Large-scale longitudinal biobank data can be leveraged to identify genetic variation contributing to human diseases progression and traits trajectories. While methods for genome-wide association studies (GWAS) of multiple correlated traits have been proposed, an efficient multiple-trait approach to model longitudinal phenotypes is not currently available. We developed GAMUT, a genome-wide association approach for multiple longitudinal traits. GAMUT employs a mixed-effects model to fit longitudinal outcomes where a fast algorithm for inversion by recursive partitioning of the random effects submatrix is introduced. To evaluate performance of the algorithms introduced and assess their statistical power and type I error, stochastic simulation was conducted. Consistent with our expectation, power was greater for cross-sectional (CS) than longitudinal (LT) effects, particularly with a diminishing LT/CS ratio. With a minimum minor allele count of 3 within genotype by time categories, observed type I error was roughly equal to theoretical genome-wide significance. Additionally, 28 blood-based biomarkers measured at 2 time points on participants of the UK Biobank were used to compare GAMUT against single-trait standard and longitudinal GWAS (including rate of change). Across all biomarkers, we observed 539 (CS) and 248 (LT) significant independent variants for the GAMUT method, and 513 (CS) and 30 (LT) for single-trait longitudinal GWAS, respectively. Only 37 variants were identified by modeling rates of change using standard GWAS. Genome wide association studies have traditionally been carried out using cross-sectional data and one outcome at a time. With the advent of global biobanks and direct-to-consumer genomic testing, longitudinal health data recorded on diverse clinical aspects of each individual have opened the door for more statistically rigorous genome-wide association modeling. The availability of such data makes it possible to discover novel genetic variants associated with trajectories of trait changes or disease progression within individuals, as well as pleiotropic variants impacting multiple phenotypes.2. Mixed models are a commonly used approach for handling relatedness in conventional GWAS, e.g. as implemented with EMMA3, GEMMA4, and SAIGE5. While mixed models may also be used to model multiple-traits or longitudinal phenotypes, computational efficiency may be difficult to achieve for analysis of large-scale cohorts. This is due to the fact that mixed model approaches require the complex task of estimating variance components before setting up and solving a large system of equations, which requires the inversion of large matrices associated with the random components in the mixed model. Sikorska et al.6 introduced computationally efficient approaches to utilize mixed models in longitudinal GWAS. Their approximate approaches were shown to be precise in estimating cross-sectional and longitudinal effects of each SNP, where cross-sectional effects are those comparable to conventional GWAS and longitudinal effects define how each SNP may impact the change in a trait over time. In an effort to simplify computations for longitudinal GWAS, researchers have often collapsed the multiple measurements into one rate of change between the earliest and the latest measurement of each individual. The rates of change were then fed into conventional GWAS workflows where associations with each phenotype were investigated independently. The collapsing approach was historically discussed and found to be theoretically problematic due to loss of information8.Mixed models are the approach of choice for the analysis of longitudinal data because they account for variability between measurements across multiple time points on one phenotype as well as variability among measurements on multiple phenotypes10. In addition, the availability of repeated measurements of the same phenotype on each individual affords an opportunity to assess genetic effects on changes in disease traits or biomarkers over time. Despite the advantages longitudinal and multiple-trait approaches each provides for identifying genetic determinants of disease and disease progression, implementation of such methods is also known to be computationally burdensome.When individuals are assessed for multiple correlated phenotypes, a multivariate approach to jointly evaluate those phenotypes was shown to be more statistically rigorous1, for longitudinal phenotypes to a multiple-trait setting. The approach efficiently models multiple correlated phenotypes as well as multiple measurements recorded through time. To evaluate the computational efficiency of the approach as well as its ability to identify variant associations what would otherwise be missed with a conventional GWAS framework, 28 quantitative traits with repeated measures in up to 16,622 UK Biobank participants were assessed. Methods to obtain the inverse of large matrices are also introduced in an effort to maximize the computational efficiency of multiple-trait longitudinal GWAS.In the current study, we extend the mixed model approach, GALLOPIn standard GWAS, we test the association of each variant with the risk to a disease or to an extreme value of a quantitative trait, such as high cholesterol. In longitudinal GWAS, we test the association of the genetic variant with the progression of the disease or with the change in a quantitative biomarker for the disease over time. Single-trait longitudinal GWAS fits genetic variants one-by-one in the mixed model  which inFollowing from a standard GWAS of a single longitudinal phenotype, multiple-trait longitudinal GWAS have been explored and shown to improve statistical power for discovery. In this type of analysis, multiple correlated traits are jointly modeled and missing records are implicitly inferred based on the covariance between traits. Multiple-trait longitudinal GWAS is typically performed in 3 steps: estimating variance components, then constructing the components of the mixed-model equations, and performing the association analysis. First, genetic and environmental variances and covariances are estimated once using a null model without genotypes, and then these are utilized in a transformed version of Henderson’s mixed model equations that are used with minor modifications for the hypothesis testing of each variant separately.10, both the single- and multiple-trait mixed models can be written as in  was utilized in estimating variance components, in which a single covariance between effects of every 2 traits but Computationally efficient average information restricted maximum likelihood , where A and D are square submatrices, the inverse is obtained using the identity,To simplify computations for the single-trait case, Sikorska et al.ystem in  so that ystem in  by \docuD in the next round. As shown in Fig. A, B, C, and D at round 0 and used that as the inverse of the larger D submatrix at round 1, i.e. D submatrix of the next round; note that the D matrix itself was not needed in the next round.For more than 4 blocks, the same identity in  were appFor the current multiple-trait model, the inverse was obtained using our proposed recursive partitioning in less computing time and resources than with other direct sparse inverse techniques see “” for the1. In principle, the linear system in 10) inclujth time point of individual For individual Three correlated traits were considered in the current simulation with 3 CS and 3 LT effects for each SNP. We use the vector In our simulation strategy, we chose to reduce the covariance between cross-sectional and longitudinal effects. We tested multiple ratios for the cross-sectional to longitudinal effects by setting the value of The UK Biobank is a prospective cohort study with rich genetic and health data from half a million participants. The UK Biobank study was carried out in accordance with relevant guidelines and regulations as approved by the NHS National Research Ethics Service . Participants in the study gave full informed signed consent.First and second measurements of 28 blood biomarker traits of UK Biobank participants of European ancestry were evaluated for cross-sectional and longitudinal associations with imputed array genotypes. Phenotypes assessed were from UK Biobank field IDs  and heart disease who may need more than statin.To compare GALLOP and GAMUT with an approach that evaluates SNPs for their associations with rates of change, a single value per individual was derived as the difference between the two measurements divided by the time interval between measurements. The GWAS model for rates of change did not include time but included the baseline measurement as a scaled covariate. All other covariates were the same across the methods studied. Models for rates of change included those without both baseline and time, as well as an addition of both baseline and time.To test the impact of comorbidity and the number of repeated measures on the longitudinal GWAS outcomes, triglycerides (TG) were extracted from the primary care data, referred to as general practice or GP data. TG measures on 8968 participants of European ancestry who were diagnosed with coronary artery disease (CAD) at any age using all available clinical history information were extracted from the UK Biobank GP data. The dataset included nearly 99,000 observations on 8968 individuals with 3 to 35 TG measures per participant over an average period of 6 years. A comparable group of 8587 individuals with 3–35 repeated measures per individual was randomly selected from the UK Biobank cohort for the purpose of comparison. The objective was to evaluate the reliability and benefit of controlling for diseases interfering with biomarker measures on the quality of longitudinal effects. With the onset of such diseases, trait trajectories might be perturbed in a way that impairs the detection of true genome-wide longitudinal signals. A single trait longitudinal GWAS was run on each dataset and the impact of comorbidity on the quality of estimates was reported. Mixed models for the two datasets were adjusted for cholesterol-lowering drugs as they might lower triglycerides as well.Four sample sizes of one, two, three and ten thousand individuals were simulated. In addition, 5 longitudinal to cross-sectional ratios of 1, 2, 5, 20, and 100% (LT ratio scenarios) were simulated and replicated 100 times. LT ratio scenarios were simulated with 3000 individuals each; and sample size scenarios were simulated with LT effects that were 20% of the CS effects (LT to CS ratio of 1:5). Data sets simulated were analyzed with GAMUT and GALLOP. As shown in Fig. Bonferroni-corrected significance threshold was calculated as 0.05 divided by the total number of variants scanned. Type I error realized in the simulation was taken as the proportion of non-causal variants with p-values below the Bonferroni-corrected significance threshold. Table To study statistical power for the multiple- versus single-trait approach, standard errors of all variants in two simulation runs were compared. In the first run, 15% of values were assigned to be randomly missing for one of the three phenotypes and in the second run, 50% randomly selected records were set to be missing for the same phenotype. Standard errors were consistently smaller with the multiple trait analyses, especially when a greater proportion of data was missing. In addition, Fig. Because standard errors were shown to be reduced for all variants, both causal and noncausal, power and type I errors associated with multiple- vs. single-trait approaches were studied in a scenario where 15 and 50% of individuals were missing for their baseline and second records. Figure 14. Variants detected by the 3 approaches are reported in the following sections. Benchmarking data included 28 blood biomarkers in the UK Biobank, where all individuals with 2 measurements for at least one biomarker were utilized in the analyses.Performance of GAMUT vs. other approaches in the context of real-world data is shown next. GAMUT results are compared with a single-trait longitudinal approach using GALLOP and a single-trait conventional GWAS on each phenotype rate of change using REGENIEZ’R–1Z + G–1 took 59 s vs. 21.6 min using direct sparse inversion. For 20 traits, inversion by recursive partitioning took 2.2 min vs. extrapolated 130 min consumed by direct sparse inversion /2 = 378 pair-wise variance component sets of estimates were obtained using AI-REML. Although the variance of each trait was estimated multiple times when paired with other traits, covariances estimated were unique. CS and LT variances of each biomarker trait in a genetic variance–covariance matrix of order 56 were averaged across all other traits. Pairwise covariances were unique. Residual variances and covariances between traits were similarly summarized, where variances were averaged while pairwise covariances were unique. Although variances were estimated multiple times, they were consistent, especially for the CS components, as the boxplots of Fig. *27/2 = 3The weakest variance components were estimated for Testosterone, measured in both sexes. Figure Similarly, genetic and residual correlations were positive and strong among LDL direct, Apolipoprotein B and Cholesterol of cluster-4 Fig. . Results2 of 0.4, a window of 500 kb, and a p-value threshold of 5e-8. Numbers of significant SNPs in the table were taken from the number of clumps found across the genome and summed for all biomarkers.Results of longitudinal genome-wide association scans performed on biomarker phenotypes as 4 multiple-trait longitudinal runs using GAMUT, 28 single-trait longitudinal runs using GALLOP, and 28 conventional runs using conventional GWAS on biomarker rates of change between first and second measurements were presented in Table Collectively, GAMUT resulted in a total of 539 significant cross-sectional and 248 longitudinal associations, whereas the single-trait longitudinal GWAS resulted in 513 cross-sectional and 30 longitudinal associations. The conventional GWAS on rates of change yielded 37 significant longitudinal associations across the 28 biomarker phenotypes. Agreement between GAMUT and GALLOP was consistent, i.e. 98 of cross-sectional and 90% of longitudinal variants identified by GALLOP were a subset of those identified by GAMUT. There was little overlap between conventional GWAS and the other two methods. All variants identified by rate of change GWAS were observed as cross-sectional associations in the other two approaches. For example, the 9 and 11 variants associated with rate of change in direct and total bilirubin were, in fact, identified as having cross-sectional, but not longitudinal associations with these traits using GALLOP and GAMUT. Additionally, variants identified by rate of change GWAS were generally the most significant cross-sectional associations uncovered by the other two approaches . Longitudinal or progression effects on the other hand were more difficult to validate but still provided multiple clinical insights. In particular, a variant on chromosome 15 (rs117268014), captured only by GAMUT as cross-sectionally and longitudinally associated with both direct and total bilirubin. The variant is in the vicinity of Gremlin1 gene (GREM1) and shown by QTLbase16 to be a blood expression QTL for Rho GTPase activating protein 11A (ARHGAP11A), a gene positioned near GREM1 and a methylation QTL for Eukaryotic Translation Initiation Factor 5A2 (EIF5A2), a gene whose overexpression correlates with multiple cancers including colorectal, gastric, and esophageal cancers. Overexpression of the gene was reported to correlate with cancer progression and poor survival17. Note also that the role of GREM1 itself is well established in the onset and prognosis of colorectal and gastric cancers. In particular its role in promoting colorectal cancer cell metastasis, motility, and invasion18.Cross-sectional effects were well powered and consistent with the biomarker GWAS literature (e.g. Ref.Unlike the single trait outcomes across the random UK Biobank sample with 2 and 0 cross-sectional and longitudinal genetic signals in Table In the current study, an efficient and powerful approach to identify genetic variants associated with cross-sectional and longitudinal effects was developed. The approach capitalizes on the availability of broad phenotyping, in which individuals are assessed for multiple clinical traits, as well as deep phenotyping, characterized by multiple measurements of these traits over time. GAMUT successfully modeled both the multiple-trait and the longitudinal aspects of the data while maximizing computational efficiency, outperforming single-trait longitudinal analysis when up to 16 traits were examined. It is imperative to emphasize that a multivariate analysis is inherently more computationally costly than the cumulative cost of the equivalent univariate analyses. The computational advantage of our multivariate approach up to 16 traits is explained by, first, the algorithmic differences between GAMUT and GALLOP in transforming the major random effects matrix of the mixed model equations into an identity, and second, having to build and transform the coefficient matrix only once for all traits. The approach is particularly useful for joint analysis of strongly correlated traits measured in real-world data collected over time. While handling missing values in longitudinal data can be cumbersome, GAMUT provides a framework in which missing records in one or more traits are implicitly estimated based on genetic and environmental covariances with the traits that have more complete data.Because longitudinal effects are likely to be much smaller than cross-sectional effects as seen in the UK Biobank blood biomarker data, sample sizes required to detect genetic variants associated with disease progression are expected to be manyfold greater than what is required for cross-sectional disease association, as was shown in our simulation. In order for the current approach to be useful, it is essential to have sufficient data at multiple time points per individual, e.g. primary care data or repeated measures in extended large-scale clinical trials. In addition, the UK Biobank continues to assess participants over two time points for thousands of traits such as the repeat MRI imaging study of brain, heart, and abdomen. Although our simulations suggest that multiple-trait approaches offer limited advantages over single-trait analysis in the presence of complete data with no missing observations, such a scenario is unlikely to exist in either observational real-world data collection or prospective clinical trials.In the current Biomarker analysis, longitudinal variant associations above genome-wide significance suffered from inflation for 3 possible reasons. First, longitudinal effects were orders of magnitude smaller than their cross-sectional counterparts; second, the number of individuals with repeated measurements was limited, and finally time points within individuals were ≤ 2 visits in the current UK Biobank phenotypic data. Inflation was shown to be controlled when a sample of participants, similar in their clinical history and with many more repeated measures, was extracted from the UK biobank primary care data and utilized in longitudinal GWAS.In this paper, we demonstrate that GAMUT is a computationally efficient framework that accounts for genetic and environmental co-variability in multiple longitudinally assessed quantitative traits. Although univariate modeling approaches have been successfully used to identify important disease associations, they also miss novel insights as they fail to leverage the correlation among pathophysiological processes that lead to disease onset or progression. We have shown this to be the case in an analysis of real-world data from the UK Biobank, where multiple cross-sectional and longitudinal insights would have remained undetected using other standard statistical methods and assert that our approach has broad applicability to large-scale collections of data available in global biobanks and prospective clinical studies today.Supplementary Tables.


HR, SBP, DBP, SpO2, BIS score and CLs were compared at the baseline, immediately before-, during-, and immediately after surgery between the test and control groups. We found that the MM resulted in significant decrease in BIS together with positive effects on hemodynamic parameters , psychological findings (improvement on STAI-S scores) and biochemical outcomes (decreased CL). In conclusion, the results demonstrate that MM appeared to be a reliable strategy for managing stress during dental implant operation with benefits in psychological, physiological and biochemical outcomes.Dental implant surgery is almost always associated with patient anxiety. Anxiety during dental surgical procedures triggers an increase in sympathetic activity. Mindfulness meditation (MM) is often associated with high levels of relaxation in the form of increased parasympathetic tone and decreased sympathetic activity. However, the effect of MM on dental anxiety is not clear. The current study aimed to show the effects of a MM as a sedative technique during dental implant surgery by examining the State-Trait Anxiety Inventory (STAI-S), bispectral index (BIS), cortisol levels (CL), systolic (SBP) and diastolic blood pressure (DBP), heart rate (HR) and saturation (SpO The physiology of the stress response includes fast and slow components. A fast response results in increased secretion of norepinephrine and epinephrine from the adrenal medulla into the circulation leading to increased blood pressure, heart rate, cardiac output, behavioral activation  and this pathway is regulated by the sympathetic-adreno-medullar (SAM) axis. A slow response, mediated by the hypothalamic pituitary adrenal (HPA) axis results in the release of corticotrophin-releasing hormone into the circulation and eventually adrenocorticotrophin hormone (ACTH) into the bloodstream. ACTH stimulates the adrenal cortex to secrete glucocorticoid hormones, such as cortisol into the circulation. The SAM pathway is the route through which the brain directs the sympathetic branch of the autonomic nervous system (ANS) to activate in response to short-term stress9 The ANS plays a key role in stress reactivity via its two main divisions, i.e. the Sympathetic Nervous System (SNS) and the Parasympathetic Nervous System (PNS)10.Dental implant surgery often lasts 1 to 2 h and is almost always associated with patient anxiety and psychological stress. The term “stress” describes the effects of psychosocial and environmental factors on physical and/or mental well-being3. Therefore, intravenous sedation has been reported as clinically useful in preventing sympathetic hyperactivity15. Although the ideal sedation agent used in dental surgery should provide a rapid onset of action and easily reversible, there have always been some serious potential risks associated with sedation such as airway obstruction16, hemodynamic changes17, amnesia effect19, bradycardia, agitated behavior15. Dental anxiety can also be managed by non-pharmacological anxiety alleviation strategies including behaviorally or cognitively oriented psychotherapeutic interventions such as relaxation techniques, guided imagery, hypnotherapy, acupuncture, cognitive behavioral therapy20.Acute stress and anxiety during dental surgical procedures trigger an increase in sympathetic activity that is particularly undesirable in patients with cardiovascular diseases22. Mindfulness is practiced to achieve enduring happiness and to gain insight into the view of the true nature of existence23. Bishop et al. (2004) suggested a two-component model of mindfulness, where the first component is the regulation of attention, and the second component involves approaching one’s experiences with an orientation of curiosity, openness, and acceptance24. The practice of MM encompasses focusing attention on the experience of thoughts, emotions, and body sensations, simply observing them as they arise and pass away resulting in body awareness, attention regulation, emotion regulation and change in perspective on the self22. Body awareness is considered an integral part of the mindfulness construct and it has been proposed as one of the major mechanisms of mindfulness interventions27.In current research contexts, the mindfulness meditation (MM) technique is defined as nonjudgmental attention to experiences in the present moment29 including anxiety31, depression32 and chronic pain33 management. Furthermore, MM positively influences aspects of physical health (i.e. improved immune function35 reduced BP and cortisol levels34) and psychological well-being37 and cognitive functioning38.In the medical and psychological research, MM has been reported to produce beneficial effects on a number of psychiatric and stress-related symptoms and has therefore increasingly been incorporated into psychotherapeutic programs39. Peripheral physiological changes have been observed with some consistency, including decreased HR40, decreased BP41 and decreased cortisol levels34. Cortisol is the end product of HPA axis functioning and salivary cortisol is a commonly used measure of physiological stress responses and provides information about cortisol levels and changes over periods of time43.MM is often associated with high levels of relaxation in the form of increased parasympathetic tone and decreased sympathetic activity45. However, previous studies on the relationship between mindfulness and dental anxiety only included trait measures of dental anxiety and the results have been inconsistent and limited to specific samples and have not investigated possible mechanisms that may be involved in the relationship between mindfulness and dental anxiety47.Mindfulness-based techniques have been suggested to be useful for managing dental anxiety including implant surgery2) parameters.We hypothesized that patients randomly assigned to receive pre-operative mindfulness meditation would show a greater improvement in psychological distress and the vital signs than non-meditated patients. Therefore, the aim of the current study was to evaluate the effects of a brief mindfulness meditation (short body scan) as a sedative technique during dental implant surgery via examining the subjective (State-Trait Anxiety Inventory) and objective  between December 2021 and August 2022. The study protocol was approved by the Institutional Ethical Committee (03.06.2022 123/53), registered on ClinicalTrial.gov  and performed in accordance with the Helsinki Declaration of Human Studies. Before the procedure, the patients were given full and adequate verbal and written information about the purpose of the study and written informed consent was obtained.Age between 25 and 65 years old,Single tooth loss in the maxillary premolar–molar region with partial bone healing (type 3) or fully healed ridge type 4,Sufficient bony ridge and keratinized tissue for implant placement without the need for simultaneous bone grafting or sinus lift procedures,No signs of acute oral infection,Periodontally and systemically healthy.Individuals were eligible to participate and included in the study if they were:Diabetes mellitus,Smoking,Pregnancy,Diagnosis of anxiety disorders/psychiatric disorder/depression and whether or not they were seeking any form of treatment for these conditions,History of radiation therapy at head and neck area and chemotherapy.The exclusion criteria were:Patients were assigned to one of the two treatment groups with the use of computer-generated randomization table (test group: mindfulness meditation (MM); control group: non-meditated. Treatment assignment was concealed to the meditator (O.U.T.) by opaque envelopes.All patients were scheduled between 9 and 11 am to avoid any variations in the anxiety and salivary cortisol levels due to circadian rhythms. One patient from the test group did not show up at the surgery appointment. Therefore, the data from 41 participants was analyzed.48. Training was conducted by one expert periodontist who is also an internationally certified MBSR instructor (O.U.T.). In session 1, the participant sat in a chair, and was instructed to close his/her eyes and relax and to focus on the flow of his/her breath. If a random thought arose, the patient was told to passively notice and acknowledge that thought and to simply let ‘‘it’’ go, by bringing attention back to the sensations of the breath. The last 7 min of day 1 were held in silence, so that the participant could effectively practice mindfulness meditation. In session 2, the facilitator instructed participants to focus on the ‘‘full breath,’’ (sensations in the nostrils and abdomen) and the last 7 min of the session 2 were held in silence. Session 3 was an extension of sessions 1 and 248. As a manipulation check, each subject was asked ‘‘if they felt that they were truly meditating’’ after each meditation session.Each participant assigned to the MM group individually received MM training for 20 min daily for 3 days before surgeryOn the day of the surgery, all patients in the test and control groups were instructed to arrive 20 min earlier than the surgery appointment hour in order to complete the forms  and to record the baseline measurements . This period was standardized at 20 min for each participant. After the first 20 min of the pre-operative measurement period, the control group was taken into operation and the test group was taken into meditation.21 was performed for 20 min (by O.U.T.). Participants were instructed to focus their awareness on sound guided by the meditator and sensations felt by the body, and merely observe non-judgmentally for any sensations, emotions, or thoughts that may arise during the meditation practice.The participants of the test group were further informed about MM and they were reassured that the meditation would not have any direct impact on the surgical procedure and that they could discontinue the session at any given point. They were subjected to MM in a room specially allocated for study purposes. Care was taken to ensure that no disturbance occurred or that any third person entered the room throughout the intervention. Shortened guided body scan meditation adapted from the MBSR program by Jon Kabat-Zinn49 by an expert periodontist (F.D.).One week before surgery, all patients received an explanation about the dental implant procedure, which lasted for 20 min. Implant placement was performed under infiltration local anesthesia . All patients received only one carpule of the anesthetic solution immediately at the beginning of the procedure. This amount was sufficient for all patients and no further anesthesia was needed. Surgeries were performed according to the standard single implant placement guidelines19. The BIS values range between 100 and 0 and give clinicians objective information about the depth of sedation. General anesthesia comprises values ranging of 40 to 60, while deep sedation is within 60 to 70 and 70 to 90 represents light to moderate sedation. The patient is considered awake for values over 90 and adequately sedated consciously for a BIS range of 80 to 85 for oral and maxillofacial surgeries17.The primary outcome of the study was the level of sedation achieved by MM, which was assessed with BIS device . BIS index, which provides a quantitative measure of sedation to analyze the sedation effect of MM in patients, was used before and during surgery2) and heart rate value (HR)  at baseline. All values were recorded at 5-min intervals:During 20 min of preparation time (in which the preoperative forms were completed) for both groups;During MM for the test groupDuring surgery for both groupsThe BIS electrodes were placed at the attendance of the patient together with monitorization for systolic (SBP) and diastolic blood pressure (DBP), arterial blood oxygen saturation , saliva samples were collected at each STAI completion phase. Prior to the collection of saliva, subjects were asked to rinse their mouths with plain water to clear away any debris or food. Care was taken to ensure the non-contamination of saliva with blood.Salivary cortisol was estimated by solid phase enzyme-linked immunosorbent assay (ELISA)  in ng/ml. The procedure was done as per the manufacturer guidelines on saliva samples.50 was used to assess the self-reported anxiety levels immediately at the attendance of the patient in the clinic (T0), immediately before (T1) and after (T3) surgery for both groups. STAI comprises separate self-report scales for measuring two distinct anxiety concepts: state anxiety and trait anxiety. The STAI-T scale consists of 20 statements that ask people to describe how they generally feel. The STAI-S scale also consists of an additional 20 statements, but the instructions require subjects to indicate how they feel at a particular moment in time. The STAI-S scale can be used to determine the actual levels of anxiety intensity induced by stressful procedures. Each question is rated on a 4-point scale . The range of possible scores is between 20 and 80. STAI scores are commonly classified as “no or low anxiety” (20–37), “moderate anxiety” (38–44), and “high anxiety” (45–80).The validated Turkish version of State-Trait Anxiety Inventory (STAI) form51.Subjective well-being was assessed . In addition, the education level of the participants was recorded MDAS is a modification of Corah’s Dental Anxiety Scale consists of 5 questions with scores ranging from 0 (not anxious) to 5 (extremely anxious) for each answer for a total score ranging between 0 and 25. The score of 0 to 5 is considered not anxious, 6 to 10 is low anxiety, 11 to 14 is moderate anxiety, 15 to 18 is high anxiety and 19 to 25 is extreme anxiety/phobic.2, SBP, DBP) and BIS monitoring data (M.O.).Three different periodontists who did not participate in the procedure; checked the completion of the forms  (M.C.H.), took the salivary samples (B.A.) and recorded the vital signs .Categorical variables were expressed as numbers and percentages, and Chi Square test was used to compare the groups. Continuous variables were summarized as mean and standard deviation and as median and quartiles (Q1–Q3) where appropriate. The normality of the distribution for continuous variables was confirmed with the Kolmogorov–Smirnov test. For comparison of continuous variables between two groups, Mann–Whitney U test was used and Friedman test was used for time-dependent measures. Spearman correlation test was used between 2 continuous variables. The F1-LD-F1 design was used for longitudinal data analysis because the data were not normally distributed. The F1-LD-F1 model generates an ANOVA-type statistic for group, time, and group-time interaction. The F1-LD-F1 model, in addition to the ANOVA-type statistic, provides relative treatment effects (RTE) as a unitless measure of effect size. The RTE has values ranging from 0 to 1 and can be interpreted as follows: An RTE of 0.25 for a specific subgroup indicates that the probability of a randomly selected person from this subgroup outperforming a randomly selected person from the entire dataset is estimated to be 25%. The probability of a randomly selected person from this subgroup scoring lower than a randomly selected person from the entire dataset, on the other hand, is estimated to be 75%. An RTE of 0.50 indicates that there is no tendency for a higher or lower score in any subgroup. All statistical analyses were carried out using IBM SPSS 20  and RStudio . The npar LD package was used in RStudio for the F1-LD-F1 design. The statistical level of significance for all tests was considered to be 0.05.A total of 60 participants were screened and 42 eligible patients were scheduled for the surgery. Of these participants, 21 patients were randomized each for the test (MM) and control group  levels were higher at all evaluation time periods in this group. Although the group effect was not significant for overall in SBP and DBP, time and group*time interaction were statistically significant  after MM (T1) and this reduction lasted during and after surgery Fig. A. The DSnt Table .  in the MM group, it decreased in the controls. The saturation levels stayed higher during and after surgery in the MM group Fig. D.The cortisol levels and STAI were in accordance with the BIS values showing a significant decrease after meditation and after surgery in the MM group and the change over time was statistically significant Table . The con2 and HR.Overall, the statistical relative treatment effect analysis showed that the MM had significant effects on BIS, Cortisol, STAI, SBP, DBP, SpOThis is the first randomized controlled clinical study that demonstrates the efficacy of a 20-min mindfulness meditation intervention in rapidly reducing the surgery anxiety of patients during dental implant operations. The findings suggest that the mindfulness meditation had significant positive effects on cardiovascular parameters, physiological findings and biochemical outcomes.15. Anxiety toward surgical procedures is usually associated with a suppressed fear of pain. These patients not only find these procedures unpleasant but they may also exhibit peripheral manifestations such as sympathetic overactivity.Dental implant surgery under local anesthesia comprises a major part of oral surgery. Although local anesthesia usually provides adequate analgesia for surgery, patients usually suffer from the discomfort and pain associated with surgical procedures53. The recent meta-analysis of RCTs demonstrated that mindfulness meditation is associated with reduced physiological markers of stress52 such as decreased HR40 decreased BP41 and decreased cortisol levels34. To this extent, we examined whether 20 min of mindfulness meditation would improve physiological variables i.e., cardiovascular parameters . The results we present are encouraging in that they suggest certain immediate physiological effects of MM and that meditation may have an effect on decreased activation of the SNS.Mindfulness as a state, trait, and clinical intervention has been extensively researched over the last two decades55. Similar to the findings of previous research, this study showed that the patients in the test group had significantly lower cortisol levels immediately after MM and after surgery. A recent systematic review highlights some potential causes of inconsistent and contrary findings of mindfulness effects interventions on cortisol levels such as; research design , sampling time and protocols, cortisol measurement (baseline effects on cortisol), participation standardization (effects of different subgroups)43. In this randomized controlled clinical trial, cortisol samples were collected at the same time of the day and analyzed by the same Elisa protocol in terms of standardization.Previous studies have demonstrated reductions in cortisol subsequent to mindfulness interventions53. Yao et al.44 used the STAI and suggested that mindfulness training may potentially help alleviate patients’ dental anxiety. Similarly, we evaluated psychological outcomes using the STAI-S. MM was found to be effective in significantly reducing the STAI-S when compared to the control group before and after surgery, suggesting that MM can provide both immediate and lasting effects on anxiety during dental surgery.Growing evidence has indicated that mindfulness practice induces both state and trait changes; that is, it temporarily changes the condition of the brain and the corresponding pattern of activity or connectivity17 found that the BIS gradually decreased during initial medicated sedative loading and ranged between 80 and 85 at the time of optimal sedation, which is similar to our findings in the test group. In our study, there was significant difference in the recorded bispectral analysis values between two groups in two time periods (immediately before and during surgery). It was observed that the BIS value gradually decreased during MM in the test group (to 73- 80). However, when surgery was started, various stimuli were added, which affected patients’ sedation levels. Therefore, the BIS values tended to increase during surgery (85 to 87.5). At the optimal sedation level, BIS was significantly lower than pre-surgery (97) and during surgery values compared to the control group (96.3–97). The significant decrease of BIS values in the MM group  may be related to a diminished activation of the amygdala in response to emotional stimuli during mindful states as well as in a resting state, suggesting increased parasympathetic tone and decreased sympathetic activity57.Taniyama et al.40, the advantages of the mindfulness meditation technique can be experienced immediately after a short training program58. One of the most intriguing findings in the field of brain structure and meditation is the minimal number of hours of meditation practice needed to induce neuroplastic changes60. These studies suggest that even short-term meditation practice can involve and modify many of the same brain regions that exhibit variations in long-term practitioners. Early-onset structural changes may endure following initial meditation practice, be maintained through ongoing practice, or result from a combination of both. However it has been reported that the response to MM variess significantly among individuals within the range of minutes to months. Although a training program was conducted for the participants of the MM group, there was no data regarding their level of MM experience. This may effect the outcomes of this randomized controlled study61.We provided a brief mindfulness training for the MM group. According to Zeidan et al.53 and Hölzel22 have reported that: “practitioners could have pre-existing differences… which might be linked to their interest in meditation, personality.” This highlights a notable confound in previous research on mindfulness literature i.e., the MM instructors’ individualistic disparities, such as the educational level and experience of the meditator. In the current study, the brief body scan meditation was performed by an MBSR instructor with 10 years of experience. In addition, individual differences in personality are likely to contribute to how people respond to and benefit from mindfulness practice, similar to the differences in brain function and structure, genetic predisposition, life experiences and environmental factors. In order to overcome this factor, the test and the control group were matched for age, sex, socio-economical status and educational level.TangThis study has some limitations. First, the study was conducted in a single center with a relatively small sample size. Second, anxiety and hemodynamic parameters are complex multicausal conditions; thus, there may well be more confounding factors that explain the variance in anxiety and hemodynamic parameters  than those found in this study. Although a large number of studies have used hemodynamic parameters in the physiological assessment of anxiety, it should be taken into account that there are also a few studies with contradictory results in this regard. Third, the local anesthesia during the procedure might affect hemodynamic parameters, which could have potential consequences for both physiological and psychological status.63. Successful relationships require physicians to practice a welcoming stance, participatory decision-making, and mindfulness about both the patient's and the dentist's inner lives64. It was also reported that dental clinics are encouraged to incorporate mindfulness interventions into their routine daily practice44. The results of this study also highlighted that mindfulness is not only associated with a reduction of general anxiety, but also with dental anxiety specifically. These findings show that clinical practitioners treating dental anxiety provide more emotional comfort for patients. Therefore, it may be suggested that if the dentist who is actively carrying out the surgical procedure also directly performs the MM, this may eventually result in better outcomes.In addition to these limitations, the patients in the control group knew that they would not receive pre-operative meditation, which may potentially affect their anxiety levels. As the patients of the control group were immediately taken to surgery after T1 while the patients of the test group received MM after T1, this additional time period may introduce a confounder factor for the outcomes. Furthermore, MM was performed by another dentist other than the operator in the current study. It has been reported that the dentist-patient relationship is another important issue, which is the core of dentistry. A good therapeutic alliance during dental therapy, including adequate, individualized, and nonjudgmental education, is of the utmost importance in order to decrease the patient's negative emotions, allow the patient to gain insight about the disease condition, and increase their feeling of control over the situationAs compared with the non-meditated group, mindfulness meditation appeared to be a feasible and reliable strategy for managing stress during dental implant operations with benefits in psychological, physiological and biochemical outcomes. Overall, the results provide evidence that brief mindfulness meditation is effective for reducing dental anxiety. We believe that these findings will provide valuable evidence to guide future mindfulness-based intervention research in the dental field.Supplementary Information.


Mutations in the anti-NTM drug target regions promote NTM evolution to mutant strains that are insusceptible to NTM drugs leading to treatment failure. We, therefore, described the mutation patterns of anti-NTM drug target genes includingrrs, and the 68 SGM were sequenced forrpoB genes using ABI 3730XL analyzer. The obtained sequences were aligned to their wild-type reference sequences for each gene using Geneious then mutations were identified. Pearson chi-square at a 95% confidence interval tested the association of NTM to mutation patterns for each gene. Methods: We carried out a cross-sectional study that included 122 NTM obtained from the sputum of symptomatic tuberculosis-negative patients in Kenya. All 122 NTM underwent targeted sequencing of the rrl gene. The 54 RGM were also sequenced forrrl gene with 58.3% (7/12) comprising RGM and 41.7% (5/12) being SGM. Mutation at position 2058  of therrl gene was seen for 83.3% (10/12) of NTM, while 16.6% (2/12) harbored a A2059G mutation. Of the 54 RGM included forrrs characterization, 11.1% (6/54) exhibited mutations  at position 1408(A1408G), while 14.7% (10/68) of the SGM had mutations in therpoB gene at positions S531W, S531L, S531Y, F506L, E509H withM.gastri having multiple mutations at positions D516V, H526D and, S531F. Results: NTM harboring mutations associated with resistance to at least one of the antibiotics used in the macrolide-based therapy were 23% (28/122). Of these NTM, 10.4% (12/122) had mutations in the Conclusion: We demonstrated a significant level of mutations associated with drug resistance for macrolides, aminoglycosides, and rifampicin in NTM isolated from symptomatic TB negative patients in Kenya. Mycobacterium tuberculosis andMycobacterium leprae (Mycobacterium avium complex (MAC),Mycobacterium chimaera,Mycobacterium kansasii,Mycobacterium malmoense, andMycobacterium xenopi, whereas RGM comprise species from theMycobacterium abscessus andMycobacterium fortuitum complexes (Non-tuberculous Mycobacteria (NTM) are a group of about 170 Mycobacteria species that do not includeNTM treatment consists of a macrolide-based antibiotic regimen which include clarithromycin or azithromycin, combined with other selected antibiotics which are given for 12 to 18 months until culture negativity is achieved. . In addiAnti-mycobacterial drugs attach to their binding sites with a high affinity, preventing the target gene product from functioning normally  gene which encodes a ribosomal methyltransferase that methylates therrl thus blocking the drug-binding site of the macrolide  within therpoB gene. Mutations in this region account for 95% of rifampicin resistance in mycobacteria. The commonly observed mutations within the RRDR ofrpoB are often seen at codons 526, and 531   through broth microdilution, .In this study, the 122 NTM including 54 RGM and 68 SGM underwent targeted sequencing of theThe sputum samples were decontaminated using the N-acetyl-L-cysteine 2% NaOH (NALC-NaOH) procedure, then inoculated into Mycobacteria Growth Indicator Tube (MGIT) and Lowestein-Jenseen (LJ) medias, incubated at 37°C and monitored for growth for up to eight weeks respectively. At the same time, sputum smears were prepared, air dried, heat fixed then fluorochrome stained with auramine O where mycobacteria appeared as bright yellow fluorescent rods when viewed under a light emitting diodes (LED) microscope. Fluorescent microscopy was performed by NTRL laboratory technologist then smears were reviewed by the section head before reporting.The culture growth in MGIT and LJ underwent the Mtb identification testing using the SD Bioline TB Ag MPT64 assay (capilia)  and capilia positive samples were excluded from the study. The capilia negative samples underwent ZN microscopy with presence of AFB indicating a possible NTM.GenoLyse®  according to the manufacturer's instructions. Briefly, 100 ul of lysis buffer (A-LYS) buffer was added to each cryovial containing the resuspended colonies and incubated for five minutes at 95oC after which 100 ul neutralization buffer (A-NB) was added and centrifugation done at 5000G for ten minutes. The supernatant was transferred to a newly labelled cryovial awaiting PCR.Mycobacterial DNA was extracted from 500 μL of re-suspended colonies using usingrrl, rpoB and rrs genes of NTM were conducted using the Horse-Power™ Taq DNA Polymerase MasterMix  in a final reaction volume for each gene of 13 μl comprising 6.25 μl of 2X Horse-Power™ Taq DNA Polymerase MasterMix, 2.5 μl DNA template, 0.25 μl of each of both forward and reverse primers , and results were visualized with an UltraViolet gel viewer . For the corresponding runs, a positive control containing drug-resistant NTM with mutations in therrl, rpoB andrrs genes was used, as well as a negative control containing drug sensitive NTM without mutations in the drug target genes were included.Conventional PCR targetingprimers  at a finprimers . ThermalThe PCR products were enzymatically purified using ExoSAP IT . Purification conditions were done at 37°C for fifteen minutes followed by a second incubation at 80°C for fifteen minutes and a final cooling step at 4°C for five minutes.The purified amplicons were sequenced in the forward and reverse directions by Sanger sequencing using Big Dye™ Terminator Version 3.1 Cycle Sequencing Kit  and the forward and reverse primers. The sequencing reaction for each gene was a 10 μl reaction comprising 1.25 μl of Big Dye Terminator, 3 μl of 5× Sequencing Buffer, 1 μl of 1 pmol of the sequencing primer, and 1.5 μl of the PCR product. The reaction volume was made up by adding 3.25 μl of nuclease-free water. The reaction proceeded through 96°C for 1 minute then 25 cycles of 96°C for 10 seconds, 50°C for five seconds, and 60°C for four minutes.Purification of cycle-sequencing products was done using the BigDye X Terminator™ purification kit following the manufacturer’s instructions  and purified products were loaded onto the ABI 3730 genetic analyzer  for capillary electrophoresis.The obtained sequences were first assembled into contigs and the consensus sequences aligned to their wild-type reference sequences for each gene using Geneious version 11.0 . Mutations in the drug resistance genes were identified visually. STATA version 14  was used to test the association of NTM species to mutation patterns using Pearson chi-square at 95% confidence interval.M. abscessus had mutations conferring resistance in bothrrl andrrs genes.Our study established that 23% (28/122) of NTM harbored mutations associated with resistance to at least one of the antibiotics in the macrolide-based therapy. One isolate (C47) containingrrl,rrs, orrpoB genes (p=0.012))  of the NTM isolates with mutations in the drug target genes and indicated a strong statistical significance (p=0.000). The majority of NTM with these mutations were detected in new  and TB relapse patients  patients, which had a significant statistical significance (p=0.001)  NTM showing mutations in the0.012)) . The age=0.001) .rrl gene for the NTM were highly significant with a p value of <0.001. Of the NTM with mutations in their drug target genes, 10.4% (12/122) had mutations in therrl gene (rrl gene was seen for 83.3% (10/12) of NTM includingM.intracellulare,M. abscessus subsp abscessus, M. nebraskense, M. massiliense, M. kumamototense, M.heraklionense, andM. bourgelatii. Arrl A2059G mutation was seen for M. abscessus subsp abscessus representing16.6% (2/12) of the NTM  exhibited mutations at position 1408(A1408G), The NTM species presenting with these mutations includealsense . The NTMrpoB mutation in SGM was observed. Mutations within codons between 503-533 of therpoB were seen for 14.7% (10/68) of SGM. These SGM includedM. avium subsp avium, M. intracellulare (F506L),M. yongonense (E509H) andM. gastri which had multiple mutations at positions D516V, H526D and, S531F (A low association (p=0.89) for, S531F .NTM) are an important cause of pulmonary disease worldwide, and are being isolated increasingly . The 21-35 years age group had the highest number of NTM isolates presenting with drug resistance associated mutations while males comprised the majority  of cases with NTM showing these mutations. These mutations were associated with a history of previous TB infection as seen in the high number of TB relapse cases recorded in this study . This is a common observation in sub-Saharan Africa given the high incidenceof M. tuberculosis disease and the frequent misdiagnosis of NTM infection with TB seen in this region  with the bulk of the isolates originating from the Lake Victoria , Coastal , and Nairobi  regions. The observed regional diversity of NTM harboring drug resistance mutations across Kenya could be attributed to NTM evolution to evade natural antibiotics secreted as secondary metabolites by nearby environmental bacteria in various geographical landscapes (Moore>80% of isolates showing susceptibility (MAC andM. abscessus have been associated with increased resistance to macrolides leading to treatment failure (M. intracellulare (A2058G),M. nebraskense (A2058C),M. massiliense (A2058T),M. kumamototense (A2058T),M. heraklionense (A2058T), andM. bourgelatii (A2058C) had a point mutation at position 2058 in the rrl, while two (16.7%)M. abscessus subsp abscessus isolates had mutation at position A2059G. The increased potential for development of these mutations inMAC species includingM. intracellulare, andM. abscessus could be attributed to inherent factors such as a high propensity for genetic mutation in the drug target region, and high drug tolerance levels  on 30https://doi.org/10.6084/m9.figshare.20331378.v2 .Data are available under the terms of theZMM - Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & EditingGN - Investigation, Writing – Review & EditingMWM - Supervision, Writing- Review and EditingFGO - Conceptualization, Methodology, Supervision, Writing- Review and EditingWDB - Conceptualization, Methodology, Supervision, Writing- Review and Editing The authors have revised the manuscript as per the recommended changes. The revised manuscript is adequate in form and contentIs the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:TB molecular biology and immunologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you for your approval General comments The manuscript addresses an important area in Tuberculosis research particularly analysis of drug resistant mutations that have been linked to treatment failure.The authors to include a hyphen in Non-tuberculous Mycobacterium (NTM).Use upper case for the first letters of the following acronyms, SGM and RGM.In the results section, percentages should not be in brackets. The proportions to indicated in brackets e.g. 23% (23/100). This should be done all the data presented in this section. When reporting on the mutations, also indicate the amino acid or nucleotide change as well. For example, which mutation is present on codon 2058? Change from what to what? Abstract:Second sentence, the authors to describe in detail the type of culture used to grow NTMs for 40 days. MGIT?Second paragraph, authors to indicate the duration of treatment of NTM infections. Also the length of treatment with Rifampicin to be mentioned. Is it the same for patients infected with comorbidities?The authors have listed several drugs used for the treatment of NTMs but described only one resistance mechanisms for NTMs, do they all have the same type of resistance mechanism? (Macrolides - Clarithromycon and Azithromycin).Paragraph seven, the authors to indicate that Rifampicin is recommended for first-line treatment of MTB alongside other drugs. Introduction:The authors to estimate the volume of sputum sample collected from each participant.For the FM microscopy, how many readers were used before reporting the smear results?What volume of DNA was finally collected after extraction? Was quantification of the DNA material done? Laboratory procedures:The authors to mention the table with results indicating that the bulk of the samples were obtained from Lake Victoria, Coastal and Nairobi region. Results:The authors to refer to my previous comments on Non-tuberculous mycobacterium.The authors to critically discuss the relevance of the rrl significant mutations reported in the study sites. What are the implications of these finding? Discussion:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:TB molecular biology and immunologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Thank you for your valuable feedback. No further comments. Please see previous comments about the need to correlate findings with MIC.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Infectious diseases and antibiotic resistanceI confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. I have read the revised version, but I still see that the manuscript has misleading information that needs to be re-written. The data provided need to associated with phenotypic data on resistance. Identification of mutations in genes is not sufficient to prove resistance, as the researchers have not confirmed any silent mutations.  The positive controls used in the methodology should be isolates that are confirmed resistant and harbour these mutations. This is the definition of a positive control. The negative control is an isolates that is genotypically confirmed having no mutations in the target genes.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Infectious diseases and antibiotic resistanceI confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Thank you for the feedback. Thank you for revising the manuscript and incorporating the comments into the text. However I would highly suggest that you revise the sections where you correlate the genotype with phenotype, since you have not done the AST (MIC) in this study. If you have not done the AST testing, you cannot state that they are resistant. Your study is primarily genotype based, and you have detected mutations in target resistance determinants, and I therefore suggest you change the wording of the results and discussion to reflect that. These mutations may be silent mutations, and if you do not have phenotype data to support it, you cannot state the fact that they are resistant. You can only say that they harbour the mutations which in previous reports have led to resistance.  These statements "Twenty-eight (23%) of the NTM were resistant to at least one of the antibiotics used in the macrolide-based treatment " and "Ten (14.7%) of the 68 SGM were resistant to rifampicin, with 40 percent having mutations at codon 531 in the rpoB gene" cannot be proven without AST. You should re-word it to say that 23% harboured mutations associated with resistance to macrolide antibiotics.  Same for the conclusion: you have not demonstrated a significant level of drug resistance for macrolides, aminoglycosides and rifampicin in NTM - you have detected mutations in gene targets that have previously been associated with drug resistance.  I hope my point is clear. I am not doubting your results, and I think it's interesting that you have detected these mutations, however it's not scientifically sound to assume resistance based on genotype only, and you must phrase your results and conclusions according to the methods used and data generated. The way it is written now indicates that you have the AST data for the isolates and you are sure that they are rifampicin or aminoglycoside resistant. I would strongly recommend you perform the AST - even for a subset of isolates - to confirm that the observed genotype correlates with the phenotype.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Infectious diseases and antibiotic resistanceI confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. I appreciate your re-evaluation and the way in which you made the issues that need to be addressed evident. I revised the paper in accordance with your advice and resubmitted it for review. The manuscript can be accepted now.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Infectious diseases with special reference to tuberculosis and leprosy, Cell Biology, Microbiology, Immunology and Biotechnology.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We sincerely appreciate your second reading and approval. M. tuberculosis-negative patients. The authors have investigated their resistance patterns on the isolated strains using gene sequencing of the drug-resistant genes and noted the mutations in the respective codon positions.The manuscript has been written on the findings of antimycobacterial resistance of NTM isolated from 122 sputum Materials and methods have been well described. Data were properly analyzed and presented adequately in the tables. However, there were a lot of discrepancies in Table 2 in the first column where the total sample number was shown to be 135 instead of 122 and therefore the percentage expressions were wrongly mentioned in the table and in the text. The percentages have to be calculated correctly and Table 2 has to be modified and described accordingly in the text. The discussion has been adequate, citing adequate references. However, the discussion with respect to the results of Table 2 has to be modified and discussed for a correct understanding of the reader.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Infectious diseases with special reference to tuberculosis and leprosy, Cell Biology, Microbiology, Immunology and Biotechnology.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. I appreciate your review. The issue mentioned above has been addressed. This study characterises mutation patterns for resistance genes in NTM from Kenya. Identification: 122 isolates were collected, but no details in the results on how many were slow-growing and rapid-growing NTM. MIC: No mention of methodology for MIC and results.The PCR: Did you include any positive or negative controls in the experiments?The discussion on mutations and their role in resistance is quite vague. It's not clear whether the mutations identified can be linked to the observed MIC. Have the researchers checked that these are not silent mutations? Although the study is interesting, I think the authors need to give more information in the methodology and results section on:Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Infectious diseases and antibiotic resistanceI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Under methods, it has been indicated that of the 122 NTM isolated, 54 were rapid-growing NTM while 68 were slow-growing NTM.The phenotypic drug sensitivity by MIC analysis was not done in this study.  We performed genotypic characterization of the drug target genes by describing the mutations associated with drug resistance through target sequencing of the drug target genes.Positive and negative controls were included during PCR and subsequent sequencing of respective target genes. The controls were based on Genotype NTM-DR results.In the discussion, the mutations identified in this study have been compared to those seen in previous studies that had also included MIC in their analysis. The point mutations identified in the drug target genes were seen to be associated with phenotypic drug resistance.I appreciate your review.


Multiple myeloma (MM) is a common hematological malignancy that has fostered several new therapeutic approaches to combat newly diagnosed or relapsed MM. While the field has advanced over the past 2 decades, the majority of patients will develop resistance to these treatments, causing the need for new therapeutic targets. SLAMF7 is an attractive therapeutic target in multiple myeloma, and a monoclonal antibody that targets SLAMF7 has shown consistent beneficial outcomes in clinical trials to date. In this review, we will focus on the structure and regulation of SLAMF7 and its mechanism of action. The most recent clinical trials will be reviewed to further understand the clinical implications and improve the prognosis of MM. Furthermore, the efficacy of anti-SLAMF7 monoclonal antibodies combined with standard therapies and possible resistance mechanisms will be discussed. This review aimed to provide a detailed summary of the role of SLAMF7 in the pathogenesis of patients with MM and the rationale for further investigation into SLAMF7-mediated molecular pathways associated with MM development. MM currently makes up 10% and is the second-highest of all hematological malignancies in the United States . MM can Signaling lymphocyte activation molecule (SLAM) family receptors regulate the immune system through expression on hematopoietic cells. There are six SLAM family members: SLAM, CD229, 2B4, CD84, NTB-A, and CS1 . Th. Th61]. Another possible resistance mechanism could be the upregulation of other cell surface proteins or pathways  61]. Th. Th61]. Multiple myeloma is an incurable disease that impacts a significant portion of the population. SLAMF7 is upregulated in MM and plays a role in the proliferation of cancer cells. SLAMF7 interacts with other proteins on NK cells to regulate tumor response. SLAMF7 can also interact with different proteins to control the regulation of T cells, B cells, and macrophages. While there has been increased interest in and research on this protein, the specific pathways of SLAMF7 need further investigation. The role of SLAMF7 in myeloma cell proliferation isn’t entirely clear yet. SLAMF7 likely promotes bone marrow adhesion, by interacting with the bone marrow microenvironment to hide from the body’s immune system, resulting in the proliferation of MM cells.There have been many clinical trials of elotuzumab combined with other anti-myeloma therapeutics. Elotuzumab combined with pomalidomide has shown the most promising results in relapsed and refractory multiple myeloma. More research should be done to understand the mechanism behind this drug-inhibiting multiple myeloma. It is suspected that the mechanism involves the soluble form of SLAMF7, which can also be used as a biomarker since it is elevated in MM patients but not present in healthy patients. Identifying biomarkers to monitor treatment efficacy can improve treatment response. While sSLAMF7 shows promise as a biomarker of MM, more research has to be done to clarify the threshold between sSLAMF7 high/low patients.While novel therapeutics such as elotuzumab and CAR-T have improved patient prognosis, MM is still incurable due to most patients developing resistance to the treatments. The resistance mechanisms still need to be fully investigated. Researching them could provide opportunities to combat this resistance or target new molecules in order to improve the prognosis of MM patients.Further research can optimize the use of SLAMF7-targeted therapies and improve the outcomes of MM patients. BLIMP-1 has been shown to be a transcriptional factor for multiple myeloma. Targeting transcriptional factors could lead to a decrease in the expression of SLAMF7 in multiple myeloma cells. Therefore, new therapies targeting this or other downstream signaling pathways could improve outcomes for multiple myeloma patients.Overall, while SLAMF7 shows a lot of promise in the treatment of MM, more research must be done to develop new and lasting treatments.


Social communication is fraught with ambiguity. Negotiating the social world requires interpreting the affective signals we receive and often selecting between channels of conflicting affective information. The affective face-word Stroop (AFWS) provides an experimental paradigm which may identify cognitive-affective control mechanisms underpinning essential social-affective skills. Initial functional magnetic resonance imaging (fMRI) study of the AFWS identified right amygdala as driving this affective conflict and left rostral anterior cingulate cortex (rACC) as the locus of conflict control. We employed electroencephalogram (EEG) and eLORETA source localization to investigate the timing, location, and sequence of control processes when responding to affective conflict generated during the AFWS. However we designated affective word as the response target and affective face as the distractor to maximize conflict and control effects. Reaction times showed slowed responses in high vs. low control conditions, corresponding to a Rabbitt type control effect rather than the previously observed Grattan effect. Control related activation occurred in right rACC 96–118 ms post-stimulus, corresponding to the resolution of the P1 peak in the Visual Evoked Potential (VEP). Face distractors elicit right hemisphere control, while word distractors elicit left hemisphere control. Low control trials require rapid “booting up” control resources observable through VEPs. Incongruent trial activity in right fusiform face area is suppressed 118–156 ms post stimulus corresponding to onset and development of the N170 VEP component. Results are consistent with a predicted sequence of rapid early amygdala activation by affective conflict, then rACC inhibition of amygdala decreasing facilitation of affective face processing . Negotiating the complex, often contradictory world of social communication requires skillful understanding not only of the goals, thoughts, and intentions of others, but also of their emotions expressed across both verbal and non-verbal channels of communication. Due to the pragmatic nature of social communication, emotional signals may be both consistent and inconsistent across verbal and non-verbal channels, and this may change rapidly across time.In turn, the perceived emotions of others often evoke corresponding emotions, which play a powerful role in motivating and regulating our own social responses . Consequcolor of the word while ignoring the written word, which may be congruent  or incongruent  with the required behavioral response. Similarly, the Eriksen Flanker task requires participants to respond to the central letter or symbol in a row of symbols, where it is flanked by characters which may be congruent or incongruent with the required behavioral response  in response to various manipulations of task conflict in order to identify and parse the neural responses underlying these well-known behavioral effects. These paradigms pitch various aspects of the experimental stimulus , selection of response category, motor response , and task requirements  to create conflicts affecting selection of the required behavioral response with observable effects on reaction time and accuracy. For example, the color-naming Stroop paradigm requires participants to respond to the response .Then as now, theories of top-down regulation (control) posit that processing conflicts elicited by these paradigms mobilize resources which regulate these conflicts in the service of task performance . Thus, nearlier at the level of stimulus processing (which underscores the importance of determining the timing of such effects). However, I trials are also expected to elicit activation of control resources to maintain the accuracy of responses in the face of Stroop type response conflicts, whereas C trials do not. Taken alone, regions of increased activation in the I vs. C comparison could indicate either or both the effects of task conflict (during stimulus or response processing) or the control processes (at the locus of either their origin or their expression) elicited to regulate this conflict.One approach taken to identify the locus of conflict processing effects in the Stroop or related paradigms is to identify brain activity following the presentation of stimuli combining task relevant and irrelevant features with conflicting task response mappings , and contrast that with brain activity following stimuli combining such features but with identical task response mappings  to interfere with the speed and accuracy of behavior. Such interference between affect and cognition has been investigated using “emotional Stroop” paradigms, in which affectively arousing stimuli are presented in conjunction with (or incorporated within) stimuli to be processed in some cognitive paradigm. Affective stimuli employed in such paradigms are normatively graded according to valence and intensity  with botFace-word Stroop paradigms present visual stimuli where a word is superimposed upon a face, and participants respond to either the word or the face by identifying a salient stimulus dimension of the target feature such as whether the target is an actor or a politician  or whethAt a behavioral level, II trials (compared to CI) produced greater activation in the left rACC, which was interpreted as control related effects . This interpretation was bolstered by a series of further analyses; activity in medial PFC and right DLPFC during prior incongruent trials was found to predict rACC activation in the current trial. Psychophysiological interaction analysis  found anThe amygdalae (left and right) are amongst the most omniconnected regions in the brain . These cInitial activation of the amygdala by affectively salient facial expressions (fear and happiness) occurs rapidly via magnocellular pathways from the superior colliculus and pulvinar nucleus of the thalamus . SynaptiBased on these findings, it is expected that amygdala activation triggered by processing conflicts between incongruent sensory affective signals in the affective face-word Stroop task will drive the amplification of conflicting representations of affective meaning generated at higher levels of visual (face and word) processing hierarchies. In which case, the down-regulation of conflict-related amygdala activation by rACC reported by We considered that application of EEG source localization methods to the averaged visual evoked potentials (VEPs) generated by a version of face-word affective Stroop may permit us to test aspects of the functional interpretation developed above. VEPs provide the fine-grained temporal resolution to allow us to establish the timing and temporal sequence of functional events with great precision. Source localization, guided by prior results and specific predictions, permits us to identify whether the cortical regions expected to be engaged in those events match the actual regions involved.The features of the VEP of interest for the current study are early waveforms generated by occipital and temporal cortex, which are specifically responsive to face processing. These are the P1 wave  and the immediately following N170 wave peaking about 150–170 ms post-stimulus . There iA tentative model of the temporal dynamics in control responses to affective stimulus conflict may be drawn: initial conflicting affective signals are relayed rapidly to the amygdalae. Conflict sensitive  amygdala activation triggers a rapid inhibitory control response from  BA32 and enhances processing of affective face features in (likely right) inferior temporal lobe (associated with the FFA). This facilitation is downregulated as a consequence of BA32 regulation of the amygdala in a later time window of the VEP see . The likWe set out to test predictions drawn from this model which extend the temporal detail reported by We chose to depart from the paradigm employed by Etkin et al. in one very important respect: while they employed the face as the response target, we employed the word. In the results of the initial (non-affective) face-word paradigm reported by The issues addressed in the current study  are driven by unanswered questions arising from the rationale, findings and conclusions lying in and behind the original paper of less negative N170 in I-trials. This indicates that the N170 is indeed sensitive to conflict in affective stimulus, and that modulation (control) of task relevant and task irrelevant affective stimulus features have been triggered by this very early processing stage, supporting our choice to examine these early time windows. Behavioral findings from the fMRI study by Following these considerations, we sought to test the following predictions:1.We predicted that, when identifying the word of the affective face-word Stroop task, participants would still show the classic Stroop effect; that is, faster reaction times to congruent face-word affective signals than to incongruent signals.2.a)We also expected conflict related control effects in the form of significant reaction time differences between II and CI trial types.b)We expected these differences to follow the Gratton effect with reaction time for II trials faster than for CI trials.3.We sought to employ EEG source localization of VEP components to determine the timing of rACC affective-conflict related control activity as reported by a)We expected eLORETA estimates of source localized activity during the VEP to face-word stimuli, in the rACC, to be significantly greater in II than CI trials.b)Due to the reversal in the laterality of stimulus processing for the incongruent distractor  we expected rACC control activation to be switched from left rACC in  to rightc)We expected control related rACC activation to occur in a time window following expected amygdala activation (80 ms) but prior to control induced downregulation of (conflicting) affective-face processing.4.We propose that affective face-word conflict triggers a rapid control response that for word targets leads to reduced facilitation of the affective component of face processing. Therefore, we expected a reduction in activity in the right FFA in the time window of the N170 for I trials compared to C trials.Male = 19, Female = 29) from a local government authority at a regional Australian city volunteered for this study. Ages ranged from 25 to 65 years . A total of 38 participants self-reported right-handedness and 10 self-reported left-handedness. Eight participants declared they were taking prescription medication for either a neurological or psychological condition. Ethics approval was granted by the responsible Human Research Ethics Committee. All participants provided written informed consent prior to the experiment and were debriefed on completion of their testing. Permission to conduct the study was granted by the local government authority’s Chief People and Culture Officer.Forty-eight English speaking employees , 51 incongruent-incongruent (II), 44 incongruent-congruent (IC), and 14 congruent-congruent (CC). As the planned analyses was the CI vs. II comparison  the stimulus sequence was constructed to maximize the occurrence of CI and II trial types. CC and IC trial types as such are not analyzed. The number of fearful/happy faces for each relevant condition can be seen in The task consisted of the presentation of 154 images across two blocks of trials, separated by a break. The first block comprised of 78 trials and the second block of 76 trials. The first trial of each block is not assigned a stimulus sequence. Stimuli were presented for 1000 ms and were separated using an interstimulus interval (ISI) that jittered between 1104 and 5992 ms to decrease expectancy effects. These ISI are longer than typical in an ERP paradigm and were chosen to match those reported by Electroencephalogram was recorded using a 64-channel (channels M1 and M2 were not used in the recording) Neuroscan QuikCap  arranged in accordance with the international 10/20 system and aligned with the anatomical nasion and inion points see . ElectroAfter providing written informed consent, participants completed a demographic information form and all cap electrodes were prepared using an electrolyte solution administered with an electronic pipette into the QuikCell sponge of the electrodes. Impedance levels were below 10 kΩ at the commencement of recording.Participants were instructed to relax into a comfortable position approximately 1 m from the computer screen, and to remain as quiet and still as possible for the duration of the experiment. All participants commenced EEG recordings with 2 min of eyes closed rest followed by 2 min of eyes open rest. EEG recording was paused while participants were provided with instructions for the Stroop task. Participants were instructed to respond to the emotional word and not the facial expression as quickly and as accurately as possible and to avoid missing a response. They were instructed to nominate their response by using their right hand only on the numeric keyboard, pressing number 1 to indicate the word “FEAR” or number 3 to indicate the word “HAPPY.” In order to counterbalance the response type with motor preparation, these numbers were reversed for each alternate participant. The break allowed between the two experimental blocks was up to 20 s.Recording recommenced at the completion of the verbal instructions immediately prior to commencement of the first block of the emotional Stroop task. Testing took approximately 90 min per participant per session, including equipment set up.Fully Automated Statistical Thresholding for EEG artifact Rejection  was conducted via ejection  to rejecEvent Related Potentials (ERPs) are a measure of cortical activity and are time and phase locked to a stimulus. EEG waveforms were epoched between 250 ms pre-stimulus and 500 ms post-stimulus. EEGLAB version 14.1.1  was used3 gray matter voxels throughout the cortex. The weights utilized by eLORETA yield images of current density in a standardized realistic head model  and the associated probabilities. In each case, when the maximum voxel statistic was significant [as determined by non-parametric randomization testing; see d values are based upon the threshold t value.Mean reaction times for correct responses were calculated for each individual in each of the I, C, CI, and II trial types. RTs faster than 200 ms and slower than 1050 ms were excluded from the mean RT calculation. Mean RT and response accuracy rate differences between trial types were analyzed in SPSS v27 using paired samples Data obtained were analyzed using reaction times and error rates . The asst-test was used to compare participants’ mean reaction times on incongruent trials and congruent trials (see t(47) = 2.080, p = 0.021 (one tailed), Cohen’s d = 0.28.A paired samples ials see . There wt-test was used to compare participants’ mean reaction times on II trials and CI trials (see t(47) = −2.386, p = 0.021 (two tailed), Cohen’s d = −0.32. A two-tailed p-value is reported in this case because the direction of the significant difference is the reverse of that described by the Grattan effect.A further paired samples ials see . There wz = −0.062, p = 0.950, two-tailed, r = 0.009.Mean accuracy was consistently high across all trial types. A Wilcoxon signed rank test indicated that II trials had a non-significant influence on participant accuracy scores compared with CI trials; Averaged VEPs for C and I trials are shown for electrodes Fz, PO7, and PO8 in Global Field Power (GFP) is the spatial variance of the EEG/ERP signal across all electrodes at each time point in the signal. As EEG/ERP topography is essentially the information on which source analysis operates, GFP provides an objective method by which to establish time windows for source analysis that are fitted to a particular data set but do not pre-empt the results of the analysis. The GFP time-series for C and I trials is shown in Averaged VEPs for CI and II trials are shown for electrodes Fz, PO7, and PO8 in https://aus01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fhdl.handle.net%2F1959.11%2F54805&data=05%7C01%7Cgjamieso%40myune.mail.onmicrosoft.com%7C6257298be77e460f0cb608db572371ec%7C3e104c4f8ef24d1483d8bd7d3b46b8db%7C0%7C0%7C638199581972550173%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=B3UkQRqoqGIXkA%2Fm5%2Ba76k30rNYuu0Xg8lMP7b%2Bk2r0%3D&reserved=0.Event related potential source analyses were conducted on averaged waveforms at all 62 recording electrodes. The grand averages at each electrode, in each condition, as well as individual averages and behavioral data for each participant are available for inspection and may be downloaded from the following RUNE link Based on the GFP data, four time windows were established for the eLORETA analysis of cortical source activity differences between CI and II trials and between C and I trials. The first window was defined by the onset of the first GFP peak until the first peak (50–98 ms post stimulus). Note that the termination of this time window closely corresponds both to the P1 peak and to the expected magnocellular activation of the amygdala. The second window was defined by interval between the first GFP peak and the following GFP minimum (98–118 ms post-stimulus). The third window is defined by period from the GFP onset of the N170 component in the VEP until the second GFP peak (118–156 ms post-stimulus). The final window corresponds to the resolution of the N170 component and is defined by the time from the second GFP peak until the second GFP minima (154–204 ms post-stimulus).t(41) = −3.847 with a probability of p < 0.002 for the set. At the threshold value, Cohen’s d = 0.64 (a medium effect size).For the time window 96–118 ms post stimulus the exceedance proportion test identified a group of 26 voxels above the threshold value of t = −4.27) was located at MNI  right BA32. Supra threshold voxels are shown in blue in The maximum voxel statistic (The full list of suprathreshold voxels is presented in The II vs. CI contrast found no significant voxels in any other time window.t(41) = −3.537 with a probability of p < 0.003 for the set. At the threshold value, Cohen’s d = 0.559 .For the time window 118–156 ms post stimulus, the exceedance proportion test identified a group of 20 voxels above the threshold value of t = −3.39 was located at MNI  at right BA20. Supra threshold voxels are shown in blue in The maximum voxel statistic The full list of suprathreshold voxels is presented in The I vs. C contrast found no significant voxels in any other time window.We sought to extend the understanding of how conflicts in affective signals between verbal and non-verbal channels are resolved in the brain. We utilized the temporal resolution of the EEG to explore within-trial dynamics controlling the processing of these conflicts in the affective face-word Stroop paradigm developed by Etkin et al. In doing so, we adapted the original paradigm so that participants must respond to the word  rather than the face cue. Our first concern was to establish that this paradigm would produce both Stroop interference and control type effects at the behavioral level. This was confirmed with RTs for I trials found to be significantly longer than C trials.Next, we sought to establish the presence of a control effect at the behavioral level between II (considered to be high control) and CI (considered to be low control) trials. There was a significant difference in RT between these two conditions, however, it was the CI trials which showed faster responses than the II trials. This contradicted our expectation of a Gratton type control effect. Instead of the high control condition being faster than the low control condition, it was slower. In fact, the effect size for this difference was larger than the effect size for the Stroop effect itself.This pattern is similar to another widely studied behavioral control effect known as the Rabbitt effect  also kn. In the This could be explained if the response criteria of participants was biased toward accuracy rather than speed. slower to respond (the Gratton effect) but in this case they are faster therefore following that deeper logic we continue to interpret this finding as a control response induced by affective–conflict.The comparison of cortical source activity between high control (II) and low control (CI) trials confirmed a highly significant cluster of affective-conflict control related voxels centered in the right rACC, with lower activity on the slower II trials and higher activity on the faster CI trials. Applying the logic of Botvinick et al. from brain imaging studies to these ERP source analysis results, this CI > II activation difference would be taken to indicate conflict (rather than control) related activity, whereas II > CI voxels are taken to indicate control related activation. That is, based on the reasoning that CI trials are lower in control (hence higher in conflict) than II trials because they are This affective-conflict-stimulus evoked control response was observed only in the 98–118 ms post-stimulus time window. This time window lies in the resolution of the P1 component of the VEP and immediately prior to the onset of the N170 component (which in this case peaked at 156 ms post stimulus). This occurs very early in the stimulus processing response, prior to the completion of sensory stimulus analysis, and hence prior to response selection and motor preparation. It precedes, rather than follows response conflict within the trial, yet it is sensitive to congruency in the previous trial. It may be that it is the discrepancy between prior trial stimulus-conflict and the current trial stimulus conflict (a form of expectancy violation) that is triggering the control response in this case.The timing of BA32 control activity in this study is too early to be triggered by either sensory analysis of stimulus conflict or response conflict. In the current study, control related rACC activity was found only in the right hemisphere, unlike In addition to the specific predictions made concerning rACC activation in relation to affective control, there were further unanticipated findings in our contrast of II and CI trials in this time window. While the locus of significant voxels was clearly right rACC (BA32), fully half of the significant group of voxels identified lay in right orbitofrontal cortex . Amongst many effects this early amygdala response is expected to prime extrastriate visual cortex for processing salient stimulus features, thus enhancing later processing in these regions. From the findings of Current findings in the I vs. C contrast correspond closely with the ERP results reported by There are two important ways in which the experimental paradigm of the current study differ from Secondly, the ratio of I to C trials in this study was approximately 2:1 whereas in It may also be useful for researchers to examine the differences in control elicited when participants are instructed on the importance of accurate responses, speedy responses, and when they receive balanced instructions. In order to unambiguously interpret I vs. C source localization differences as evidence for facilitation, absence of facilitation or conflict interference, it is necessary to include affectively neutral face and word stimuli in the AFWS paradigm [as employed for example by This research compliments and extends the findings of At a behavioral level, we were able to identify a novel Rabbitt type control effect. The Rabbitt effect has been shown to be modulated by the same conflict related activity  as the Gratton effect in a cognitive Stroop color naming task . These rWe have confirmed the role of rACC in the control response to affective conflict and identified that these effects are lateralized. Considering these findings with those of Current results support the view that rACC regulation of affective-conflicts are triggered within trials by early conflict-related amygdala activation, leading to down regulation of the amygdala response and hence to down regulation of the amygdala’s attentional facilitation of selected affective signals (in this case targeting the right FFA). While the current findings extend our understanding of how the brain responds to affective conflicts, the ultimate utility of such knowledge lies in understanding the real world of complex social interactions in which the management of affective conflicts plays a key role in negotiating the complex interactions between self and others.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Human Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.GJ contributed to planning, writing, and analysis. JP contributed to planning, writing, analysis, and data collection. IE contributed to writing and analysis. AH contributed to planning and writing. All authors contributed to the article and approved the submitted version.