The purpose of this study is to provide Level-1 objective, real-world outcome data for patients with lumbar spinal stenosis suffering from neurogenic claudication secondary to hypertrophic ligamentum flavum.mild® Procedure  as a first-line therapy in combination with nonsurgical conventional medical management (CMM) vs CMM alone as the active control.The MOTION Study is a prospective, multicenter, randomized controlled trial comparing the mild Procedure at baseline. Both the mild+CMM group and the control group were allowed unrestricted access to conventional real-world therapies. Patient-reported outcomes included the Oswestry Disability Index, the Zurich Claudication Questionnaire, and the Numeric Pain Rating Scale. A validated Walking Tolerance Test, the incidence of subsequent lumbar spine interventions, and the occurrence of adverse events were used to measure objective outcomes.Patients in the test group received the mild+CMM.Sixty-nine patients in each group were analyzed at 1-year follow-up. No device- or procedure-related adverse events were reported in either group. Results from all primary and secondary outcome measures showed statistical significance in favor of mild+CMM over CMM alone for patients with lumbar spinal stenosis who were suffering from neurogenic claudication secondary to hypertrophic ligamentum flavum. Use of the validated Walking Tolerance Test to objectively measure increased ability to walk without severe symptoms provided evidence of statistically significantly better outcomes for mild+CMM than for CMM alone. With no reported device or procedure-related adverse events, the long-standing safety profile of the mild Procedure was reaffirmed. mild is a safe, durable, minimally invasive procedure that has been shown to be effective as an early interventional therapy for patients suffering from symptomatic lumbar spinal stenosis.One-year results of this Level-1 study demonstrated superiority of  Lumbar spinal stenosis (LSS) is a degenerative spinal condition caused by narrowing of the spinal canal, which often results in compression of neural elements and symptoms of neurogenic claudication (NC) . Patientmild® Procedure  [mild Procedure provides minimally invasive decompression of the spinal canal for patients with LSS with NC secondary to HLF. The mild Procedure has demonstrated a better safety profile than have other spinal interventions, including surgical decompression, interspinous spacers, and fusion [mild has been shown to be similar to that of epidural steroid injections, with more durable results [Early treatment for patients with LSS generally begins with conservative measures, which can include physical therapy, home exercise programs, and oral analgesics, followed by low-risk interventional therapies . First-lCA, USA) . The mild fusion , 10. Fur results .mild in combination with conventional medical management (CMM) (mild+CMM) vs a CMM-only control group (CMM-Alone) in patients with LSS with NC. The study is designed to reflect real-world practice by allowing the use of standard-of-care CMM in both study groups at the discretion of the investigators. The only difference between the study groups is the use of mild, which is allowed in the mild+CMM group and not allowed in the CMM-Alone arm. In this study, the mild Procedure is used as first-line therapy together with other low-risk CMM treatment options. MOTION provides Level-1 evidence of the superiority of mild+CMM over CMM-Alone, as evaluated by multiple validated patient assessments and objective real-world outcome measures. Six-month results have been previously reported [The MOTION study compares reported . Primarymild treatment in combination with CMM (mild+CMM) vs a CMM-only control group (CMM-Alone). The study is registered with the US Clinical Trial Registry as the MOTION Study (NCT03610737). It is being conducted at 19 interventional pain management centers throughout the United States. The study was approved by an Institutional Review Board for each participating site before patient enrollment, and it followed the Consolidated Standards of Reporting Trials (CONSORT) [The MOTION study is a prospective, multicenter, randomized controlled trial comparing the CONSORT) . RecordsPatients ranging in age from 50 to 80 years and experiencing NC symptoms for a duration of at least 3 months were included in this study. Confirmation of patient eligibility involved an evaluation of medical history, including comorbidities, history of symptoms, and prior treatments, such as surgery and interspinous spacer. Baseline function was evaluated with the Oswestry Disability Index (ODI), Numerical Pain Rating Scale (NPRS), and Zurich Claudication Questionnaire (ZCQ) scores. Magnetic resonance images or computed tomographic images (when magnetic resonance imaging was not possible) of the spine were assessed by an independent medical monitor. MOTION study selection criteria are presented in mild+CMM, whereas patients in the control group were treated with CMM alone. Study investigators at each site determined the appropriate CMM regimen for each patient. All primary and secondary endpoint outcomes were assessed at 1 year with comparisons to baseline. Crossover was allowed for patients in the CMM-Alone control group after 1-year follow-up. Subjects will continue study follow-up regardless of any crossover.Patients with LSS were randomized in a 1:1 ratio to one of two parallel interventions for treatment of NC. In the test group, patients were treated with The appropriate CMM regimen for each MOTION patient was determined by the site investigator throughout the course of the study. As a reflection of real-world practice, CMM included any conservative or low-risk interventional therapies that are standard of care for early treatment of NC. CMM may be comprised of home exercise, walking aids, and physical therapy, as well as early interventional therapies, such as epidural steroid injections, medial branch injections, radiofrequency ablation, and facet joint injections.mild Procedure provides minimally invasive lumbar decompression performed from the posterior lumbar spinal approach with local anesthetic and light sedation. The procedure removes small portions of the lamina and preferentially resects and debulks the thickened ligamentum flavum, leaving no implants behind. The mild Procedure has been previously described in detail [mild+CMM group underwent the mild Procedure after baseline assessment. The use of any additional CMM therapies was determined by the study investigator.The n detail . PatientMultiple objective outcome measures were used to assess levels of function and pain for study patients. Objective outcome measures provide quality-of-recovery evaluations that are independent from judgment and are therefore less susceptible to bias . These omild Procedures. “Safety data” for this study refers to device- or procedure-related adverse events. All serious adverse events (SAEs) were reported, regardless of relationship. SAE was defined according to the CFR  Title 21 definition. All reportable adverse events were adjudicated by an independent clinical event adjudicator.Additional objective outcome measures included the number of subsequent lumbar spine interventions and safety data. Subsequent lumbar spine interventions that were defined to indicate study treatment nonresponse included laminectomy, laminotomy, lumbar fusion, interspinous spacers, neurostimulators, adhesiolysis, kyphoplasty, and additional Subjective patient-reported outcomes included both primary and secondary patient-reported outcome measures. The primary efficacy endpoint was mean improvement in ODI score at 1-year follow-up compared with baseline. ODI is used to evaluate functional disability related to lower back pain . SecondaTo test the primary superiority hypothesis, sample size was calculated to a threshold of 90% power. A sample size of 75 subjects in each group was considered sufficient under the assumption of a two-sided hypothesis with type 1 error of 0.05, power (1–β) of at least 90%, and randomization ratio of 1:1, with accounting for up to 10% data attrition. The randomized study population of N = 155 meets these criteria.t test at a 0.05 level of significance and with a Fisher exact test for frequency in Microsoft Excel  and VassarStats .Continuous data are summarized with means and standard deviations. Categorical variables are summarized with frequency counts and percentages. For multiple possible events occurring within a single patient , the percentage is based on the number of patients experiencing the event. In this case, both patient and event counts are reported. Mean comparisons were performed with a two-tailed mild+CMM group and 78 to the CMM-Alone group. Five of 77 patients in the mild+CMM group and two of 78 patients in the CMM-Alone group were not treated. In the mild+CMM group, one patient withdrew for unrelated reasons, one missed a follow-up visit, and one died due to COVID-19. In the CMM-Alone group, five patients withdrew for unrelated reasons, and two missed follow-up visits. As a result, 69 patients were included in the analysis for each group at 1-year follow-up. See One hundred and eighty-one (n = 181) patients across 19 sites were evaluated for eligibility. Twenty-six (n = 26) did not meet the selection criteria and were excluded. The remaining 155 patients were randomized, with 77 assigned to the mild+CMM and CMM-Alone patients were 64.7 years and 66.8 years, respectively. Patient age was not statistically different between the groups (P = 0.077). Other patient characteristics and baseline metrics are listed in P values ranging from 0.246 to 1.000). A complete list of spinal comorbidities was previously reported in the MOTION 6-month report [At baseline, the average ages of h report .mild in the mild+CMM group were treated at one level only (59.7%), and L4–L5 was the most commonly treated level (76.4%), followed by L3–L4 (52.8%). Most patients were treated bilaterally (90.3%). Nearly two thirds of mild Procedures were performed in the hospital outpatient setting (65.3%), and the balance (34.7%) were conducted in an ambulatory surgery center. The large majority of mild patients received monitored anesthesia care sedation (93.1%), which includes light sedation and monitored anesthesia care with local anesthesia. mild Procedure characteristics for patients treated in the mild+CMM group.The majority of patients who received mild, who generally have no activity restrictions within 24 hours of treatment and can immediately proceed with reconditioning. Overall, the CMM-Alone group received a higher rate of interventional therapies than did the mild+CMM group (61.3% vs 47.9%), but this difference was not statistically significant.All CMM therapies performed in this study are listed in mild+CMM group achieved a mean improvement of 258% in walking time to onset of severe symptoms, compared with a 64% mean improvement for patients in the CMM-Alone group. This difference between groups was statistically significant (P < 0.001)  .mild+CMM patients (P = 0.002) (mild Procedures (n = 9), laminectomies (n = 2), fusion (n = 1), stimulator (n = 1), and spacer (n = 1). In the mild+CMM group, subsequent lumbar spine interventions were spacers (n = 2), surgical decompression (n = 1), and stimulator (n = 1).At 1-year follow-up, 26.1% of CMM-Alone patients had undergone a subsequent lumbar spine intervention, compared with 5.8% of = 0.002) . This remild+CMM group, eight patients experienced 13 unrelated SAEs (11.7%) (P = 0.793).There were no device- or procedure-related adverse events reported in either study group, and therefore safety was similar between the groups at 1 year. In the CMM-Alone group, seven patients experienced eight unrelated SAEs (11.5%), and in the mild+CMM group experienced a 16.1-point composite ODI mean improvement, compared with a 2.0-point mean improvement for patients in the CMM-Alone arm. This difference was statistically significant and indicates superiority of mild+CMM (P < 0.001)  . See Tabmild+CMM vs CMM-Alone . ZCQ patient satisfaction also indicated that mild+CMM patients were statistically significantly more satisfied with their treatment than were CMM-Alone patients at 1-year follow-up (P < 0.001). For ZCQ patient satisfaction, lower scores indicate greater patient satisfaction with their treatment, and scores ≤2.5 indicate that the patient is satisfied [mild+CMM at both the 6-month and 1-year follow-ups. Within-group mean improvements for certain outcome measures for CMM-Alone patients did not reach statistical significance, including ODI and ZCQ physical function at 1-year follow-up and ZCQ symptom severity and physical function at 6-month follow-up. See Mean improvement from baseline to 1-year follow-up for all secondary outcome measures, including NPRS for back and leg and the ZCQ physical function and symptom severity domains, demonstrated statistical significance in favor of atisfied . A withimild+CMM with CMM-Alone provides Level-1 evidence that the mild Procedure together with CMM is superior to CMM alone in improving function and decreasing pain for patients with LSS suffering from NC and HLF. The mild Procedure removes a major root cause of LSS by debulking the thickened ligamentum flavum, preserves baseline spine stability, and leaves no implants behind. The mild Procedure has a safety profile similar to that of epidural steroid injections, and patients typically resume normal activity within 24 hours with no restrictions. Measurement of increased ability to walk without severe symptoms via the validated Walking Tolerance Test, incidence of subsequent lumbar spine interventions, and frequency of adverse events provide objective assessments of patient quality of recovery in the real-world clinical environment. The outcomes of this study reflect routine use of the mild Procedure in a typical clinic setting.This prospective, multicenter, randomized controlled trial comparing mild+CMM showed a statistically significant improvement over CMM-Alone and within group for the ODI composite primary endpoint and secondary NPRS and ZCQ endpoints, along with sustained functional improvement at 1 year. This sustained improvement from baseline to 1 year within mild+CMM, while maintaining a wide margin of difference between the groups, shows the durable impact of the mild Procedure together with adjunctive conservative therapies. This durability has been seen in many previous studies reporting on similar outcome measures at 1-, 2-, and 5-year follow-up [mild treatment. One year after treatment with mild, Mekhail and colleagues at Cleveland Clinic reported a seven-fold improvement in standing time, from 8 minutes to 56 minutes, and a 16-fold increase in walking distance, from 246 feet to 3,956 feet [ollow-up , 24–29. 956 feet . The MOTmild+CMM felt that they needed additional interventions beyond conservative therapies over the course of a year, 26.1% of patients in CMM-Alone sought subsequent lumbar spine interventions during the same time period, of which half were mild Procedures. The greater level of satisfaction with mild+CMM is reflected in the significantly better satisfaction scores for this treatment arm in the ZCQ questionnaire. The rate of crossover to subsequent lumbar spine interventions in the CMM-Alone group reinforces the position of mild as first-line therapy in real-world settings.The incidence of subsequent lumbar spine interventions can be considered as a measure of patient dissatisfaction with the initial treatment and continued need for symptom relief. Although only 5.8% of patients in mild Procedure with epidural steroid injection, including the ENCORE randomized controlled trial, in which there was no difference in the rate of adverse events between the two treatment groups  [mild Procedure has also been proved extensively in multiple studies that reported no major device- or procedure-related SAEs [No device- or procedure-related adverse events or SAEs occurred in the MOTION study. This level of safety reflects the low rates of complications in previous studies comparing the  = 1.00) , 30–32. ted SAEs .mild Procedure is not intended to directly treat lateral or foraminal stenosis, but reports have indicated that related bony anomalies were actually a positive predictor of success with mild [mild Procedure has been shown to be safe and effective for patients with multiple comorbidities, such as foraminal narrowing, lateral recess stenosis, bulging disc, and facet arthropathy, in addition to HLF [mild Procedure can have on patients with typical constellations of related spinal comorbidities.The ith mild . The MOTn to HLF , 24, 30.mild Procedure in a typical clinic setting. In day-to-day practice, the mild Procedure is not used alone but in conjunction with other conservative therapies. The nonblinded nature of the study can also be considered a limitation. The use of objective real-world outcome measures, together with independent physicians in the role of medical monitor, clinical events adjudicator, and study principal investigator, were intended to limit study bias.Although the MOTION study was designed to include a patient population commonly seen every day in the clinic, the inclusion of numerous CMM treatment options chosen at the investigator’s discretion provided a broad range of treatment options and sequencing, as is encountered in the real world. This limited control over the use of CMM, though intended in the study design, may be viewed as a study limitation. The use of CMM in both arms of this study simulates real-world practice, but it also may result in confounding, as patients are treated on the basis of routine use of the mild Procedure in combination with CMM over the use of CMM alone for patients with LSS suffering from NC secondary to HLF. The mild Procedure with CMM was shown to be durable to 1 year through validated, patient-reported outcomes and objective real-world measures. Use of the validated Walking Tolerance Test to objectively measure increased ability to walk without severe symptoms provided evidence of statistically significantly better outcomes for mild+CMM than for CMM-Alone. With no reported device- or procedure-related adverse events, the long-standing safety profile of the mild Procedure was validated. The results of this study confirm the use of the mild Procedure as a safe and effective first-line treatment for the indicated LSS patient population.The 1-year results of the Level-1 MOTION randomized controlled trial demonstrate the superiority of the


A total lockdown for pandemic SARS-CoV-2 (Covid-19) entailed a restriction of elective orthopedic surgeries in Switzerland.  While access to the hospital and human contacts were limited, hygiene measures were intensified. The objective was to investigate the impact of those strict public health guidelines on the rate of intra-hospital, deep surgical site infections (SSI), wound healing disorders and non-infectious postoperative complications after orthopedic surgery during the first Covid-19 lockdown.In a single-center study, patients with orthopedic surgery during the first Covid-19 lockdown from March 16, 2020 to April 26, 2020 were compared to cohorts that underwent orthopedic intervention in the pre- and post-lockdown periods of six months each. Besides the implementation of substantial public health measures (promotion of respiratory etiquette and hand hygiene), no additional infection control bundles have been implemented.5791 patients were included in this study. In multivariate Cox regression analyses adjusting for the large case-mix, the lockdown was unrelated to SSI (hazard ratio (HR) 1.6; 95% confidence interval (CI) 0.6–4.8), wound healing disorders  or other non-infectious postoperative complications  after a median follow-up of seven months.The risks for SSI, wound healing disorders and other complications in orthopedic surgery were not influenced by the extended public health measures of the total Covid-19 lockdown.Trial registration BASEC 2020–02646 .Level of evidence: Level III. On March 11, 2020, the World Health Organization declared a worldwide pandemic due to the extremely rapid expansion of SARS-CoV-2 (Covid-19) infections. Besides its highly contagious profile, severe consequences due to acute respiratory syndrome and unpredictable sequelae on the overall health status, the socio-economic impact and financial burden on healthcare systems have been significant and will keep challenging societies all over the world . On the To the best of our knowledge, no study to date has investigated the impact of the Covid-19 lockdown on postoperative infections, wound- and other surgery-related complications in orthopedic surgery. Hence, we aimed to fill this gap with a single-center cohort and aspired to elucidate the impact of enhanced hygiene measures and limited human contacts on many important complications after orthopedic surgery. We renounced on reporting HAI among hospitalized Covid-19 patients and avoided to analyze the nosocomial proportion of Covid-19, for which a broader literature is available.The Balgrist University Hospital prospectively registers all moderate to severe infections, including many postoperative complications, since July 2018. The immediate pre-lockdown period witnessed no specific Covid-19 policies besides the promotion of the "respiratory etiquette" and HH –9. DurinThis study followed the ethical principles of the Helsinki Declaration and approval of the project was obtained from the Cantonal Ethics Commission. A one-year study period  was arbitrarily chosen, was divided into three periods and included all surgeries performed in the operation theater: pre-lockdown period with 2688 interventions from October 1, 2019 to March 15, 2020; the Covid-19 lockdown period with 230 surgeries from March 16, 2020 to April 26, 2020 , and requiring revision surgery.The primary outcome was the incidence of deep SSI –12 afterSecondary outcomes were the incidence of non-infectious postoperative wound healing disorders, postoperative local complications other than infection or visible wound problems, and the epidemiology of other non-surgical HAI between the three study periods. Wound healing disorders were defined as substantial necrosis, uninfected dehiscence without typical infectious signs and/or hematoma necessitating surgical drainage. Other causes for revision surgery were recurrence of disease, residual symptoms and/or intervention-specific complications. Key HAI were grouped as urinary tract infections, bacterial pneumonia, Covid-19 disease, and bloodstream infections . NosocomOn the hospital's level, three collaborators with experience in infection control observed the HH compliance  at the b2 , the Wilcoxon-rank sum-test or the Kruskal–Wallis-test for non-parametric, continuous variables. To adjust for the heterogeneity of the surgeries and the large case-mix imposed by the ban of elective surgeries, three multivariate Cox regression analyses were performed. The three final Cox regression models targeted three different outcomes: "SSI", "wound healing disorders" and "other complications". In the first multivariate model, the group of SSI was compared to all other non-infected surgery episodes. In the second model, non-infectious wound problems were compared to all episodes without infection or wound problems, and in the last analysis, all local complications were compared to all uneventful surgeries. Most variables were analyzed as a continuum, but stratifications were added for the ASA-Score, the duration of surgery and the study period. The cut-off values for these strata relied on the 25%, 50% and 75% percentiles of the distribution of values of that variable. To adjust for the case-mix of the surgeries within the three study periods, we recurred to a multivariate Cox regression analysis by inserting stepwise independent variables with a p value ≤ 0.05 from the univariate analysis into the final model. The software IBM SPSS Statistics 25 and STATA™  were used and p values ≤ 0.05 (two-tailed) were considered significant.Group comparisons were performed using the Pearson-χ2 (SD 5.3) and the mean ASA-Score 2 points. The foot and ankle was the predominant surgery site , followed by the knee , the spine , the shoulder , the hip , the hand , tumor surgery , and the diabetic foot . Of all index interventions, 47% were primary surgeries, the operative interventions lasted on average 85 minutes (SD 4) and the hospital stay averaged 3 days (SD 5). Considering re-interventions, 43 patients (0.7%) required revision surgery due to SSI, 39 patients (0.7%) due to wound healing disorders and 225 patients (3.9%) due to other causes. The mean time interval from index to revision surgery was 54 days and the average number of re-interventions was 1.2 surgeries.Within the one-year study period, 5791 orthopedic interventions were performed with a median follow-up of 218 days (SD 118) after the index surgery. The mean age of the overall study cohort was 55 years  and 51% were males. 521 patients (9%) had diabetes mellitus, the mean BMI was 26.3 kg/mp = 0.02), the three time periods were comparable for demographics, BMI, ASA-Score, primary surgeries, the duration of surgery as well as the length of hospital stay (Table p < 0.01) because of emergency operations performed in the Spine Center. Additionally, in crude group comparisons, the incidence of revisions for SSI was also significantly higher during lockdown  because of the emergency nature of surgical interventions. Non-infectious complications appeared significantly more often in the pre-lockdown period  whereas wound healing disorders were equally distributed between all three periods .The overall incidence of other HAI was 1.7% (99 infections per 5791 surgeries), including cutaneous or oral mycosis, urinary tract infections  and  value 0.72–0.76, reflecting an acceptable accuracy of the multivariate models. Table This large-scale study including 5791 adult orthopedic surgeries over one year in a tertiary university hospital was the first one to investigate the impact of Covid-19 lockdown on deep SSI, wound healing disorders and non-infectious complications following orthopedic surgery. The six-weeks total lockdown did not influence the risks of SSI, wound healing disorders, HAI or other postoperative complications after orthopedic interventions. We continued to witness HAI and observed a continuum of the HH compliance rather than an increased compliance several months after the lockdown [The impact of strict lockdown measures has not been investigated in orthopedic surgery yet. Generally, the literature is sparse and the opinions divided considering the influence of a lockdown including intensified hygiene awareness, increased hand-rubbing and reduced human contacts on SSI and HAI. Earlier publications advocated a favorable outcome on SSI. To cite examples, a general surgery cohort compared 123 patients operated in the Covid-19 lockdown to 400 patients operated in the same time period one and two years earlier. The SSI risk was significantly lower during the lockdown . SimilarSimilar to our findings, other research groups found the same SSI and overall HAI risk before and during the lockdown periods  or even Unterfrauner et al., unpublished data). In cardiac surgery, the wound infection rate in daily surgical routine is higher compared to our institution (3% vs. 0.7%). Since infections after orthopedic surgery  are known to ensue dramatic secondary complications and inflict long-term deficits on function and general health [Although intensified hand hygiene is known to reduce SSI rates , there wl health , well-trAdditionally, there is no strong rationale that public health measures reduce intra-hospital HAI without a concomitant specific prevention program . AlternaIt would be of great interest to link an observed HH compliance to the incidence of HAI during the pandemic wave , 18–20. The study population only concerned adult orthopedic patients and SSI. We might have missed superficial infections, or wound disorders treated in the ambulatory setting without the need for revision surgery or surgical consultation.We lack a continuous monitoring of the HH compliance during the lockdown. It is clear that during the initial phase of the lockdown, the HH compliance consequently improved , 27. TheDue to the relatively short period of the lockdown period, we could only assess key HAI such as bacterial pneumonia, urinary tract infections and bloodstream infection. These are the same HAI reported in other publications regarding the Covid-19-lockdown , 19, 21.Statistically, we compared a lockdown period including political ban of elective surgeries with normal periods. This study is the largest regarding Covid-19 lockdown and orthopedic surgery. The number of surgeries in the control periods are sufficient, but the lockdown period of six weeks was marked with a ban of elective surgeries. Hence, no single center with a ban of elective surgeries can catch up to an equivalent sample size in the lockdown and control periods. Such a formal analysis is only possible in multicenter evaluations or in settings without ban of elective surgeries during the first lockdown.Finally, we did not monitor antibiotic consumption , 28–30 oStrengths of our study include the high number of 5791 surgeries, a prospective register of infections, two control cohorts pre- and post-lockdown and adjusted analyses. Besides its retrospective design, it has some limitations:In a large Swiss single-center cohort of 5791 adult orthopedic surgeries in a tertiary university hospital, the risks for deep SSI, wound healing disorders, HAI and other complications during the first total Covid-19 lockdown was not different to time periods pre- and post-lockdown. The public health measures did not influence our intra-hospital nosocomial epidemiology.


Is the type of cigarette initially smoked associated with suicidal behaviors among adolescents in Korea?In this cross-sectional study of 255 887 Korean adolescents who participated in the nationwide Korea Youth Risk Behavior Web-Based Survey from 2015 to 2018, those who used electronic cigarettes when they started smoking had a higher risk of suicidal behavior than did those who used conventional cigarettes when they started smoking. Adolescents who changed from using electronic cigarettes to conventional cigarettes were more likely to exhibit suicidal behaviors than were those who changed from using conventional cigarettes to electronic cigarettes.In this study, initial cigarette type was associated with suicidal behaviors among adolescents, which may have implications for public health policies and educational programs regarding electronic cigarette use. Assessment of whether past electronic cigarette (e-cigarette) use or initiating smoking with e-cigarettes is associated with suicidal behaviors among adolescents is needed to inform future research and public health interventions.To evaluate the association between starting smoking with e-cigarettes or conventional cigarettes and suicidal behaviors among Korean adolescents.This cross-sectional study analyzed data on adolescents in grades 7 through 12 who participated in the nationwide Korea Youth Risk Behavior Web-Based Survey between 2015 and 2018.Type of cigarette initially smoked: electronic or conventional.Multiple logistic regression analysis was performed to examine the association between initial cigarette type and suicidal behaviors, including suicidal ideation and suicide planning and attempts. All participants completed questionnaires about their history of suicidal behavior and were categorized into groups according to the type of cigarette used at initiation of smoking and any subsequent change (or lack of change) in the type of cigarette used.P < .001; girls: AOR, 1.55 ; P < .001) and suicide attempts  compared with those who initially used conventional cigarettes. Changing from e-cigarettes to conventional cigarettes was associated with a higher risk of suicide attempts among both boys  and girls  compared with changing from conventional cigarettes to e-cigarettes.A total of 255 887 Korean adolescents  age, 15.0 [1.8] years) were included in the primary analysis. Among 131 094 male adolescents, 3310 boys (2.5%) initially used e-cigarettes and 27 368 boys (20.9%) initially used conventional cigarettes. Among 124 793 female adolescents, 952 girls (0.8%) initially used e-cigarettes and 9296 girls (7.4%) initially used conventional cigarettes. Of those who initially used e-cigarettes, 178 of 3310 boys (5.4%) and 134 of 952 girls (14.1%) attempted suicide. Of those who initially used conventional cigarettes, 946 of 27 368 boys (3.5%) and 911 of 9296 girls (9.8%) attempted suicide. Adolescents who initially used e-cigarettes had a higher risk of suicidal behaviors, including suicide planning (boys: adjusted odds ratio [AOR], 1.63 ; In this study, the initial use of e-cigarettes vs conventional cigarettes was associated with suicidal behaviors among adolescents. In future research on the association of e-cigarette use with adolescent mental health and interventions for suicide prevention, the type of cigarette initially used and changing the cigarette type should be considered. This cross-sectional study uses data from the Korea Youth Risk Behavior Web-Based Survey to examine the association between the type of cigarette initially smoked and suicidal behaviors among Korean adolescents from 2015 to 2018. It is unclear whether e-cigarettes are safer than conventional cigarettes because the toxic effects of e-cigarette components are not fully understood.Electronic cigarettes (e-cigarettes) are portable devices that deliver nicotine through the battery-powered vaporization of a solution of nicotine, propylene glycol, vegetable glycerin, and flavor chemicals. They are sometimes introduced as harmless or safer alternatives to conventional cigarettes. Although e-cigarettes deliver nicotine without the thousands of toxic substances contained in conventional cigarettes,3 Among Korean adolescents, current conventional cigarette use increased from 6.4%  in 2017 to 6.7%  in 2019, and current e-cigarette use increased from 2.2%  in 2017 to 3.2%  in 2019.4 Statistics indicate a high smoking rate among boys and an increasing smoking rate among girls; thus, smoking among adolescents is a public health concern in South Korea.The use of e-cigarettes among adolescents has increased exponentially worldwide. In the US, current e-cigarette use among adolescents in grades 6 to 8 increased from 0.6% in 2011 to 4.9% in 2018; among adolescents in grades 9 to 12, e-cigarette use increased from 1.5% in 2011 to 20.8% in 2018.5 and suicide is the leading cause of death among Korean adolescents. According to the Korea National Statistical Office, the suicide rate among youths increased from 7.7 per 100 000 people in 2017 to 9.1 per 100 000 people in 2018.7 Recognizing the factors associated with suicide is important; however, the differences in sociodemographic factors associated with suicidal behaviors, such as increased nonfatal suicidal behaviors among women and higher suicide rates among men,8 need to be considered. Of note, as part of the guidelines for clinical suicide assessment, physicians should specifically inquire about suicidal ideation and suicide planning and attempts.10South Korea has the highest suicide rate among the member countries of the Organization for Economic Cooperation and Development,17 Previous studies,15 including longitudinal studies, have indicated that smoking during adolescence is associated with depression and suicidal behaviors, including suicidal ideation and suicide planning and attempts. However, most studies evaluating this association have focused on conventional cigarettes. Despite the increasing use of e-cigarettes and e-cigarette use involving inhaled nicotine, few studies have examined the association between using e-cigarettes  and suicidal behaviors.19The association between smoking conventional cigarettes and mental health is well known.20 have evaluated the association between e-cigarette use and mental health. A recent study17 of Korean adolescents found that youths who used both conventional cigarettes and e-cigarettes and those who used e-cigarettes only were more likely to exhibit suicidal ideation and suicide planning and attempts. Substances contained in e-cigarettes, such as e-liquids, are sometimes used in suicide attempts.22 However, most of these studies19 only examined the association between current or lifetime use of e-cigarettes and suicidal behaviors.Some studies23 Furthermore, the distinct characteristics of adolescents who initially use e-cigarettes and their increased risk of subsequent use of conventional cigarettes26 warrants investigation into whether the initial use of e-cigarettes and the continuation of smoking using conventional cigarettes are associated with mental health. To our knowledge, no previous studies have investigated the association between the initial use of e-cigarettes and suicidal behaviors.Adolescents have the highest rates of e-cigarette use, and unlike adults, they do not typically begin using e-cigarettes to quit smoking but are instead motivated by curiosity or appealing e-cigarette flavors.Therefore, we assessed the association between the type of cigarette initially used and suicidal behaviors among Korean adolescents. Furthermore, we examined whether switching the cigarette type after smoking initiation and the timing of the change were associated with suicidal behaviors.27 All data used in this study are publicly available on the KYRBWS website.4 The KYRBWS complied with the Declaration of Helsinki,28 and all individuals who participated in the KYRBWS provided informed consent. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cross-sectional studies.29Data for this cross-sectional study were obtained from the Korea Youth Risk Behavior Web-Based Survey (KYRBWS), which was conducted annually from 2015 to 2018 by the Korea Centers for Disease Control and Prevention (KCDC). Ethics approval for the KYRBWS was waived by the KCDC’s institutional review board in accordance with the Bioethics and Safety Act of 2015.The KYRBWS was an anonymous, self-administered, structured questionnaire that had a complex research design, which included multistage sampling, stratification, and clustering. The KYRBWS used an online survey system that did not allow respondents to proceed to the next section of the questionnaire unless all questions were answered in the current section. Responses that had logical errors and responses that were outliers  were processed as missing values. The questionnaire contained approximately 120 items in 15 categories, including demographic characteristics and health-associated behaviors. Students in grades 7 to 12 were the target population. The question, “When did you start vaping e-cigarettes?” was included in the KYRBWS from 2015 to 2018. Therefore, data from only these years (comprising 255 887 adolescent participants) were analyzed; there were no missing values.The main dependent variables were suicidal behaviors, comprising suicidal ideation, suicide planning, and suicide attempts. Suicidal ideation was assessed on the KYRBWS through the question, “Have you seriously considered suicide in the past 12 months?” Suicide planning was assessed by the question, “Did you make any specific plans for suicide in the past 12 months?” Suicide attempts were assessed by the question, “Have you attempted suicide in the past 12 months?” Suicidal ideation, suicide planning, and suicide attempts were categorized based on whether respondents answered these questions with yes or No. Each of the 3 questions about suicidal behaviors was composed of 3 separate variables .The independent variable was the initial type of cigarette used, which was determined by yes or no answers to the questions, “Have you ever smoked even 1 puff in your lifetime? and “Have you ever used e-cigarettes?” Participants who answered no to both questions were classified into the group that had never smoked, and participants who answered yes to only 1 of the questions were classified into the group that started smoking using that type of cigarette . Those who answered yes to both questions were categorized according to the initial type of cigarette used and the school year  during which they started smoking either e-cigarettes or conventional cigarettes. The school year of smoking initiation was used to classify participants who initially used e-cigarettes into the group that started smoking with e-cigarettes, and participants who initially used conventional cigarettes or both conventional cigarettes and e-cigarettes during the same school year were classified into the group that started smoking with conventional cigarettes. This categorization allowed us to differentiate the association between initial use of e-cigarettes vs initial use of conventional cigarettes and suicidal behaviors.Based on these categories, the independent variable was divided into 3 groups: never smoked, started with e-cigarettes, and started with conventional cigarettes. For the secondary analyses, we divided the group of participants who had smoked at least 1 e-cigarette or 1 conventional cigarette into subgroups based on whether they had changed the cigarette type used. The KYRBWS did not assess whether participants smoked both cigarette types after changing their initial cigarette type; thus, all participants who reported smoking a cigarette type different than the type they initially smoked were included in the group that switched cigarette type. For the subgroup analyses, participants who changed cigarette type were divided according to their current smoking status and whether they changed cigarette type within 2 years of starting to smoke that cigarette type.30The covariates were school grade (7-12), self-reported economic status , living situation , self-reported academic achievement , alcohol use (ever or never), physical activity level , self-reported health status , and perceived stress level .8 Differences in the frequency and proportion of categorical variables were evaluated using χ2 tests. Multiple logistic regression analysis was performed to examine the association between initial cigarette type and suicidal behaviors, with adjustment for covariates in the primary analyses. Adjusted odds ratios (AORs) and 95% CIs were calculated. Secondary analyses were performed to investigate the association between changing cigarette type and suicidal behaviors, and subgroup analyses were conducted to evaluate the association between the timing of the change in cigarette type and suicidal behaviors. All statistical analyses were performed using SAS software, version 9.4 (SAS Institute), and a weighted logistic regression procedure was used to account for the complex and stratified sampling design. Bonferroni correction was applied to handle type 1 errors owing to multiple comparisons, with a 2-sided significance threshold of P < .02  for the primary analysis and P < .002 for the secondary and subgroup analyses .All analyses were conducted separately by sex to account for sex-specific differences in smoking rates and suicidal behaviors.The primary analysis included 255 887 adolescents . Among male participants, 3310 (2.5%) initially used e-cigarettes, 27 368 (20.9%) initially used conventional cigarettes, and 100 416 (76.6%) never smoked. Among female participants, 952 (0.8%) initially used e-cigarettes, 9296 (7.4%) initially used conventional cigarettes, and 114 545 (91.8%) never smoked. Among male participants, rates of suicidal ideation (500 [15.1%]), suicide planning (271 [8.2%]), and suicide attempts (178 [5.4%]) were higher among those who initially used e-cigarettes compared with those who initially used conventional cigarettes  and those who never smoked . Among female adolescents who initially used e-cigarettes, rates of suicidal behaviors were also higher  than rates among those who initially used conventional cigarettes  and those who never smoked  .P < .001), and among those who initiated smoking with conventional cigarettes, the AOR was 1.88 . Among girls who initiated smoking with e-cigarettes, the AOR was 4.04 , and among those who initiated smoking with conventional cigarettes, the AOR was 2.46 . Other factors associated with suicidal behaviors are shown in Participants who started smoking using either type of cigarette were more likely to attempt suicide than were those who never smoked. Among boys who initiated smoking with e-cigarettes, the AOR was 2.90 , suicide planning , and suicide attempts  compared with those who initially used conventional cigarettes. Among female adolescents, the initial use of e-cigarettes was significantly associated with suicide planning  and suicide attempts  compared with the initial use of conventional cigarettes  among female adolescents who initially used e-cigarettes and changed to conventional cigarettes. Adolescents of both sexes who initially used e-cigarettes and changed to conventional cigarettes had a higher risk of suicidal behavior than did those who initially used conventional cigarettes and changed to e-cigarettes. Among boys, the AOR for suicidal ideation was 1.88 ; for suicide planning, 2.29 ; and for suicide attempts, 1.89 . Among girls, the AOR for suicidal ideation was 1.84 ; for suicide planning, 2.22 ; and for suicide attempts, 2.36 . Statistical significance remained after Bonferroni correction.The demographic characteristics of participants included in the secondary analyses are presented in eTable 1 in the garettes . The higP < .001) and among girls  and with suicide planning among boys  and among girls . The association between initial cigarette type and suicidal behaviors stratified by current smoking status is presented in eTable 2 in the Two sensitivity analyses were conducted to confirm the robustness of the results. Similar results were found with the exception of adolescents who started smoking or changed cigarette type within 12 months of completing the survey. In the analysis conducted using age rather than school grade as a covariate, similar results were observed . Furthermore, the data were obtained from approximately 250 000 respondents of a national survey based on random cluster sampling,Of note, adolescents who initially used e-cigarettes had a higher risk of suicide planning and suicide attempts than did those who initially used conventional cigarettes. In the secondary and subgroup analyses, the group that initially used e-cigarettes and switched to using conventional cigarettes was the most likely to exhibit suicidal behaviors. Furthermore, switching from e-cigarettes to conventional cigarettes after 2 years had the strongest association with suicidal ideation and suicide planning compared with changing from smoking e-cigarettes to conventional cigarettes within 2 years. Among male adolescents who currently smoked, those who initially used e-cigarettes had a higher likelihood of suicidal ideation, suicide planning, and suicide attempts.32 However, to our knowledge, no studies have examined the association between the use of e-cigarettes before conventional cigarettes and mental health.Adolescents who use e-cigarettes before conventional cigarettes have been reported to have better school performance and higher intake of caffeinated drinks.35 Even nicotine-free e-cigarettes contain nicotine in their aerosols, and discrepancies between the labeled and actual amounts of nicotine have been found.37 Continuous exposure to nicotine may reduce serotonin and its metabolites, which can diminish the protection that serotonin provides from stress-induced processes that cause disease.40 In addition, prolonged nicotine administration can disrupt the brain's dopamine pathway, amplify sensitivity to stress, and distort mechanisms that alleviate depressive symptoms.41The present findings may be explained by several biological and behavioral mechanisms. E-cigarette aerosol contains highly oxidizing free-base nicotine, which is easily absorbed by the body and is the most addictive form of nicotine.42 nicotine withdrawal phenomenon or nicotine dependence can also occur in adolescents who initially use e-cigarettes. This withdrawal or dependence may lead adolescents to seek other nicotine-containing products, such as conventional cigarettes (the gateway theory26), and further increase their impulsivity.43 Furthermore, the different efficacy of nicotine vaporization and the perception that e-cigarettes are safer and healthier than conventional cigarettes may encourage adolescents to smoke e-cigarettes more frequently, resulting in excessive nicotine exposure.44 Substances in e-liquids may also alter the brains of adolescents and may be associated with suicidal behaviors. The flavor chemicals in e-liquids, which are not used in conventional cigarettes, are associated with increases in inflammatory cytokines, such as interleukin 1β and interleukin 6, which have important roles in the pathophysiology of suicidal behaviors.46 In addition, through the use of e-cigarettes, adolescents can absorb various trace metals, including arsenic, aluminum, and lead, which can affect the central and peripheral nervous systems and mental health.49 E-cigarette components, such as e-liquids, flavoring, and metallic coils, also provoke oxidative stress, which harms developing brains.50 Therefore, initial or current use of e-cigarettes may be associated with aggressive and impulsive behavior, impaired cognition, and increased suicidal ideation. Although it is difficult to acquire standardized toxicologic data regarding e-cigarettes,51 future studies are warranted to investigate the molecular composition of the toxicity profile of e-cigarettes.Given that the nicotine concentration in e-cigarettes is generally lower than that in conventional cigarettes,53 Moreover, e-cigarette users have an increased risk of other drug use and mental health problems, including attention-deficit/hyperactivity disorder, anxiety, low self-esteem, and high impulsivity.55 The toxicity of e-cigarettes, in conjunction with the aforementioned characteristics of adolescents who used e-cigarettes before conventional cigarettes, may have been associated with increases in suicidal behavior.The distinct characteristics of adolescents who initially use e-cigarettes may also be associated with suicidal behaviors. Adolescents who first smoked with e-cigarettes have lower levels of rebelliousness, and they may be sensitive to nicotine or nicotine withdrawal.Owing to the cross-sectional nature of this study, we could not establish causality or determine the direction of the association. However, we used all applicable measures to estimate the direction of the association, including adjustment for sociodemographic characteristics and established factors associated with adolescent suicidal behaviors, and conducted sensitivity analyses that excluded adolescents who started smoking or changed cigarette type within 12 months of the KYRBWS evaluation period for suicidal behaviors.Our findings suggest that caution should be taken regarding the risk of suicidal behaviors associated with the initial use of e-cigarettes and the subsequent change in cigarette type among adolescents; this is an important hypothesis to be confirmed in future longitudinal studies. If longitudinal clinical trials are able to determine causality, the findings may indicate that e-cigarettes are as unhealthy and unsafe as conventional cigarettes, at least with regard to suicidal behaviors among adolescents. The findings may also justify changes in public health policy and practice, such as the inclusion of questions about e-cigarette history when screening adolescents for suicide risk, the regulation of e-cigarette advertising, and the adoption of educational programs that address the adverse effects of e-cigarettes. Moreover, this study’s results suggest that both current and previous use of e-cigarettes is associated with suicidal behaviors among adolescents; therefore, e-cigarette use should not be overlooked.This study has limitations. We could not identify the direction of the associations or establish causality, although appropriate methods were used to mitigate these limitations. Furthermore, the KYRBWS data were collected anonymously online and self-reported, which may have produced nonrandom misclassification. We could not evaluate the concentration levels of nicotine or other e-liquid components or the dose-response relationship because the KYRBWS did not assess that information. The possibility of residual confounding also cannot be eliminated. In particular, although previous exposure to secondhand smoke may be associated with the initiation of smoking or suicidal behaviors, the KYRBWS did not include variables that could be used to fully assess the respondents’ history of exposure to secondhand smoke. The subgroup analyses showed significant associations even after adjusting for multiple comparisons through Bonferroni correction; however, some associations had wide 95% CIs owing to the small sample.In this cross-sectional study, adolescents who started smoking using e-cigarettes had a higher risk of suicidal behavior than did those who started smoking using conventional cigarettes. In addition, adolescents who initially used e-cigarettes and changed to conventional cigarettes were more likely to exhibit suicidal behaviors than were those who initially used conventional cigarettes and changed to e-cigarettes. These findings suggest that initial cigarette type is associated with suicidal behavior among adolescents and that consideration of initial cigarette type and subsequent change in cigarette type is warranted when conducting future research and formulating public policy.


A decrease in hippocampal neurogenesis is considered an important cause of cognitive impairment, while changes in mossy fiber sprouting are closely related to development of spontaneous recurrent seizures in chronic temporal lobe epilepsy (TLE). Racemic l-3-n-butylphthalide (DL-NBP) can alleviate cognitive impairment in ischemic stroke and Alzheimer’s disease by promoting neurogenesis. DL-NBP treatment can also improve cognitive function and reduce seizure incidence in chronic epileptic mice. However, the mechanisms of action of DL-NBP remain unclear. The aim of the present study was to examine the effects of DL-NBP on mossy fiber sprouting, hippocampal neurogenesis, spontaneous epileptic seizures, and cognitive functioning in the chronic phase of TLE.Nissl staining was used to evaluate hippocampal injury, while immunofluorescent staining was used to analyze hippocampal neurogenesis. The duration of spontaneous seizures was measured by electroencephalography. The Morris water maze was used to evaluate cognitive function. Timm staining was used to assess mossy fiber sprouting.TLE animals showed reduced proliferation of newborn neurons, cognitive dysfunction, and spontaneous seizures. Treatment with DL-NBP after TLE increased the proliferation and survival of newborn neurons in the dentate gyrus, reversed the neural loss in the hippocampus, alleviated cognitive impairments, and decreased mossy fiber sprouting and long-term spontaneous seizure activity.We provided pathophysiological and morphological evidence that DL-NBP might be a useful therapeutic for the treatment of TLE. Temporal lobe epilepsy (TLE) is the most common type of intractable epilepsy, the major characteristic of which is spontaneous recurrent seizures (SRS). Hippocampal injury and reduced neurogenesis caused by recurrent or uncontrolled seizures are related to cognitive dysfunction and can markedly affect the quality of life of patients –3. FurthL-3-n-butylphthalide (NBP) is the extraction of Apium graveolens Linn seeds (Chinese celery). One isomer of NBP, DL-NBP, has been synthesized and is currently in clinical trials for treatment of acute ischemic stroke. Recent studies have shown that DL-NBP improves cognitive function in traumatic brain injury, ischemic stroke, and Alzheimer’s disease, likely by reducing neural loss and promoting neuronal regeneration –8. HowevIn the present study, we examined effects of DL-NBP on mossy fiber sprouting, hippocampal neurogenesis, spontaneous epileptic seizures, and cognitive function in the chronic phase of TLE in rats.n = 20), epilepsy , and epilepsy plus DL-NBP treatment  groups. Each rat was housed separately during postoperative status epileptics, and then each group was fed in separate cages (five rats per cage) after status epileptics. All animals were housed in standard humidity and temperature conditions in a 12 h light/dark cycle, with ad libitum access to food and water. All experimental procedures were approved by the Animal Care and Use Committee of China Medical University. All animal experiments complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and were carried out in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People’s Republic of China.Adult male Wistar rats (220–250 g) were divided randomly into sham-operated  into the right lateral ventricle . Rats weSeizure severity was graded using five levels according to the Racine scoring system. Grade IV animals with more severe attack severity and animals with an attack duration >2 h were used in this study. After approximately 2 weeks of recovery, animals developed focal SRS accompanied by interictal spikes and waves (chronic phase). SHAM animals were injected with normal saline using the same procedures described above, with no deaths.th day post-surgery for 2 weeks.The purity of DL-NBP was >99.5% . DL-NBP was diluted to 7 mg/mL in vegetable oil and administered orally at a dose of 70 mg/kg/day from the 7Rats were subjected to the water maze test on days 31–33 after seizure initiation. As we reported, the match-to-place version was used to evaluate spatial learning . On the n = 5 per group). The electrodes were embedded within the CA3 region of the right hippocampus  and the regio nasalis using coordinates derived from the Paxinos and Watson atlas. After two days of recovery, the total duration of spontaneous EEG seizures over 2 h was recorded from freely moving animals. Digital acquisition software  was used to record the EEG signals, which were stored for off-line analysis.Electroencephalograph (EEG) was recorded from rats in the EP group and the EP+NBP group on days 34–36 after surgery. Intracranial EEG electrodes were placed into the right hippocampus  on a cryotome , when they were in the bottom. For each animal, five from every series of ten sections were used for immunofluorescent staining.https://imagej.nih.gov/ij).Brain sections were incubated with primary antibodies against guinea pig anti-doublecortin  at 4 °C overnight, followed by incubation in Alexa Fluor 488 goat anti-rabbit secondary antibodies  for 2 h. Images were collected by microscopy  using a ×20 or ×40 objective. The number of DCX-positive cells was counted using NIH ImageJ software (2) in both cerebral hemispheres was counted by microscopy with a high magnification objective.Brain tissues were fixed with paraffin and then cut into a series of coronal sections (10 μm thick). Brain sections were then incubated in toluidine blue solution for 20 min at 56 °C, quickly washed in distilled water, incubated in 95% alcohol for 5 min, dehydrated in increasing alcohol concentrations for 5 min, cleared for 5 min with xylene, and then mounted in neutral gum solution. The number of CA3 pyramidal neurons in the hippocampus (per 1 mmn = 5 per group) were deeply anesthetized and then perfused intracardially with sodium sulfide solution  and normal saline. The brains were then removed and post-fixed in paraformaldehyde solution. Coronal sections were stained with Timm staining solution for 90 min at room temperature. Images were taken using an Olympus BX51 microscope.Animals in the three groups . Water maze data were analyzed by repeated-measures or one-way analysis of variance. The remaining data were analyzed using the Kruskal–Wallis or Mann–Whitney U non-parametric tests. All data are presented as mean ± standard error of the mean. Significance was set at P < 0.05). DL-NBP treatment was associated with a significant increase in the number of DCX-positive newborn neurons compared with the EP group . These data indicate that DL-NBP treatment increased the number of newborn neurons in the DG following KA-induced epilepsy.Immunofluorescent staining of DCX-positive neuroblasts was performed to examine whether DL-NBP treatment could regulate neurogenesis in the dentate gyrus (DG) Fig. . At the P < 0.01). DL-NBP treatment was associated with a significant increase in the number of Nissl-positive neurons in the CA3 compared with the EP group . These data suggest that DL-NBP treatment attenuated neuronal loss caused by KA-induced epilepsy.Next, we performed Nissl staining to evaluate hippocampal neuronal damage Fig. . There wP < 0.01) and duration  of spontaneous EEG seizures following KA-induced epilepsy.Spontaneous EEG seizures were assessed on day 36 after surgery , which was significantly reduced following DL-NBP treatment .Mossy fiber sprouting in the hippocampus was evaluated using Timm staining Fig. . CompareP = 0.041), while DL-NBP treatment was associated with a significant decrease in the escape latency compared with the EP group . There were no differences in swimming speed, the total swimming path, and the total swimming time between the different groups. In the probe test, animals in the EP group showed a significant reduction in the number of crossings compared with the SHAM group . Treatment with DL-NBP was associated with a significant increase in the number of crossings compared with the EP group .We performed the Morris water maze test to evaluate changes in cognitive function Fig. . CompareIn the present study, adult rats were randomly assigned to control rats, rats with epilepsy, or rats with epilepsy treated by DL-NBP for 30 days. The key findings of the study were that, in the chronic phase of seizures in TLE rats, DL-NBP treatment could reverse neural loss and reduce mossy fiber sprouting in the hippocampus and increase neurogenesis in the DG. Furthermore, DL-NBP treatment was associated with an improvement in spatial learning cognitive function and a decrease in long-term seizure activity.Chronic epilepsy can cause changes in the hippocampal microenvironment , which cDL-NBP is a multiple growth factor activator that shows neuroprotective effects in ischemia-reperfusion injury  and chroWith respect to chronic epilepsy, DL-NBP was found to increase levels of brain-derived neurotrophic factor, neuroprotective factor, and Klotho level . HoweverEpilepsy is the result of an imbalance between excitatory and inhibitory neuronal activity. Recurrent seizures are a major cause of cognitive impairment . InteresWe found that DL-NBP treatment can improve cognitive function in adult rats after TLE, as well as inhibit long-term seizure activity, reverse neural loss, increase neurogenesis, and reduce mossy fiber sprouting. Thus, DL-NBP may be a useful therapeutic agent for TLE. Nevertheless, direct evidence and specific mechanism are needed to provide in future research, that is, enhanced neurogenesis and decreased mossy fibers are the reason for the antiepileptic effect of DL-NBP in TLE.


Background: Disruptions in central autonomic processes in people with epilepsy have been studied through evaluation of heart rate variability (HRV). Decreased HRV appears in epilepsy compared to healthy controls, suggesting a shift in autonomic balance toward sympathetic dominance; recent studies have associated HRV changes with seizure severity and outcome of interventions. However, the processes underlying these autonomic changes remain unclear. We examined the nature of these changes by assessing alterations in whole-brain functional connectivity, and relating those alterations to HRV.Methods: We examined regional brain activity and functional organization in 28 drug-resistant epilepsy patients and 16 healthy controls using resting-state functional magnetic resonance imaging (fMRI). We employed an HRV state-dependent functional connectivity (FC) framework with low and high HRV states derived from the following four cardiac-related variables: 1. RR interval, 2. root mean square of successive differences (RMSSD), 4. low-frequency HRV  and high-frequency HRV . The effect of group (epilepsy vs. controls), HRV state (low vs. high) and the interactions of group and state were assessed using a mixed analysis of variance (ANOVA). We assessed FC within and between 7 large-scale functional networks consisting of cortical regions and 4 subcortical networks, the amygdala, hippocampus, basal ganglia and thalamus networks.Results: Consistent with previous studies, decreased RR interval (increased heart rate) and decreased HF-HRV appeared in people with epilepsy compared to healthy controls. For both groups, fluctuations in heart rate were positively correlated with BOLD activity in bilateral thalamus and regions of the cerebellum, and negatively correlated with BOLD activity in the insula, putamen, superior temporal gyrus and inferior frontal gyrus. Connectivity strength in patients between right thalamus and ventral attention network (mainly insula) increased in the high LF-HRV state compared to low LF-HRV; the opposite trend appeared in healthy controls. A similar pattern emerged for connectivity between the thalamus and basal ganglia.Conclusion: The findings suggest that resting connectivity patterns between the thalamus and other structures underlying HRV expression are modified in people with drug-resistant epilepsy compared to healthy controls. Heart rate varies on a moment-to-moment basis in response to changing physiological demands, and is regulated by sympathetic and parasympathetic components of the autonomic nervous system (ANS). Evaluation of the momentary changes in heart rate variability (HRV) can provide insights into the interplay of central mechanisms controlling sympathetic and parasympathetic  activity , 2. A shEpilepsy is accompanied by significantly different patterns of HRV . A lowerFunctional magnetic resonance imaging (fMRI), a non-invasive tool for probing brain activity and functional connectivity (FC), has been used to study the neural substrates of autonomic regulation –20. InitFunctional connectivity measures between brain sites are altered in people with epilepsy –36; howeThirty-two (32) patients with drug-resistant epilepsy were selected from an ongoing investigation into the localization of epileptic activity in the brain using simultaneous EEG-fMRI with ECG , with a 2, slice thickness = 2.4 mm with 0.6 mm gap, 44 slices, and voxel size = 3.75 × 3.75 × 3 mm3. Subjects were instructed to keep their eyes closed, avoid falling asleep, and not think about anything in particular. A T1-weighted image was also acquired using an FSPGR  sequence, with the following parameters: matrix size = 256 × 256, FOV = 24 × 24 cm2, slice thickness = 1.5 mm, 150 slices, and voxel size = 0.94 × 0.94 × 1.5 mm3.Scanning was performed at the Epilepsy Society  on a 3.0 Tesla GE (Signa excite HDX) scanner. A 20-min (400 vol) Scalp EEG signals and an ECG signal were simultaneously acquired during fMRI scanning at 5 kHz using a 64 channel MR-compatible EEG system with ring Ag/AgCl electrodes . The electrodes were placed according to the 10/20 system and referenced to electrode FCz.http://www.fil.ion.ucl.ac.uk/spm)   in a MatTo account for anatomical variability across participants and reduce thermal noise, all individual functional volumes were smoothed using a 5 mm full-width half-maximum (FWHM) Gaussian kernel. Subsequently, the Brainnetome atlas was used to extract mean fMRI time-series from 210 cortical and 36 subcortical parcels . The parWe used the framewise displacement (FD) as defined in Power et al.  to identFinally, to further mitigate the effects of motion as well as reduce the effects of physiological processes and scanner artifacts, we regressed out the following nuisance regressors from all parcel time-series: the first ten principal components from voxel time-series within the white matter , six regThe ECG was corrected for gradient artifacts using adaptive template subtraction  implemenThe RR intervals were used to obtain time-series of the root mean square of successive differences in RR intervals (RMSSD), and the normalized low (0.04–0.15 Hz) and high (0.15–0.40 Hz) frequency components of HRV. The aforementioned three HRV measures were computed in adjacent time windows of 100 s each, and a timestep of 1 s, to probe changes in sympathetic and parasympathetic activity during the 20-min resting-state scan. RMSSD is a time-domain HRV measure that is believed to reflect parasympathetic activity , the lowt-test on the associated beta parameters derived from the general linear model. Differences in the activation maps between epilepsy patients and healthy controls were assessed with a second-level, mixed-effects analysis with subjects as the random-effects factor, using a two-sample t-test on the associated beta parameters. To control for potential effects, sex, age and levels of head motion  were treated as covariates. Moreover, to account for false positives, the statistical maps were thresholded with a voxel-wise threshold of family-wise error (FWE) rate p < 0.05 corrected for multiple comparisons using the random-field theory , moving-average RR interval and instantaneous heart rate were convolved with the canonical hemodynamic response function (HRF) from SPM12  prior tod theory  and an ehttps://atlas.brainnetome.org/), and the subcortical parcels were grouped into the following four networks: amygdala (four parcels), hippocampus (four parcels), basal ganglia (12 parcels) and thalamus (16 parcels) . To better understand how FC depends on the state of autonomic activity, we estimated FC in each individual considering low or high HRV states separately. The low and high HRV states were defined as the timepoints in a scan at which an HRV measure  had values in the lowest and highest quartile range for that given scan, respectively.To investigate the effects of HRV state on brain functional organization, a whole-brain state-dependent FC analysis was performed on the parcellation of the Brainnetome atlas that includes 246 parcels covering the neocortex and sub-cortical regions . The conp < 0.0125  adjusted for multiple comparison with respect to pairs of networks using false discovery rate (FDR).A mixed analysis of variance (ANOVA) was conducted with the group  as a between-subject factor and HRV state (low/high) as a within-subject factor, which allowed us to examine the effect of the group and HRV state on FC within and between networks as well as their interactions. Potential effects of sex, age and levels of head motion on FC were regressed out through linear regression at the group level before conducting the mixed ANOVA. The levels of head motion for the low and high HRV state were determined separately considering only the timepoints corresponding to each state. The connectivity strength between pairs of networks that was used in the mixed ANOVA was defined as the mean correlation averaged across all pairs of parcels that belonged to the two corresponding networks. The HRV state-dependent FC analysis was performed for the three HRV measures and the moving-average RR interval. Statistical significance was set at n = 28, mean age of 28.7, 14 women) and healthy controls   there were no significant differences in the number of volumes between healthy controls and epilepsy patients (p > 0.10), and the two groups exhibited similar levels of motion during the fMRI scan . The sex and age distributions were similar between the epilepsy patients  after regressing out potential effects of sex and age. The mean RR interval during the 20-min scan was significantly lower in patients compared to healthy controls  (p < 0.05) whereas RMSSD and LF-HRV were similar in the two groups. The LF-HRV (0.04–0.15 Hz) and HF-HRV (0.15–0.40 Hz) measures demonstrated a strong negative inter-correlation  . HF-HRV p < 0.05; FWE-corrected) with only one of the cardiac dynamic metrics, namely instantaneous heart rate, in the following regions: positive correlations in the thalamus and several regions of the cerebellum ; negative correlations in the bilateral inferior frontal gyrus, orbitofrontal cortex, middle temporal gyrus, precentral gyrus and claustrum, as well as right insula and putamen  and the connectivity between the left posterior parietal thalamus and left ventromedial putamen . For both connections of the thalamus, healthy controls exhibited a lower  connectivity strength in the high LF-HRV state compared to the low LF-HRV state, whereas epilepsy patients exhibited the opposite trend . Therefore, it is unclear whether the stronger interactions observed between the right caudal temporal thalamus and left dorsal granular insula compared to ipsilateral interactions have some biological significance. Similar altered interactions emerged for changes in LF-HRV levels and connectivity between the thalamus and basal ganglia, with more pronounced effects for the connectivity of left posterior parietal thalamus and ventromedial putamen.We examined the association of autonomic cardiac regulation with spontaneous fluctuations in fMRI and whole-brain FC in people with drug-resistant epilepsy, compared to healthy controls. In both groups, heart rate was positively correlated with fMRI signal intensity in bilateral thalamus and regions of the cerebellum, and negatively correlated with lateral regions, including bilateral inferior frontal gyrus, orbitofrontal cortex, middle temporal gyrus and right insula and putamen . In addiThese findings support the role of thalamus, insula and putamen in autonomic control, as shown in previous studies , 30, 53,In healthy controls, a seed-to-voxel connectivity analysis revealed that thalamic activity was anticorrelated with core regions of the ventral attention network such as the insula, anterior cingulate cortex (ACC) and supramarginal gyrus, and this anti-correlation was enhanced during elevated levels of HRV . HoweverThe thalamus consists of a series of nuclei which are responsible, among others, for the relay of information from cardiovascular receptors to the insular cortex . In turnA growing body of evidence from functional and structural studies suggests thalamic dysfunction in epilepsy which may underlie the abnormal connectivity of the thalamus with the ventral attention network and the basal ganglia observed in our study , 5. AlleRR intervals and, to a less extent, the high frequency component of HRV , were on average lower in patients compared to controls  which isThis study has limitations that should be considered. While cardiovascular mechanisms are likely impaired in epilepsy and contribute to SUDEP , breathiSeveral studies describe an inverse relationship between heart rate and HRV measures –76. Howe2*) contrast mechanism used in fMRI is a measure sensitive to changes in blood oxygenation induced by local neuronal activity  impedes investigations in relation to sleep effects. The potential for more exaggerated changes in FC during sleep mandates further studies on this issue.HRV impairment in epilepsy is more pronounced during nocturnal periods , 15 and This study represents an exploratory, data-driven approach to investigate whether large-scale networks are involved in cardiac regulation, and is hampered by potential sleep, breathing, and circadian interactions that could interfere with understanding of important brain interactions. A hypothesis-driven analysis, controlling for these interactions may elucidate more precisely the key disruptions in autonomic processes found in epilepsy.In healthy controls and people with drug-resistant epilepsy, fluctuations in heart rate covaried with brain activity in key regions of the central autonomic network and in regions associated with cardiac regulation. Functional connectivity of the thalamus with the basal ganglia, a major autonomic regulatory site, and the ventral attention network was strongly linked to levels of LF-HRV, and that relationship differed between healthy controls and epilepsy patients. These findings support a significant role for thalamic contributions to cardiovascular impairments in epilepsy which may lead to cardiac rhythm and blood pressure failings implicated in SUDEP. Because activity in regional thalamic structures can be so readily modified by somatosensory peripheral stimulation, we speculate that the findings suggest a means to interfere with the deficient functional connectivity patterns in epilepsy.louis.lemieux@ucl.ac.uk.The datasets presented in this article are not readily available because there is not written informed consent from the patients/participants to share their data with external investigators. Requests to access the datasets should be directed to LL, The studies involving human participants were reviewed and approved by the National Research Ethics Committee . The patients/participants provided their written informed consent to participate in this study.MK preprocessed and analyzed data, interpreted the findings, and drafted the initial manuscript. RH interpreted the findings, revised the manuscript, and refined the clinical and physiological interpretation of findings. MG advised on methodological aspects of the study and revised the manuscript. LL and BD designed and conceptualized study, interpreted the findings, and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chronic myeloid leukemia (CML), a hematopoietic neoplasm arising from the fusion of BCR (breakpoint cluster region) gene on chromosome 22 to the ABL (Abelson leukemia virus) gene on chromosome 9 (BCR-ABL1 oncogene), originates from a small population of leukemic stem cells with extensive capacity for self-renewal and an inflammatory microenvironment. Currently, CML treatment is based on tyrosine kinase inhibitors (TKIs). However, allogeneic hematopoietic stem cell transplantation  is currently the only effective treatment of CML. The difficulty of finding a compatible donor and high rates of morbidity and mortality limit transplantation treatment. Despite the safety and efficacy of TKIs, patients can develop resistance. Thus, microRNAs (miRNAs) play a prominent role as biomarkers and post-transcriptional regulators of gene expression. The aim of this study was to analyze the miRNA profile in CML patients who achieved cytogenetic remission after treatment with both HSCT-allo and TKI. Expression analyses of the 758 miRNAs were performed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Bioinformatics tools were used for data analysis. We detected miRNA profiles using their possible target genes and target pathways. MiR-125a-3p stood out among the downregulated miRNAs, showing an interaction network with 52 target genes. MiR-320b was the only upregulated miRNA, with an interaction network of 26 genes. The results are expected to aid future studies of miRNAs, residual leukemic cells, and prognosis in CML. Chronic myeloid leukemia (CML) was the first leukemia described in the literature, by Rudolf Virchow and John Hughes Bennett in 1845 . FolloweCML is a clonal myeloproliferative neoplasm of hematopoietic stem cells with an incidence in adults of 1–2 cases per 100,000 inhabitants . Three mThe identification of BCR-ABL1 tyrosine kinase as a therapeutic target has made possible the development of specific target drugs, administered orally, that molecularly control the tumor burden in CML. Imatinib mesylate (IM) was the first tyrosine kinase inhibitor (TKI) that was developed and approved for therapeutic use. IM acts as a competitive inhibitor of the ATP site on the BCR-ABL1 protein, blocking its tyrosine kinase activity and preventing the substrate from being phosphorylated. This block obstructs the transduction of signals necessary for cell proliferation . CurrentHSCT-allo from a healthy allogeneic donor has been shown to consistently eradicate leukemic stem cells in most patients. The successful outcome of this treatment is related to the effectiveness of the leukemia graft reaction (LGR) orchestrated by allogeneic T cells .In the last two decades, the advent of TKIs has changed the indication of HSCT-allo from an early and curative intervention to a salvage treatment, recommended for patients resistant to ITKs or in the advanced stages of the disease ,8.Given the evidence of therapeutic resistance to ITKs, recent advances in gene expression technology mechanisms have shown that microRNAs (miRNAs) have great potential both as therapeutic targets and as prognostic markers in different types of cancer ,10,11.MiRNAs are small non-protein coding RNAs that act as post-transcriptional regulators of gene expression . RecentlFrom the first descriptions, in 2002, of the relationship between miRNA and cancer, it was observed that cancer patients exhibit dysregulated expression levels of miRNAs, both positively and negatively . ConsequThe aim of this study was to identify a miRNA profile, target genes, and target pathways in CML-chronic phase patient groups treated with HCST-allo and imatinib mesylate, both in cytogenetic remission and over 12 months of treatment. Expression analyses of the 758 miRNAs were performed using RT-qPCR. Bioinformatics tools were used for data analysis. We detected miRNA profiles using their possible target genes and target pathways. MiR-125a-3p stood out among the downregulated miRNAs, showing an interaction network with 52 target genes. MiR-320b was the only upregulated miRNA, with an interaction network of 26 genes.Expression data for the miRNAs obtained in this study are available from the NCBI GEO database (accession number GSE 164549). A total of 758 miRNAs were analyzed using RT-qPCR array in peripheral blood samples from 14 patients with CML-chronic phase treated with imatinib mesylate and 14 patients treated with HSCT-allo, both groups in cytogenetic remission. According to the cut-off criteria , 43 differentially expressed miRNAs were identified from this microarray dataset. Of these miRNAs, one (2.3%) was upregulated and 42 (97.7%) were downregulated .http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/index.html, accessed on 27 August 2021) to identify possible miRNA target genes. Through this analysis we identified 1171 genes. These genes were verified and identified as differentially expressed using microarray data available in the Gene Expression Omnibus , accession number GSE 43225, in a study conducted on CML patients. Among the genes we found in our study, 95% were also identified among the genes in the microarray data from the Gene Expression Omnibus.The 43 differentially expressed miRNAs were analyzed using the miRWalk program .MiRNA expression in all patients treated with imatinib mesylate and HSCT-allo using heatmap is shown in http://amp.pharm.mssm.edu/Enrichr/, accessed on 24 September 2020) was used to visualize the main pathways related to the target genes of the miRNAs. The Reactome program provided a biological interpretation and provided models to visualize the results. STRING , https://string-db.org/, accessed on 27 September 2020) was performed on the target genes of upregulated and downregulated miRNAs, generating data visualized through pictures created by Cytoscape software , http://www.cytoscape.org, accessed on 28 September 2020)  er 2020) .http://geneontology.org/, accessed on 24 September 2020) to identify the pathways involved with these genes. The main terms found are listed in Based on the network results, the genes that showed the greatest interaction with each other were selected, according to the figures generated by Cytoscape. For these genes, which were considered as the main genes, an analysis was performed using Gene Ontology  is a hematopoietic neoplasm that arises from BCR-ABL1 translocation, originating from a small population of leukemic stem cells with extensive self-renewal capacity and an inflammatory microenvironment. Currently, CML treatment is based on tyrosine kinase inhibitors and target-specific drugs that are safe and effective. However, HSCT-allo was the first treatment in CML patients to eradicate the Philadelphia chromosome. In this process, after high doses of chemotherapy, transplanted hematopoietic stem cells can reestablish the recipient’s hematopoiesis .Currently, HSCT-allo is the only treatment available that promotes a CML cure; however, morbidity and mortality rates related to this treatment are high, in addition to the difficulty of finding a donor, restricting its applicability ,25,26,27The first ITK approved for use was imatinib mesylate ,30, a poIn recent years, many studies have focused on the significant role of miRNAs in physiological and pathological processes . These sIn addition to miRNA-based studies, bioinformatics has also contributed to the understanding of processes linked to the development and progression of CML, along with the discovery of possible disease markers. Recent studies have shown increasing evidence of the interaction between genes and proteins, leading to investigations into molecular cancer mechanisms. In this context, bioinformatics is essential for integrating biological systems and computational science, helping to better understand the diagnosis processes, therapy, and prognosis of cancer and other diseases .4log, MR4.5Log or undetectable) throughout the treatment and another five patients (35.7%) achieved a major molecular response (MR3Log). On the other hand, nine patients (64.3%) treated with HSCT-allo relapsed and five (35.7%) achieved major molecular response (MR3Log). By analyzing the miRNAs of these two patient groups using bioinformatics tools, it was possible to obtain a profile consisting of 43 differentially expressed miRNAs, one (2.3%) upregulated and 42 (97.7%) downregulated  patients treated with ITK achieved a deep molecular response  . CRK proOf the 43 differentially expressed miRNAs identified in our study, 42 (97.7%) were downregulated. Among the 42 downregulated miRNAs, we will highlight miR-125a-3p, miR-485-3p, miR-409-3p, and miR-574-3p, due to their relevance in the literature. This set of miRNAs was described by Xiong and collaborators as differentially expressed in the K562 cell line, BCR-ABL1 positive .Low expression of miR-125a has been detected in bone marrow samples from patients with acute myeloid leukemia (AML) compared to the control group  . PreviouMiR-485 has been described as poorly expressed in AML HL60 cell lines when compared to normal peripheral blood cells .MiR-409-3p is also a miRNA described in the literature as having reduced expression in CpG-rich methylated CML cell lines ,51. ThisThese findings are expected to contribute to the understanding of disease pathogenesis and persistence of residual BCR-ABL1 cells observed in our patient groups after different therapeutic strategies.After analyzing the target genes, we used Gene Ontology to study the enrichment of the pathways of these genes and to observe the main pathways related to these genes. Some pathways were found to be fundamental to CML development, such as regulation of protein kinase activity, processes related to cell division, and mitogen-activated-protein kinase (MAPK) activity.The MAPK pathway is an important signaling cascade found in several types of cancer  and playIn the present study, we explored through bioinformatics tools the profile of miRNAs, their target genes, and related pathways in CML patients undergoing treatment. Protein–protein interaction networks were built to relate miRNAs found with their target genes, and it was possible to obtain data that corroborate the current literature. As this is an exploratory study with real-world data and scarce samples, it was not possible to validate the results through a second round of RT-qPCR. However, we believe that this study contributes an innovative approach and useful results to the fields of oncology and bioinformatics. We stress the need for expanded studies in order to confirm the miRNA profiles discussed in this work as markers of residual disease.The study was approved by the Hospital Amaral Carvalho Ethics Committee (CEPHAC-2.917.389). Patients who were followed up at Hospital Amaral Carvalho signed a form attesting to free and informed consent (the FICF form) to participate in the study. Peripheral blood samples from 28 patients with CML-chronic phase in cytogenetic remission (Philadelphia chromosome-negative) were included in the study. The patients were divided into two groups: 14 patients undergoing HSCT-allo and 14 patients treated with imatinib mesylate. A pool of leucocytes from 14 healthy blood donors was considered as the control group. The miRNA profile was determined by comparing the control group and each patients’ group to determine which miRNAs were upregulated or downregulated. The patients’ clinical data are presented in ® RNA Blood Mini Kit  according to the manufacturer’s protocol. The total RNA amount was determined by the ratios A 260 nm/A 280 nm and A 260 nm/A 230 nm (acceptable when both ratios were greater than 1.8). RNA integrity was ensured by obtaining an RNA integrity number (RIN > 8) using the Agilent 2100 Bioanalyzer  [Total RNA was isolated from buffy coat of fresh peripheral blood using the QIAampGermany) ,66.The reverse transcription reaction was performed using the Taqman MicroRNA Reverse Transcription Kit in combination with Megaplex RT Primer Human Pool Set A and B . This reaction transcribed 758 miRNAs and three positive endogenous controls  and one negative control ,66.® MGB strategy was used for quantitative real-time PCR (q-PCR), which is based on the specific annealing of each probe with its complementary sequence. For miRNA amplification, the Taqman® Low Density Array Human MicroRNA Arrays v2.0 (A and B) kit  was used according to the manufacturer´s instructions. Analyses were performed on the ViiA7 platform (ABIV®) for a total of 758 miRNAs per patient [The TaqMan patient ,66.http://www.ncbi.nlm.nih.gov/geo/geo2r/, 23 September 2020). Differentially expressed genes were considered when they showed a fold change of at least 1.5. Gene Ontology was used to search for enriched terms among differentially expressed genes using Bonferroni´s correction and accepting only terms with p ≤ 0.05. Differentially expressed genes related to upregulated and downregulated miRNAs were analyzed according to EnrichR for enrichment analysis. The Reactome was used for data analysis, and the evaluation of protein–protein interaction network (PPI), based on a list of genes, was performed using the online tool STRING version 11.0. Experiments, databases, co-expression, neighborhoods, and co-occurrence were considered. The minimum interaction score was 0.700. Cytoscape software version 3.8.0 was used to view the results.Bioinformatic analysis programs were selected based on the options described by Reimand et al. . Expresst-test. Statistical significance was set at p < 0.05.A comparison analysis of Ct was used to quantify miRNA expression. Differences were statistically assessed using Student’s It was possible to determine the miRNA profiles of patients treated with HSCT-allo and imatinib mesylate, as well as their target genes and pathways. The results of our study are expected to contribute to further research in the field of CML prognosis, in particular, the identifying of new biomarkers for this disease. Bioinformatics analyzes predict, in silico, interactions that require biological validation before clinical application.


In vitro studies evaluating hepatic biliary transporter inhibition, mitochondrial function, and metabolic profiling were performed in hepatocytes from different species. The long term toxicity of CPL207280 was studied in vivo in rats and monkeys. Activity of CPL207280 was one order of magnitude lesser than that of TAK-875 for the inhibition of bile acid transporters. CPL207280 had a negligible effect on the hepatic mitochondria. In contrast to TAK-875, which was metabolized through toxic glucuronidation, CPL207280 was metabolized mainly through oxidation. No deleterious hepatic effects were observed in chronically treated healthy and diabetic animals. The study presents promising data on the feasibility of creating a liver-safe GPR40 agonist. Additionally, it can be concluded that DILI is not a hallmark of GPR40 agonists; it is linked to the intrinsic properties of an individual agonist.GPR40 (FFAR1) is a promising target for the managing type 2 diabetes (T2D). The most advanced GPR40 agonist TAK-875 exhibited satisfactory glucose-lowering effects in phase II and III studies. However, the phase III studies of TAK-875 revealed drug-induced liver injury (DILI). It is unknown whether DILI is a consequence of a specific GPR40 agonist or is an inherent feature of all GPR40 agonists. CPL207280 is a novel GPR40 agonist that improves diabetes in Zucker Diabetic Fatty (ZDF) rats, Goto Kakizaki (GK) rats and db/db mice. In this report, the DILI-related toxicity of CPL207280 was compared directly with that of TAK-875.  This effect was absent for CPL207280 over the entire range of concentrations  cell cultures, which allow for better recapitulation of the complex in an in vivo microenvironment than traditional 2D monolayer models [Next, mitochondrial defects caused by TAK-875 and CPL207280 in a 3D spheroid system were comprehensively analyzed. Spheroids composed of HepaRGr models . They al1/2 and slow clearance rate in humans, which resulted in its long exposure (approximately 30 h) in humans [GRP40 agonists are considered compounds that are quickly metabolized, which can be problematic for development of drugs with desired, sustainable effect. TAK-875 was designed to ensure a relatively long Tn humans . To compn humans .in vivo comparison of the toxicity of CPL207280 and TAK-875 was enabled only in rats.Based on the values obtained from different species, TAK-875 was classified as a stable compound. CPL207280 proved to be an unstable compound and was metabolized at a high rate by human and monkey microsomes. Microsomes offer only limited insight into metabolism  because they possess only a fraction of enzymes. To obtain a comprehensive insight into the metabolic rate, the next study was performed in hepatocytes, equipped with all enzymes involved in liver metabolism. Again, CPL207280 showed high clearance rates for humans and was similar for monkeys . These r1/2 of the compound for each species. Hence, the maximal incubation time was 120 min for rats, mice, and dogs, and 60 min for humans and monkeys. In all species, 23 metabolites were identified, of which 12 were produced by human hepatocytes (in vivo.A number of carboxylic acid-containing drugs have been withdrawn from the market due to adverse effects stemming from the formation and reactivity of acyl glucuronide metabolites (for review ). It is atocytes . Monkey atocytes . Glucuro+-taurocholate co-transporting polypeptide (NTCP), organic anion transporting polypeptides (OATPs), liver export transporters, bile salt export pump (BSEP), and multidrug resistance-associated proteins (MRPs). In particular, inhibition of the BSEP transporter has been well characterized in pathological states and pharmacology. Blockade of BSEP results in retention of bile acids, which culminates in cholestasis and contributes to DILI [in vitro  are produced by the liver from cholesterol and are transported to the bile and further to the upper intestines to facilitate digestion of foods. Next, they move along with the digested nutrients down the gut and are reabsorbed in the ileum, wherein they enter the blood and return to the liver. This process is called enterohepatic circulation and was found to be impaired in the livers of rats and dogs treated with TAK-875 . The cau to DILI , 34. The to DILI . We studin vitro . As expensporter . Inhibitin vivo, we first tested whether counterpart doses ensured similar drug exposure in plasma. To address this question, a pharmacokinetic study comparing both molecules in animal models was performed. Rats were administered 3 mg/kg of compounds p.o. or the same dose i.v. CPL207280 demonstrated greater bioavailability (63%) than TAK-875 (38%)  .d) of 20%, which is indicative of greater organ penetration. CPL207280 was quickly absorbed, reaching Cmax in approximately 1 h and was quickly eliminated from circulation, which was reflected by 2x greater Kel. TAK-875 reached Cmax after 2 h  to CPL207280 than to TAK-875  caused liver injury after a shorter time (13-weeks), while liver injury was observed for lower doses (80 mg/kg) after a longer exposure of 39 weeks (3 times longer) . To furtAs monkeys were the primary species whose hepatocytes showed a spectrum of CPL207280 metabolites similar to that observed for human hepatocytes, we decided to perform a repeat-dose study in a cynomolgus monkey. The doses were chosen based on the body surface area and amounted to 50, 100, and 200 mg/kg in the 14-day study and to 20, 150, and 250 mg/kg in the 56-day study . No alteT2D impairs liver metabolism. Hence, drugs that do not cause DILI in metabolically healthy subjects may still pose a risk of DILI in T2D individuals , 39. To In this study, the liver safety of CPL207280, a novel GPR40 agonist developed for T2D treatment, was evaluated with a focus on addressing all toxicity-related concerns accounting for overall hepatotoxicity identified for the compound predecessor TAK-875 . Interesin vivo studies missed an equal drug-exposures to enable fair toxicity comparison [in vivo liver toxicity comparison [in vivo comparison of both molecules to studies in rats only. The comparison in rats, however, was indirect and performed retrospectively via literature [In the current study, TAK-875 was used as a reference for CPL207280 to evaluate its safety properties: effect on viability of hepatocytes, inhibition of efflux bile acid transporters, production of reactive glucuronide metabolites, retention/deposition in the liver, and effect on liver safety markers of model animals. Recently, the liver safety of two GPR40 agonists—AMG-873 and TUG-770—was studied in mice and compared with TAK-875 . It was mparison , 44. Drumparison . Howeverterature .in vitro experiments. Notably, starting with the study of the direct effect of test compounds on hepatocytes (HepG2 and human primary cells), we found that CPL207280 is 10 times better tolerated by hepatocytes than TAK-875. In the viability study, TAK-875 showed an IC50 of approximately 50 μM, which was in line with the values reported in initial studies detailing TAK-875 toxicity  than CPL207280  [The main objective for the GPR40 agonist designers has been to devise compounds structurally different to FFAs and much more potent than FFAs , 55. Thif GPR40) , 59, butS1 File(XLSX)Click here for additional data file.