observations in this paper, however, the conclusions at present could be strengthened by increased rigor throughout as different interpretations of the data exist.This manuscript establishes a robust assay for wounding the 1) As they are establishing a novel wounding methodology and characterizing the response, increased quantitation of the dynamics and extent of responses in Figures 1-3 are required. For all of these, it would be ideal to have (in addition to the videos shown) quantitations over several videos to see the reproducibility of the responses. This will also be helpful in laying out the temporal order of changes occurring. I find this to be especially important as the authors suggest that the microtubule response occurs before the actin accumulation, though this is not clear from the video presented.We have added quantifications as mentioned above.2) Constriction of the F-actin at the wound. This is documented for a single wound in Figure 3H, but is not obvious in the videos provided. It is important to document whether this is a constriction or just a loss of F-actin over time. This is especially confusing as the Introduction states that healing hear is not driven by acto-myosin contraction, but rather by Arp2/3 mediate F-actin assembly.We provide clearer evidence for the closure of the actin ring in new figures and videos. We indeed believe that the actin ring is not closing by contraction but by inward directed polymerisation of actin. We included the recruitment of Arp2/3 complex protein ARX-2 , confirming the role of branched actin polymerisation in actin closure demonstrated in .3) The accumulation of tubulin at wound sites was dramatically less than Eb1. This is perhaps surprising and should be discussed.Please see our answer above and to point 1 of Reviewer #2.Along these lines, it was not clear whether the authors could track Eb1 puncta within the wound site due to density of puncta. Are all puncta motile and analyzable?It was indeed not possible to track EB1 comets after they reach the wound due to their dense accumulation. But we were able to track the peripheral comets in 9 wounds and 5 control regions. These analyses reveal that their trajectories were not directed toward the wound. We believe that EB1 signal do reflect the dynamics of the + tips MT until they reach the wound . As mentioned above, we have now visualised the recruitment at the wound of another MT + tip protein dynactin 2.When at the wound, EB1 comets stop, accumulate as a ring that migrates at the same speed as the actin ring to close the wound . As we now discuss, these observations are compatible with EB1 binding to actin at the wound.4) Epistasis of actin and microtubule accumulation. The authors state in the Discussion that Eb1 accumulates before actin. However, this is not clear from the videos provided.As mentioned above, in addition to new videos, we now provide kymograph analysis. Both show that EB1 associated at the leading edge of the actin while the ring forms and as the ring closes.Functionally, there is a decrease in F-actin recruitment when tubulin genes are knocked down or microtubules disrupted. However, the timepoints here were late and don't address whether initial accumulation occurs but does not increase or whether it peaks and goes down, or never increases in the first place.tbb-2 RNAi treated worms .We have added a quantification of the phenotype and show that actin is recruited later and to a lesser extent in An additional concern along these lines is that the F-actin as reported by LifeAct appears to be significantly disrupted in the unwounded tubulin mutant epidermis (S2B – to a similar extend as the Act2 mutant). This raises the caveat that it may not be a wound-induced effect, but a general effect on cytoskeletal organization.tbb-2 abrogation during the L4 larval stage. Between L4 and adult, worms molt. There is a major reorganisation of both actin and MT networks during molting ; and the reorganisation of both networks seems to highly coordinated (NP unpublished). Altering MTs could therefore impact the organisation of the actin cytoskeleton. We now mention this briefly in the Discussion. We expect that the coordination of MT and actin networks upon molting or after wounding will use the same mechanisms, and this will be the subject of future studies.We thank the reviewer for highlighting this point that we had reported but not discussed in the original submission. The actin pattern is somewhat affected in young adults after tbb-2(RNAi)-treated worms, some worms have an almost normal actin network. If we analyse these worms, we can still observe the concentric wave of actin as in the control but less actin accumulates at the wound site and wound closure is less efficient . High resolution live imaging with a confocal microscope revealed that some MT at the wound site do pattern the growing actin patch . Together with the fact that EB1 is found at the leading edge of the actin ring during its formation and closure, this suggests that MT dynamics facilitate actin accumulation at the wound. We have modified the text accordingly.Returning to the reviewer’s concern, within a population of 5) Connection to the immune response. I felt that I needed more discussion of how the SNF-12 localization connected to the phenotype. Is the localization of the SNF-12 transcription factor at the wound site necessary for its transcriptional activity? How do you imagine this occurs.Please see our answer to point 2 of Reviewer #2. We regret that we didn’t make clear that SNF-12 is an amino acid transporter that we believe is acting as a signalling protein. We have added to the Discussion “We speculate that this spatial regulation of signalling may contribute to restricting defence gene activation to the immediate vicinity of a wound or site of infection within the major epidermal syncytium.”Are microtubules required for MAPK kinase activity or DCAR-1 signaling in any way? Authors should rule out the possibility that microtubules affect this signaling axis and that they work through localization of SNF-12.We apologize if we were not clear enough in the text. DCAR-1 signalling is entirely dependent upon SNF-12, so yes, microtubules affect this signalling axis and they work through localization of SNF-12. We have changed the text in the Discussion to make it clearer. We know that DCAR-1 (the most upstream element of the MAPK pathway described so far) is not recruited to the wound site before SNF-12 but much later, one hour after wounding with a general increased expression. We believe that these observations go beyond the scope of the current paper.Along these lines, in the induction of AMP's in response to cuticle defects similarly dependent on tubulin genes?nlp AMP genes . We have found that blocking MT dynamics suppresses this high expression (N.P. unpublished). We could add these results if absolutely required but believe they are beyond the scope of the current paper.We previously reported that in furrow-less Dpy mutants there is a constitutive high expression of the 6) SNF-12 clusters were significantly lower in unwounded epidermis of tubulin mutants compared to controls. One possibility is that microtubules are required for cluster formation and not localization of SNF-12.tba-2, SNF-12 patterning and recruitment were severely compromised .” “Together, these results suggest that MTs play an important role in SNF-12 localization and dynamics and thereby in the induction of AMP gene expression.”We agree and we now write in the text, “When we disrupted MTs, via RNAi of Reviewer #2:[…] Several points need to be addressed:1) It is very difficult to tell what, exactly, is happening with the microtubules at the wound site because the different approaches seem to be suggesting different things. That is, in some cell types microtubules accumulate around wounds in a radial array such that many microtubules are focussed toward the wounds .. Such an arrangement would be consistent with the accumulation of the EB1-GFP fluorescence around wounds (but see below). However, it is not consistent with the movement of the EB1-GFP comets, which appear to move in all directions and in the absence of any obvious bias toward the wounds. The live imaging of the microtubules themselves  is also inconsistent with a radial array. As with the movement of the comets, it seems to indicate that the microtubules are randomly oriented with respect to the wound. Now, one might be tempted to imagine that the intense EB1-GFP signal that accumulates around the edge of the wound is sufficient evidence of some kind of wound-localized array of microtubules but there are two problems with this notion. First, that signal is not obviously in comet form, rather, it just appears to be a uniform slathering of GFP on the plasma membrane. Second, EB1 only associates with growing microtubules plus ends; it does not associate with stable microtubule plus ends . Thus, the apparently stable accumulation of EB1-GFP at the wound edge is rather mysterious. To settle this point the authors need to fix wounded embryos and stain for microtubules or find an alternative way to characterize the organization of microtubules around wounds.We agree with the reviewer that our observation in the nematode epidermis are not wholly consistent with what has been reported in other systems. To address the reviewer’s point, we chose not to fix the worm as explained above, but have now analysed more MT markers like DNC-2, MAPH-1 and another tubulin isotype TBA-1, in each case generating strains that allow us to visualise at the same time EB1 and/or actin recruitment. Further, we have increased the spatial resolution of our study by implementing the FRAP module for wounding on a confocal microscope, tracked EB1 comets and analysed EB1 and actin ring closure. Together these allow us to have a more complete description of the reorganisation of MTs that accompanies wounding, as explained above.2) This may reflect my ignorance, but it is not at all clear to me what links the transport of SNF-12 to wounds and the triggering of the innate wound response. Does something special happen to the SNF-12 at wound edges? I was under the impression that SNF-12 exerts its effects on transcriptional control of the response after entering the nucleus as previously reported by the authors . While it is not incumbent on the authors to trace out all of the steps in the pathway that leads to an upregulation of the immune transcriptional response, it would be helpful to have a least a little bit of information on why recruitment to the wound is important.This is a very good question. We do not know how or why the localisation of the amine transporter SNF-12 would affects its signalling. It is known that, to be functional, some signalling complexes need to be assembled on particular vesicular compartments (e.g. TLR7). In our system, SNF-12 localisation could potentially affect its interaction with the Stat transcription factor STA-2.Understanding this will be a challenge for the future. We have altered the Discussion to address the point explicitly: “The fact that overexpression in the epidermis of the MT severing protein Spastin caused a similar effect on SNF-12 dynamics as knocking down tubulin expression, supports a role for MT in positioning SNF-12-containing vesicles. […] We speculate that this spatial regulation of signalling may contribute to restricting defence gene activation to the immediate vicinity of a wound or site of infection within the major epidermal syncytium.”