Intermittent theta burst stimulation (iTBS) delivered to the dorsolateral prefrontal cortex (DLPFC) has been investigated as a promising treatment for stress and stress‐related mental disorders such as major depression, yet large individual differences in responsiveness demand further exploration and optimization of its effectiveness. Clinical research suggests that resting‐state functional connectivity (rsFC) between the DLPFC and the anterior cingulate cortex (ACC) can predict iTBS treatment response in depression. The present study aimed to investigate whether rsFC between the left DLPFC and ACC subregions could predict the degree to which the stress system is affected by iTBS. After assessment of baseline resting‐state fMRI data, 34 healthy female participants performed the Trier Social Stress Test on two separate days, each followed by active or sham iTBS over the left DLPFC. To evaluate iTBS effects on the stress‐system, salivary cortisol was measured throughout the procedure. Our results showed that a stronger negative correlation between the left DLPFC and the caudal ACC was linked to a larger attenuation of stress‐system sensitivity during active, but not during sham iTBS. In conclusion, based on individual rsFC between left DLPFC and caudal ACC, iTBS could be optimized to more effectively attenuate deregulation of the stress system. The present study aimed to investigate whether resting‐state functional connectivity (rsFC) between the left dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC) subregions could predict the degree to which the stress system is affected by intermittent theta burst stimulation (iTBS). Stronger negative correlation between the left DLPFC and the caudal ACC was linked to a larger attenuation of stress‐system sensitivity during active, but not during sham iTBS. The burFunctional connections between brain regions may prove to be important to understand TMS effects and add to heterogeneity in responsiveness. Functional connectivity between different brain areas can be quantified using resting‐state fMRI. Correlations between spontaneous fluctuations of brain activation signify a functionally connected brain network Friston, . Since rThe most prevalent stress‐related mental health problem is major depressive disorder (MDD)  and the subregions of the ACC could be predictive for rTMS efficacy in (acute) stress regulation in healthy individuals. As such, we aimed to identify a potential biomarker for rTMS‐induced stress regulation based on individual differences in neural patterns. To investigate this, we used intermittent theta burst stimulation (iTBS), an rTMS protocol that mimics endogenous theta rhythms, demonstrating similar or more potent excitatory effects than conventional high frequency stimulation  were recruited through Ghent University student fora as well as social media, based on the following inclusion criteria: (a) no current/history of psychiatric disorders, as evaluated by the Mini‐International Neuropsychiatric Interview .Thirty‐eight healthy female volunteers in their young adulthood  scan for the rsFC analysis. Thereafter, all participants were randomly assigned to active‐first or sham‐first stimulation. To avoid carry‐over effects between active and sham stimulation, a time delay of at least 1 week was respected. On each stimulation day, after an initial 25 min resting period, participants performed the TSST, followed by two iTBS or sham sessions with a 5 min intersession interval.Cortisol levels were measured immediately after the stress task, after the first iTBS/sham session, before and after the second iTBS/sham session, and 5 minutes after the second iTBS/sham session.  Furthermore, to assess changes in mood, six Visual Analogue Scales . Resting‐state functional images were acquired using a gradient echo T2*‐weighted sequence, while participants were awake and instructed to keep their eyes closed . The resting‐state scan lasted for 7 min.2.4To induce stress in our participants we evoked an acute stress response using the TSST .Saliva samples for cortisol assessment were collected using salivettes , containing a sterile polyester swab for collecting saliva, yielding a clear and particle‐free sample. Saliva cortisol levels (μg/L) were determined by Cortisol Saliva Luminescence immunoassay . Limit of Quantification was 0.12 μg/L and the within‐run and between‐run variation coefficients were less than 5%. The intraindividual stability of baseline salivary cortisol levels was reported to be more stable in women  connected to a 70 mm “butterfly‐”shaped coil. For the sham stimulation, a specially designed sham coil was used, visually identical to the active one and producing a similar sound without active stimulation. Both active stimulation and sham coils were placed on the individually located DLPFC. To accurately target the left DLPFC, we used Brainsight neuronavigation  to locate the center part of the left midprefrontal gyrus based on the individual anatomical MRI data. The individual resting motor threshold (110%) was determined by inducing a motor evoked potential on the right abductor pollicis brevis muscle. The following parameters were used for the iTBS sessions: 50 Hz frequency; 5 Hz burst frequency; 1,620 pulses in 54 cycles, each including 10 burst each 3 pulses with a train duration of 2 s and an intertrain interval of 6 s. Sessions (either sham or active) were separated by a 5 min resting period.2.8http://www.conn-toolbox.org). The software is powered by SPM12 . All functional images were first slice time corrected . Realignment parameters were estimated with respect to the first functional scan of the run. Artifact Detection Tool based outlier detection was run to identify possible outliers for first level analysis scrubbing . Anatomical images were coregistered and spatially normalized to the MNI template. Images were spatially smoothed with a 4 mm full‐width‐at‐half‐maximum Gaussian kernel. Linear detrending and band‐pass filtering of 0.01–0.08 Hz was applied on the blood oxygen level‐dependent (BOLD) signal to avoid low‐frequency noise and high‐frequency artifacts. White matter and cerebrospinal fluid principal components were regressed out from noise regions of interest (ROIs), in which signal is unlikely to be related to neural activity.rsfMRI images were preprocessed using CONN  were defined according to Kelly et al. , as 6 mm2.91, T2, T3, T4, and T5) as the within‐subject factors, Order (active‐first vs. sham‐first) as the between‐subjects factor, and the six VAS mood scales  as the multiple dependent variables . Higher scores indicate more negative affect.To examine whether the stress task provoked a similar psychological stress response during both sessions, mood changes were analyzed using a mixed MANOVA, with stimulation (active vs. sham stimulation) and time (TAUCi), as an index of the sensitivity of the HPA‐axis to the stressful event, and the area under the curve with respect to the ground (AUCg), as an index of the total cortisol release by the HPA‐axis and reflecting the intensity of the HPA‐axis response  = −2.47, p = .018) and the sham group  = −2.73, p = .010). Moreover, paired t‐tests showed that there were no significant differences between the two stimulation days in cortisol levels at baseline, immediately before the stress task , and immediately after the stress task (before the first iTBS/sham session), regardless of stimulation type (p > .459). The statistical conclusions are the same if the analyses are performed controlling for the change in cortisol levels from baseline to immediately after the stress task.Cortisol levels were log transformed because they did not show normal distributions. Following the formulas proposed by Pruessner, Kirschbaum, Meinlschmid, and Hellhammer , we compAUCi and AUCg indexes. The active and sham AUCi and AUCg values were used as the dependent variables. As a within‐subjects factor we included Stimulation (active‐iTBS vs. sham‐iTBS), and as a between‐subjects factor, we included Order (first session active vs. first session sham). In a second step, to investigate the influence of the rsFC between ACC and left DLPFC on the effects of iTBS on cortisol secretion, we used mixed ANCOVAs with AUCi and AUCg values as dependent variables, stimulation (active‐iTBS vs. sham‐iTBS) as the within‐subject factor, order (first session active vs. first session sham) as a the between‐subjects factor, and the rsFC indexes as covariates. Independent analyses were performed for each rsFC index . Given the number of analyses performed to investigate the influence of rsFC on the effect of iTBS on AUCi and AUCg, we applied a Bonferroni correction to the p‐value of the mixed ANCOVAs in order to avoid Type I error. Therefore, the significance level was set at p = .0025 (0.05 divided by 20 comparisons), two‐tailed. When analyses showed a statistically significant interaction between stimulation (active‐iTBS vs. sham‐iTBS) and the rsFC indexes, correlations were used to investigate the relationship between AUCg and AUCi for the sham and active iTBS sessions and the rsFC indexes. Importantly, the time of day when the experiment is performed may affect the stress‐induced changes in HPA axis activity . No outliers were found in this study. All the analyses were performed using SPSS 24.0 (IBM SPSS Statistics 24.0).We performed two mixed ANOVAs to investigate the effects of iTBS on the 3n = 34), we refer to Table n = 33), we refer to Table The final sample included in the rsFC analyses was 34 participants. For rsFC and cortisol values of the final sample included in the analyses  = 3.55, p < .001) and a significant interaction between stimulation and order  = 3.52, p = .011). There was no significant main effect of stimulation type  = .46, p = .834) or interaction between stimulation and time  = 1.06, p = .390). Performing univariate analyses of variance (ANOVAs) on the subscales of VAS results, we found no significant effect of time on “fatigue”  = 2.08, p = .088), “depression”  = .11, p = .979) or “cheerfulness”  = .51, p = .727). There was a significant main effect on “tension”  = 12.78, p < .001) and “anger”  = 3.14, p = .017) as it increased during the experiment, while “vigor”  = 2.88, p = .026) decreased. We only found a significant interaction effect between stimulation and order on “anger”  = 5.35, p = .028) and “tension”  = 5.78, p = .022). Although anger and tension generally increased during the protocol, participants who received active stimulation in the second session were angrier and more tense compared to participants who received sham stimulation. This effect could not be found when the active stimulation was given during the first session.Mixed MANOVA revealed a significant main effect of Time  = 0.95, p = .337; AUCg: F = 0.03, p = .871), Order  = 0.49, p = .487; AUCg: F = 0.68, p = .415), or the interaction between Stimulation and Order  = 1.10, p = .302; AUCg: F = 0.45, p = .508). These results indicate that iTBS does not affect AUCi and AUCg after being stressed.The results of the mixed ANOVA with 3.3.2AUCi  < 1.25, p > .272) and AUCg  < 0.73, p > .400). Moreover, none of the rsFC indexes showed a significant main effect  < 2.72, p > .109; AUCg: F < 0.50, p > .484).Independent mixed ANCOVAs with each rsFC index as a covariate were performed to investigate the influence of rsFC between the DLPFC and the subparts of ACC on the effects of iTBS on the activity of the HPA axis after stress. We observed no significant main effect of Stimulation and Order for all the analyses with AUCi  = 12.52, p = .001), but not for AUCg  = 4.23, p = .048). None of the other interactions between rsFC indexes and Stimulation showed a significant effect  < 1.56, p > .221; AUCg: F < 2.92, p > .098).Regarding the influence of the rsFC on the effects of iTBS on the stress response, we observed a significant rsFC interaction between the left DLPFC and the left cACC and the factor Stimulation for AUCi during the active iTBS session , but not during the sham iTBS session   after stress. These observations suggest that the incorporation of individual brain biomarkers may be of essence to optimize rTMS effectiveness in terms of stress regulation and increase its response rate.The present study is the first to investigate how baseline rsFC between the individual stimulation site on the left DLPFC and distinct parts of the ACC may predict the effects of iTBS on stress reactivity in healthy subjects. Concerning the baseline group functional connectivity analysis, we observed a negative correlation between the individual left DLPFC targets and the more dorsal parts of the ACC. In general, active and sham stimulation did not affect the stress response differently. Only when considering individual differences in rsFC strengths, and with active iTBS only, we found that stronger baseline rsFC anticorrelations between the individual stimulation site (left DLPFC) and the left cACC showed predictive value for lower cortisol levels (The dorsal and caudal components of the ACC are known to have strong connections with the DLPFC. This more “cognitive” subdivision of the ACC is part of a larger cognitive control network—including the DLPFC—that plays a central role in the neurobiology of depression (Li et al., Although this study has several important strengths, such as the use of a within‐subjects design where each participant receives both sham and active stimulation, as well as the use of a well‐validated stressor, it should be noted that our study also has several limitations. A first limitation entails the timing of our stimulation. So close to the stressor, iTBS stimulation might have affected both the peak of stress as well as the recovery speed, making it more difficult to disentangle these effects. Second, we also used a 5‐min interval in between the two iTBS sessions, contrasting with most iTBS protocols using a 10–15 min interval. Third, since we only included female subjects in our study, our results cannot be simply generalized to men as gender differences are documented in response to the TSST (Kelly, Tyrka, Anderson, Price, & Carpenter, In conclusion, the results of this study highlight the importance of considering individual differences in rsFC in order to optimize iTBS effectiveness, and to potentially increase treatment response rate for stress‐related disorders. Neurostimulation based upon rsFC between the DLPFC and cACC might prove to be a more effective method to attenuate the HPA‐axis deregulation. Nevertheless, future research is needed to further elucidate the effects of iTBS on the HPA‐axis in depressed individuals, considering rsFC parameters.AppendixS1: Supplementary materialsClick here for additional data file.


BIM deletion polymorphism with respect to responses to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs) has been controversial. The potential reasons for these inconsistent findings were unknown.In previous studies, the predictive role of EGFR‐mutant non‐small cell lung cancer (NSCLC) patients treated with first‐ and second‐generation EGFR‐TKIs were examined in the CTONG0901 and GLCI cohorts, respectively. Sixty‐eight patients were treated with third‐generation EGFR‐TKIs in the GLCI cohort. The BIM gene status was examined by next‐generation sequencing.Data from CTONG0901 clinical trial and medical records of Guangdong Lung Cancer Institute (GLCI) were retrospectively pooled. A total of 194 and 141 BIM deletion polymorphism was 11.3% and 17.0% in CTONG0901 and GLCI cohorts, respectively. For first‐ and second‐generation EGFR‐TKIs in CTONG0901 cohort, objective response (ORR) was 54.5% in BIM deletion group versus 56.4% in wild‐type BIM group (P = .87); disease control rate (DCR) was 90.9% versus 88.4% (P = 1.00); progression‐free survival (PFS) was 10.5 versus 11.2 months (P = .59); and overall survival (OS) was 20.5 versus 20.5 months (P = .73). In GLCI cohort, ORR was 54.2% versus 60.7% (P = .55); DCR was 91.7% versus 96.6% (P = .27); PFS was 10.1 versus 11.6 months (P = .63); and OS was 58.5 versus 45.0 months (P = .93). For third‐generation EGFR‐TKIs, ORR was 18.2% versus 63.2% (P = .02); DCR was 81.8% versus 96.5%, (P = .12); PFS was 5.8 versus 9.0 months (P = .13); and OS was 30.0 versus 24.8 months (P = .85). Cox regression analysis showed that concomitant genetic alterations could adversely affect the response to EGFR‐TKIs, but not BIM deletion.The frequency of BIM deletion showed no relation to an impaired response to first‐, second‐, and third‐generation EGFR‐TKIs in NSCLC patients. The factors influencing the response of EGFR‐TKIs were concomitant genetic alterations, but not BIM deletion.The presence of  CMLchronic myeloid leukemiaEGFR‐TKIsepidermal growth factor receptor tyrosine kinase inhibitorsNGSnext‐generation sequencingGLCIGuangdong Lung Cancer InstituteNSCLCnonsmall cell lung cancerPFSprogression‐free survivalOSoverall survivalORRobjective response rateHRhazard ratioCIconfidence interval1EGFR) mutations is high in Asian populations, reaching 30% in non‐small cell lung cancer (NSCLC) patients and 50% in those with adenocarcinoma.EGFR mutations can be treated with first‐, second‐, and third‐generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs), in a first‐line setting and beyond.BIM is a pro‐apoptotic member of the B‐cell lymphoma‐2 family and plays a role in regulating apoptosis during tumor formation.BIM exerts a pro‐apoptotic action through various pathways by translocation to the mitochondrial membrane.BIM contains only one BH3 domain, which is essential for the pro‐apoptotic activity of each BIM subtype.BIM expression to induce apoptosis of lung cancer cells with EGFR mutations.BIM expression in malignant tumor inhibits tumor cell apoptosis and promotes tumor development. BIM deletion polymorphisms are common in Asian populations, with an incidence of 12‐16% in lung cancer patients with EGFR mutations.EGFR mutations and BIM deletion polymorphisms are less responsive to EGFR‐TKI therapy.BIM deletion polymorphism had no effect on the progression‐free survival (PFS) or overall survival (OS) of patients treated with EGFR‐TKI therapy.Previous reports have indicated that the incidence of epidermal growth factor receptor , concomitant genetic alterations are increasingly detected. Comprehensive genetic analysis is needed to understand the inconsistent outcomes and to identify which genetic variants have an impact on the responsiveness to EGFR‐TKIs. Here, we used NGS to examine the predictive and prognostic roles of BIM deletion polymorphisms with respect to the response to first‐, second‐, and third‐generation EGFR‐TKIs in NSCLC patients from two independent cohorts.The role of 22.1EGFR‐mutant patients diagnosed with advanced‐stage NSCLC were enrolled in the CTONG0901 clinical trial. The first‐generation EGFR‐TKIs, erlotinib or gefitinib, were given to patients as any‐line treatment. Of the patients, 194 had sufficient tumor tissue at baseline for analysis by NGS using a panel of 168 genes; 22 of these patients had BIM deletion polymorphism and 172 had the wild‐type BIM.A total of 256 EGFR mutations from Guangdong Lung Cancer Institute (GLCI) were retrospectively pooled. Patients were treated with first‐ or second‐generation EGFR‐TKIs as any‐line treatment. The EGFR mutation status was identified by an amplification refractory mutation system or NGS. BIM deletion polymorphism represents a germline mutation, so the status of BIM was assessed by NGS at least once during the treatment process. Twenty‐four patients had BIM deletion polymorphism and 117 had wild‐type BIM. In later lines of treatment, 68 patients were treated with third‐generation EGFR‐TKIs (11 patients in BIM deletion group and 57 in wild‐type BIM group).From January 2016 to July 2018, the clinical data of 141 NSCLC patients with 2.2BIM deletion; 172 patients: wild‐type BIM) and 29 in the GLCI cohort  had NGS results at the baseline of first‐ and second‐generation EGFR‐TKIs. Thus, 223 patients were included in the final analysis of concomitant mutations and survival . Fresh frozen or formalin fixed paraffin‐embedded tissues from 217 patients, and peripheral plasma or cerebrospinal fluid specimens from six patients, were used for NGS testing. A total of 214 samples were analyzed with a panel of 168 genes; nine samples were analyzed with panels of 295 or 520 genes. The NGS results of 168 overlapping genes were included in the final analysis.A total of 194 patients in the CTONG0901 cohort  and 17.0% (24/141) in the CTONG0901 and GLCI cohorts, respectively. Homozygous BIM deletion only occurred in 4.3% of patients (2/46).The incidence rates of BIM deletion in the CTONG0901 and GLCI cohorts is summarized in Table BIM deletion and wild‐type BIM groups.The distribution of clinical and pathological characteristics in patients with and without 3.2BIM deletion and wild‐type BIM groups , and 54.2% versus 60.7% in the GLCI cohort, respectively (P = .55). The disease control rate (DCR) was 90.9% versus 88.4% in patients with and without BIM deletion in the CTONG0901 cohort, respectively (P = 1.00), and 91.7% versus 96.6% in the GLCI cohort, respectively (P = .27). The median PFS and OS were not significantly different between patients with and without BIM deletion in the CTONG0901 cohort   . BIM deletion polymorphism was not associated with a poorer curative effect of EGFR‐TKIs in two independent cohorts.The proportions of partial response, stable disease, and progressive disease were comparable between the s Figure . The obj Figures  and 2D.  Figures  and 2E. BIM deletion group than in the wild‐type BIM group . The DCR was 81.8% in the BIM deletion group and 96.5% in the wild‐type BIM group (P = .12)   Figure . The med Figures  and 2F.3.3BIM deletion and the response to EGFR‐TKIs. Along with the results of NGS, all genetic alterations and BIM deletion polymorphisms were input into univariate and multivariate analyses to identify which concomitant genetic variations were associated with impaired or enhanced curative effects of EGFR‐TKIs. The heat map showed the EGFR mutation subtypes, BIM status, and concomitant genetic variations detected in at least three patients . The frequency of concomitant TP53 mutation was the highest, which was 58.6% (17/29) in BIM deletion group and 68.0% (132/194) in wild‐type BIM group. Further, we screened the genes identified by NGS to determine which had an influence on the PFS and OS of patients treated with EGFR‐TKIs. Cox multivariate regression analysis showed that four genetic alterations were associated with poorer PFS—TP53 mutation , NTRK1 mutation and amplification , RB1 mutation , and PIK3CA mutation  were the strongest independent predictors of shorter PFS in patients treated with EGFR‐TKIs . KEAP1 mutation and deletion was the strongest independent predictor of a longer OS   Figure . Thus, n4BIM deletion polymorphism mediated primary resistance to TKIs in cancers including chronic myeloid leukemia (CML) and EGFR‐mutant NSCLC; this; was confirmed by in vitro cell culture experiments and clinical data.BIM deletion was associated with a reduced benefit of first‐ and third‐generations EGFR‐TKIs, based on the results of in vitro and in vivo experiments.BIM deletion polymorphism cannot account for intrinsic TKI resistance of Chinese individuals with CML.BIM deletion polymorphism with respect to the responsiveness to first‐generation EGFR‐TKIs. Two studies from KoreaBIM deletion polymorphism was not an independent predictor of a poor response to EGFR‐TKIs in EGFR‐mutant NSCLC patients, whereas other studies BIM mRNA expressionBIM deletion polymorphism was not a prognostic biomarker for OS in most studies. In the present study, BIM deletion polymorphism had no influence on the PFS or OS of patients treated with first‐ and second‐generation EGFR‐TKIs in two independent cohorts. In general, almost half of the clinical studies did not replicate the results of cell line experiments regarding the effects of BIM deletion on the response to EGFR‐TKIs. It is inevitable for the occurrence of drug resistance after patients treated with first‐ or second‐generation of EGFR‐TKIs for 9‐12 months.EGFR T790M mutation and third‐generation EGFR‐TKIs would be the optimal choice for the next‐step treatment.BIM status on the response to third‐generation EGFR‐TKIs. In our study, although patients with BIM deletion tended to have a poor response to osimertinib or avitinib, the DCR, PFS, and OS were comparable to those of patients without BIM deletion. The small sample size of patients treated with third‐generation EGFR‐TKIs likely leads to selection bias, which could explain the lower ORR in the BIM deletion groups.In 2012, Ng reported that BIM deletion polymorphism, the genetic background could also have an impact on the responsiveness to EGFR‐TKIs. The presence of TP53, RB1, and PIK3CA mutations reduced the response to TKIs. Here, we attempted to give an explanation of the inconsistent evidence regarding the role of BIM deletion in the responsiveness to EGFR‐TKIs and identify why some clinical reports could not replicate the results of in vitro and in vivo experiments. First, with the advent of NGS, the genetic background with hundred genes could be profiled and the results should be taken into account when analyzing the influential factors to clinical benefit of EGFR‐TKIs. The co‐mutation profile of NSCLC patients have been landscaped in previous study.TP53 or RB1 mutation tended to show an impaired response to EGFR‐TKIs. However, the detection method of BIM status in published studies could not profile the genetic background.BIM deletion in EGFR mutant patients with NSCLC was around 15% in these previous reportsBIM deletion group. When more patients combined with TP53 or RB1 mutation included in this group, the outcome would be that BIM deletion had a more deleterious response to EGFR‐TKIs than wild‐type BIM. Third, it is apparent that some studies with clinical data could not replicate the results of in vitro and in vivo experiments in other research. As we know, the cell lines were pure, carrying only one or two mutations. However, the situation in clinical practice is usually more complex, where the presence of certain confounding factors is inevitable.This study verified our hypothesis that, in addition to EGFR‐mutant patients with known BIM status reported to date, only 68 patients were included in the survival analysis of treatment with third‐generation of EGFR‐TKIs. Second, baseline NGS results were available for only 24 patients with BIM deletion, which may have been an insufficient sample size to examine the role of BIM status in Cox regression analysis. Third, the mechanism underlying the lack of effect of BIM deletion polymorphism on responsiveness to EGFR‐TKIs remains unclear.This study had some limitations. First, although our study had the largest sample size of 5EGFR‐mutant patients with advanced‐stage NSCLC in the CTONG0901 and GLCI cohorts had BIM deletion polymorphism, respectively, which had no relationship with any clinical or pathological factors. The presence of BIM deletion was not associated with impaired survival in patients treated with first‐, second‐, and third‐generation EGFR‐TKIs. Concomitant genetic alterations, but not BIM deletion polymorphism, had an influence on the clinical benefit of EGFR‐TKIs in patients with advanced NSCLC.Overall 11.3% and 17.0% The authors declare no conflict of interest.EGFR 19 deletion with and without BIM deletion polymorphism, in the CTONG0901 cohort  and in the GLCI cohort . BIM del: BIM deletion; BIM wt: wild‐type BIM.FIGURE S1: Survival analysis of the PFS and OS of patients with Click here for additional data file.EGFR 21L858R mutation with and without BIM deletion polymorphism, in the CTONG0901 cohort  and in the GLCI cohort . BIM del: BIM deletion; BIM wt: wild‐type BIM.FIGURE S2: Survival analysis of the PFS and OS of patients with Click here for additional data file.EGFR‐mutant patients with NGS results at baseline treated with first‐ and second‐generation EGFR‐TKIs. Only variations detected in at least three patients are shown.FIGURE S3: Genetic profiles of Click here for additional data file.


The locus coeruleus (LC) is involved in numerous crucial brain functions and several disorders like depression and Alzheimer's disease. Recently, the LC resting‐state functional connectivity (rs‐fc) has been investigated in functional MRI by calculating the blood oxygen level–dependent (BOLD) response extracted using Montreal Neurological Institute (MNI) space masks. To corroborate these results, we aimed to investigate the LC rs‐fc at native space by improving the identification of the LC location using a neuromelanin sensitive sequence. Twenty‐five healthy male participants (mean age 24.8 ± 4.2) were examined in a Siemens MAGNETOM Prisma 3 T MRT applying a neuromelanin sensitive T1TSE sequence and functional MRI. We compared the rs‐fc of LC calculated by a MNI‐based approach with extraction of the BOLD signal at the exact individual location of the LC after applying CompCor and field map correction. As a measure of advance, a marked increase of regional homogeneity (ReHo) of time series within LC could be achieved with the subject‐specific approach. Furthermore, the methods differed in the rs‐fc to the right temporoparietal junction, which showed stronger connectivity to the LC in the MNI‐based method. Nevertheless, both methods comparably revealed LC rs‐fc to multiple brain regions including ACC, bilateral thalamus, and cerebellum. Our results are relevant for further research assessing and interpreting LC function, especially in patient populations examined at 3 T MRI. Recent human fMRI studies revealed the baseline functional connectivity of the LC  and extraction of the blood oxygen level–dependent (BOLD) time series was based on Montreal Neurological Institute (MNI) space masks. The definition of LC was either based on averaging the LC location in a representative subject group measured with neuromelanin sensitive sequences  metric  were recruited by public advertisement. Inclusion criteria were defined by the absence of a current medical condition and history of major psychiatric illness as determined by medical history, physical examination, blood laboratory tests, electrocardiography, and toxicology findings. Potential healthy subjects who met the following criteria were excluded: Subjects with history of drug or alcohol dependency or abuse within the proceeding 6 months and subjects with serious unstable illness including hepatic, renal, gastroenterologic, respiratory, cardiovascular (including ischemic heart disease), endocrinologic, neurologic, immunologic, or hematologic disease, subjects with uncorrected hypothyroidism or hyperthyroidism and subjects with one or more seizures without a clear and resolved etiology. The criteria also excluded subjects with any illness likely to alter brain morphology and/or physiology like uncontrolled hypertension or diabetes and with the presence of any metallic (ferromagnetic) implants  or tattoos. The subjects were required to be off current medication for at least 6 weeks prior to inclusion. The study was approved by the institutional ethical review board of the University of Magdeburg, and all subjects gave written informed consent in accordance with the Declaration of Helsinki. The collected data of the study is available on request from the authors.2.2syngo MR E11 software and a 64‐channel head coil with the following parameters for the anatomical images: 3D‐MPRAGE sequence, echo time (TE) = 2.82 ms, repetition time (TR) = 2,500 ms, inversion time (T1) = 1,100 ms, flip angle = 7°, bandwidth = 140 Hz/pixel, acquisition volume = 256 × 256 × 192 mm3, isometric voxel size = 1.0 mm3, scan duration = 5 min 18 s.Image acquisition was performed using a Siemens MAGNETOM Prisma 3 T MRI scanner with 3, slice thickness = 2.50 mm, inter‐slice gap 0.5 mm, leading to an effective slice thickness of 3 mm, TR = 634.0 ms, TE = 10.0 ms, bandwidth = 165 Hz/Pixel, flip angle = 80°, scan duration = 10 min 50s.Since neuromelanin acts as an endogenous MR contrast agent, that is, it shortens the longitudinal relaxation time T1, the LC appears hyperintense in high‐resolution T1‐weighted MRI at 3 T. Here a T1‐weighted TSE sequence according to the original work of Sasaki et al.  with the3, slice thickness = 2.20 mm, leading to a high resolution of 2.2 mm isotropic voxels and no gap, TR = 2000 ms, TE = 30.0 ms, flip angle = 150°, 320 volumes in total, scan duration = 11 min. We visually checked raw data for each dataset and each time point.Blood oxygenation level dependent (BOLD) signals were acquired using a multi‐band accelerated T2*‐weighted echo‐planar imaging (EPI) sequence provided by the Center for Magnetic Resonance Research of the University of Minnesota. We acquired eyes‐closed resting state fMRI data with the following parameters: 66 axial slices parallel to the anterior–posterior commissure plane covering the whole brain acquired in interleaved order, acquisition volume = 220 × 220 × 146 mm2, 66 slices aligned with the fMRI slices, scan duration = 2 min 13 s).To be able to address the problem of geometric distortions in EPI caused by magnetic field inhomogeneities, a field map was acquired after the EPI sequence using a double‐echo gradient recalled echo (GRE) sequence  based on the neuromelanin contrast  in FSLview  based preprocessing steps included simultaneous realignment, unwarp and field map correction of functional data, slice time correction (with slice timings provided to SPM because of the multiband sequence), ART based outlier detection , structural and functional direct segmentation and normalization to MNI space  and smoothing with a 4 mm kernel. Denoising included regression of white matter and CSF signals  and cerebellar parcellation from AAL Atlas  to account for the total number of connections included in the analysis, as suggested by the default CONN settings. For ROI‐to‐ROI analysis, h values, as a measure of effect size, were reported, which represent the mean Fischer transformed pairwise correlations between LC and the connected ROI's (Tables 2 and 3).Cluster defining threshold was set to a t tests .The smallest subject‐specific mask contained 15 voxels, the biggest a total of 95 voxels. The mode (50 voxels) of the subject‐specific masks exactly met the voxel size of the MNI space mask.3.2p < .001). All 25 subjects clearly showed higher concordance of time series within LC after extraction of the signal at the subject‐specific location. Table We found significant greater ReHo KCC values in the subject‐specific method compared to the MNI space approach p < .00. All 25 3.33.3.1p < .001, left thalamus FDR p < .006), ACC (FDR p < .001) and cerebellar regions. Furthermore, the LC exhibited negative baseline connectivity to frontotemporal regions. A detailed description of significant connections is presented in Figures By extracting the BOLD time series out of the individual subject LC location, we found significant positive ROI‐to‐ROI LC rs‐fc including bilateral thalamus , anterior cingulate cortex (FDR p < .001) and cerebellar regions. Furthermore, the LC exhibited positive baseline connectivity to the basal ganglia, the right amygdala (FDR p < .004) and right nucleus accumbens (FDR p < .003). For a detailed description of significant connections, see Figures By extracting the BOLD time series based on the MNI space mask, we also found significant positive baseline ROI‐to‐ROI LC rs‐fc connectivity including bilateral thalamus , bilateral thalamus  and cerebellum  in the individual subject seed approach . Negative LC rs‐fc was shown to frontotemporal neocortical areas like left frontal pole, left superior, middle and inferior frontal gyrus and left lateral occipital cortex , bilateral thalamus , bilateral cerebellum  and right TPJ . Negative connectivity was shown to left lateral occipital cortex and left frontal pole , LC‐bilateral thalamus  and LC‐cerebellum  in both MNI and subject‐specific methods and they are in accordance to the ROI‐to‐ROI analyses presented before . The right TPJ is highly involved in the attention networks (Corbetta, Patel, & Shulman, There are some limitations to this study. We cannot exclude small submilimeter shifts in the registration of TSE to T1MPRAGE and in EPI to anatomical coregistration, but in our extensive visual control of preprocessing, we found no relevant displacement in the alignment of these sequences.Especially automated coregistration of the neuromelanin sensitive sequence to the anatomical sequence was challenging because of the small field of view of the T1TSE sequence. We achieved better results without coregistering T1TSE to the T1MPRAGE images, which was supported by visual inspection.The LC location was drawn by an experienced neuroradiologist, because exact definition of LC borders by an algorithm is difficult and not reliably established so far. Future studies with higher EPI resolutions may assess inter‐rater reliability to control for the impact of potential differences in LC delineation, which is unlikely to influence the results at the BOLD resolution presented here. In comparison to previous studies we examined a small sample size of 25 men, but could show a reliable and sufficient LC rs‐fc measurement in a relevant group size that may be difficult to exceed in most medicational studies.5In high resolution 3 T MRI, the MNI space masks adequately reveal LC rs‐fc to many brain regions involved in attentional processing. Nevertheless, the anatomical within‐subject approach shows a tremendous increase in ReHo, demonstrating higher specificity of LC signal extraction. Furthermore, LC‐TPJ connectivity may be driven by other brainstem nuclei than the LC.In general, our study underlines the feasibility and relevance of LC rs‐fc measurements in fMRI. We encourage LC rs‐fc measurements in surveys expecting involvement of norepinephrine or attentional networks and provide a basis for the interpretation of forthcoming results. The subject‐specific method may be suitable for future research both at 3 T and especially at higher field strengths which aim to investigate subtle changes in rs‐fc and regional activity of the LC. Since the LC is involved in Alzheimer's disease (Kelly et al., The authors declare no potential conflicts of interest.Data S1 Supporting Information.Click here for additional data file.


Vibrational modes were assigned based on the modeling results obtained at the B3LYP/6-311++G** level by density functional theory (DFT) calculations, which were performed to predict the FT‒IR and Raman spectra. Band downshifts were caused by 13C atom isotopic substitution with mass changed. Moreover, SERS spectra of 13–GLP by combining ninhydrin reaction on Ag NPs were obtained. Isotopic Raman shifts are helpful in identifying the components of each Raman band through vibrations across the molecular system. They are coupled by probe molecules and thus bind to the substrates, indirectly offering the opportunity to promote interactions with Ag NPs and reduce the complex equilibrium between different orientation/conformation of glyphosate molecules on the metal surface.Glyphosate is one of the most commonly used and non-selective herbicides in agriculture, which may directly pollute the environment and threaten human health. A simple and effective approach to its detection is thus quite necessary. Surface-enhanced Raman scattering (SERS) spectroscopy was shown to be a very effective method to approach the problem. However, sensitivity in SERS experiments is quite low, caused by different orientation/conformation of the adsorbed molecules on the metal surface, which limit its detection by using SERS. In this paper, 2‒ Glyphosate was first synthesized by the Monsanto company and marketed under the name “Roundup”, and then became well known as a common herbicide to control weeds. Based on its chemical properties, glyphosate is expected to be adsorbed by the soil; due to its widespread use, it directly pollutes the environment and contaminate foods. The detection of glyphosate residues water, food, and feed stuff is an essential step in regulating and monitoring the level. The majority of these methods are based on either gas chromatography (GC) or high-performance liquid chromatograph (HPLC) coupled to a variety of detectors, mainly mass spectrometric detectors. Most of the methods are highly sensitive, however, in most cases tedious since they include numerous steps for the purification and derivatization of the compound.Glyphosate (CSurface-enhanced Raman scattering (SERS) has been widely used as a powerful tool for ultrasensitive chemical analysis, as this technique is sensitive enough to detect glyphosate molecule . Taking The SERS detection method based on isotope labeling can achieve the purpose of defining vibration mode precisely. Isotope labeling has been reported in the detection of glyphosate residues and its transformation products based on NMR, MS, HPLC, GC, and GC‒MS ,4. The S13C‒glyphosate (13–GLP) as a model molecule for the theoretical and experimental studies of the molecule structure. Isotopic effects on Raman/FT–IR vibrational frequency and intensity due to the change of reduced masses and the vibrational coupling were verified by density functional theory (DFT) simulations and its corresponding isotopic SERS measurements. Density functional theory (DFT) calculations were performed to predict the Fourier Transform Infrared (FT–IR) and Raman spectra for the molecule. In order to reduce the complex equilibrium between different orientation/conformation of glyphosate molecules on the metal surface, a glyphosate detection method coupled by probe molecules with accurate vibration assignments thus bind to the substrates indirectly offered the opportunity to promote interactions with Ag NPs.In this paper, in order to make more clearly defined assignments to each atom, we determined 2‒13C‒glyphosate , glyphosate , potassium bromide , and silver nitrate  were purchased from Sigma-Aldrich Chemical Co. (Shanghai) . Ninhydrin  were obtained from Shanghai Aladdin Bio-Chem Technology Co., LTD . Sodium citrate , sodium molybdate , and all other chemicals were analytical-grade reagents and were purchased from Beijing Chemical Reagent Factory  and used without further purification. Ultrapure water (18.25 MΩ) was used throughout the experiments.2‒−1. Raman spectra were recorded on a Jobin Yvon/HORIBA LabRam ARAMIS Raman spectrometer  equipped with an integral BX 41 confocal microscope . Radiation from an air‒cooled internal HeNe laser (632.8 nm) and an external cavity diode laser (785 nm) were used as the excitation source. The FT‒IR spectra of ninhydrin were recorded as KBr disks at room temperature by a Bruker IFS‒66V FT‒IR spectrometer , equipped with a DTGS detector  at a resolution of 4 cm3 added into 200 mL H2O) and using sodium citrate  as reducing agent . . 12]. 13C at C7 position. The corresponding structural parameters of bond lengths, bond angles, and dihedral angles are shown in 13C‒glyphosate and glyphosate are only one isotopic atom apart, thus their structural parameters are exactly the same, as shown in The optimized geometry of 13–GLP or 12–GLP is shown in Each 13–GLP or 12–GLP molecule consists of 18 atoms, undergoes 48 normal modes of vibrations. The calculated vibration modes are listed in −1, and 0.9621 for wavenumbers higher than 1800 cm−1. The theoretical Raman spectra of the 13–GLP and 12–GLP calculated  at the B3LYP/6-311++G level of theory is presented in The absence of imaginary frequencies in the DFT calculations indicates that the calculated 13–GLP and 12–GLP structures are minima on their respective potential energy surfaces. To fit the theoretical to the experimental wavenumbers, a scaling factor of 0.980 was employed to optimize the computed data under 1800 cm−1  from δ(C7H2) or δ(C2H2). Large shifts are also observed at wavenumbers higher than 1800 cm−1, Raman bands at 2792, 2992 cm−1 for theoretical 13–GLP Raman spectrum appear at 2799, 3001 cm−1 for theoretical 12–GLP assigned to C7H8 stretching, C7H9 stretching. However, C7H2 twisting has the same Raman spectrum appear at 1237 cm−1 in both 13–GLP and 12–GLP. As we know, in similar structures, the smaller the atomic mass of the chemical bond has a higher infrared absorption frequency. The isotopic substitution is expected to cause downshifts for those bands deriving from modes which involve the 13C atom. Thus, a downshift for band involves 13C atom isotopic substitution.The calculated Raman modes of 13–GLP are observed at 412, 435, 476, 531, 589, 629, 673, 721, 806, 829, 849, 886, 978.6, 979.5, 995, 1027, 1130, 1151, 1223, 1237, 1248, 1309, 1327, 1350, 1426, 1460, 1480, 1755, 2792, 2851, 2970, 2992, 3439, 3618, 3678 and 3684 cm−1 , Table 1−1. Most of vibrational modes observed in the solid-state Raman spectrum of 13–GLP agreed well with frequencies of 12–GLP. Summary observed normal Raman scattering vibration modes of glyphosate 12–GLP and 13–GLP are listed in 13C atom isotopic substitution can be assigned more accurately. As with the results of theoretical vibration, some Raman bands downshift were caused by the isotopic substitution. Raman bands at 504, 639, 928, 1069, 1350, and 1460 cm−1 for 13–GLP Raman spectrum in solid state appear at 511, 648, 933, 1082, and 1465 cm−1 for 12–GLP assigned to O12H bending and C7H2 bending, C2N5H twisting and C7N5H twisting, and C7H2 rocking, C7H2 bending. Calculated theoretical Raman spectrum of 12–GLP/13–GLP is different from experimental Raman spectrum, the relative intensities are not precisely predicted for all the Raman bands. However, it could show structural parameters of 12–GLP/13–GLP, as they are still fairly useful for the assignments of the normal modes in the Raman spectrum. Raman spectrum will be influenced by excitation wavelength, and this may explain these discrepancies. The shapes of theoretical spectrum can well conform to experiment, but the wavenumbers are underestimated by about 15 cm−1.The observed vibration modes are listed in 2 bending downshift from 1465 cm−1 for 12–GLP to 1460 cm−1 for 13–GLP, another Raman shift is still 1482 cm−1, however, in the calculated frequencies of CH2 bending, one Raman shift is 1426, another downshift from 1464 cm−1 to 1460 cm−1. It is a remarkable fact that Raman shift of CH13C atoms . According to the baseline of vibrational spectrum −1 for 13–GLP in normal Raman spectrum in solid state to C7H2 bending, and 1482 cm−1 to C2H2 bending. Similarly, we assigned 928 cm−1 for 13–GLP in normal Raman spectrum in solid state to C7H2 rocking.Isotopic substitution (an atom is replaced by an isotope of larger mass) leads to a decrease in the wavenumber of all modes that involve the movements of the −1. Most of vibrational modes observed in the solid-state IR spectrum of 13–GLP agreed well with frequencies of 12–GLP. However, the downshifts for some bands cause by 13C atom isotopic substitution can also be observed –(CNH3) as two-mass oscillator, downshift cause by mass changed. Likewise, the IR band at 1032/1026 cm−1 assigned to HOP bending also downshifted, yet only Raman shift of 13–GLP at 1026 cm−1 is observed, Raman shift of 12–GLP is not observed.The band at 864/857 cm13C atom isotopic substitution to 12C.In conclusion, spectra shift by isotopic can be assigned vibrational modes more accurately because it does not change the symmetry of molecular structure and force constants between chemical bonds. The vibrational frequency of isotope substitution is shifted due to the difference of mass. In this paper, bands downshifts cause by Glyphosate Raman and SERS spectra have been subject of different experimental and theoretical studies, we also obtained SERS spectra of 13–GLP absorbed on Ag NPs. However, the observed SERS pattern in terms of a complex equilibrium between different orientation/conformation of the glyphosate molecules on the metal surface shows that sensitivity in SERS experiments is quite low . −1 in each spectroscopy. −1 display intensity changed due to mass changes caused by the 13C atom isotopic substitution. Pesticide molecules without SERS-active functional groups could be coupled by some probe molecules to achieve a better signal . In prev−1 in the normal Raman spectra to 657 and 784/786 cm−1, respectively. Raman intensity of the band at 662 and 791 cm−1 of normal PD product from 12–GLP/13–GLP are similar, however, SERS intensity are different, the peak at 662 cm−1 is much higher than at 784/786 cm−1. The band at 538 cm−1 shift to 521/524 cm−1 when absorbed on Ag NPs in SERS spectra. A strong peak appears at 690/689 cm−1 in SERS spectra that is not present in experimental Raman spectra . The limit of detection (LOD) is thus calculated to be 4.3 × 10−7 mol·L−1. Limit of quantitation (LOQ) is 5.0 × 10−7 mol·L−1. Recoveries for tap water  were 83.8–115.5%, relative standard deviation (RSD) were 1.1‒16.8%.We examined the SERS spectra of the PD products from 13–GLP at different concentrations shown in 13C isotopically substituted derivative is used to confirm the vibration modes in Raman and FT‒IR spectra. The isotopic substitution is expected to cause downshifts deriving from modes which involve the 13C atom, and PO symmetric stretching and PC stretching with no bond to 13C atom. This downshift caused by isotopic substitution is also present in DFT calculations, though there is no difference in space configuration between isotope labeled molecules and protomolecules. We further reported the SERS spectra of 13‒GLP by combining ninhydrin reaction on Ag NPs coupled by probe molecules and thus bind to the substrates. This indirectly offers the opportunity to promote interactions with Ag NPs and reduce the complex equilibrium between different orientation/conformation of glyphosate molecules on the metal surface with a LOD of 4.38 × 10−7 mol·L−1 and LOQ of 5.0 × 10−7 mol·L−1. 2‒13C isotopically substituted derivative is used to confirm the vibration modes in Raman and FT‒IR spectra more conveniently with higher sensitivity, better reproducibility, and lower relative standard deviation. This work will open a new promising area for isotopic SERS to investigate the mechanism of surface photoinduced reactions through tracking the fingerprint information changes of the species on metal nanostructures.Glyphosate Raman and SERS spectra have been the subject of different experimental and theoretical studies, however, sensitivity in SERS experiments is still quite low due to the complex equilibrium between different orientation/conformation of the adsorbed molecules on the metal surface. 2‒


Aging Cell, 14: 826–837. https://doi.org/10.1111/acel.12363Song, J., Kim, D., Chun, C. and Jin, E. (2015). miR‐370 and miR‐373 regulate the pathogenesis of osteoarthritis by modulating one‐carbon metabolism via SHMT‐2 and MECP‐2, respectively. In the article, “miR‐370 and miR‐373 regulate the pathogenesis of osteoarthritis by modulating one‐carbon metabolism via SHMT‐2 and MECP‐2, respectively,” the authors would like to correct two sentence, few typos, and the label of Figure 2B, the graph of Figure 3B, the image of safranin O staining in Figure 4F of DMM/anti‐miR‐370 cartilage, the bottom label of the graph in Figure 6a, and the last two images of safranin O staining in Figure 6f of DMM/MECP2 cartilage as these were misplaced. The sentence is: (b) SiRNA‐mediated knockdown of SHMT‐2 was confirmed by immunoblotting (left upper panel).It should read as: (b) SiRNA‐mediated knockdown of SHMT‐2 (siSHMT‐2) was confirmed by immunoblotting .The sentence is: Annexin V staining revealed that the induction of miR‐373 in OA chondrocytes sifnficanty reduced this apoptotic cell death to 2.26%, whereas inhibition of miR‐370 increased cell death up to 10% in normal chondrocytes.It should read as: Annexin V staining revealed that the induction of miR‐373 in OA chondrocytes significantly reduced this apoptotic cell death to 2.26%, whereas inhibition of miR‐373 increased cell death up to 10% in normal chondrocytes. In the result for Figure 2B middle panel, "significantly increased" should read as "significantly decreased", in the result for Figure 4E, "reduced miR‐370 level and apoptosis" should read as "reduced miR‐370 and induced apoptosis, and in the legend of Figure 6F, miR‐370 should read as miR‐373. The correct version of the Figures 3B, 4F, 6A, and 6F are shown below:The authors would like to apologize for the inconvenience caused.


Several studies have described the effectiveness of mesenchymal stem cell (MSC) transplantation in patients with liver cirrhosis (LC). However, in the majority, biochemical tests, clinical features, and pathologic results were used rather than radiologic tests to compare treatment outcomes. A 57‐year‐old male visited a stem cell clinic with a diagnosis of LC attributed to hepatitis B virus. This patient took tenofovir and diuretics at the initial presentation and was administered bone marrow‐derived MSCs twice via hepatic intra‐arterial infusion. Subsequently, the patient's clinical symptoms and biochemical tests  improved. Computed tomography findings showed loss of ascites, reduced nodularity, and especially increased liver volume, which suggested that MSCs have meaningful effects on liver volume, as well as improving liver function. Indeed, many studies have reported the effectiveness of mesenchymal stem cell (MSC) transplantation in patients with liver cirrhosis (LC). However, in most of these studies, biochemical tests, clinical features, and pathologic results were used rather than radiologic tests to compare treatment outcomes. This case report demonstrates the effectiveness of MSC infusion based on computed tomography findings of an increase in liver volume in a patient with hepatitis B virus associated LC. Liver cirrhosis (LC) is the end result of long‐term liver damage, and its associated morbidities impose significant health‐care burdens. Liver transplantation is the only definitive therapeutic option for these patients. However, the paucity of donors, rejection, and the high cost are hindering factors. In addition, lifelong immunosuppression is a worrisome issue. Cell‐based regenerative therapies, particularly adult stem cell‐based therapies, are evolving as viable clinical alternatives.A 57‐year‐old Korean man presented at a stem cell clinic with a diagnosis of HBV‐associated LC with abdominal distension and peripheral pitting edema. Eight weeks before this visit, the patient was admitted to a local medical center with the same symptoms, and biochemical tests and radiologic findings resulted in a diagnosis of LC. At this presentation, the symptoms had not improved despite the patient being on tenofovir and diuretics  for 8 weeks, and the patient had Child‐Pugh (CP) grade B (score 9) disease with gross ascites, spider angioma, umbilical hernia, and peripheral pitting edema but no overt jaundice. The liver was hardly palpated in the epigastric area. Endoscopy showed esophageal varix and CT revealed liver shrinkage, a huge amount of ascites, and splenomegaly.5 cells/cm2 into 75‐cm2 flasks and maintained in a humidified 5% CO2 atmosphere at 37°C for 5–7 days, after which nonadherent cells were removed by replacing the medium. Cells were then cultured for another 2–3 days, and when near confluent (70–80%), adherent cells were detached using trypsin containing ethylene diamine tetra‐acetic acid  and then replated at 4–5 × 103 cells/cm2 in 175‐cm2 flasks. Cells were serially subcultured to passage 4 or 5 for infusion (4.4 ± 0.5 passages [mean ± SD]).Following the provision of informed patient consent, MSC transplantation was conducted on two separate occasions. A total of 20–25 milliliters (mean ± SD: 23.1 ± 11.5 mL) of bone marrow (BM) aspirates were obtained under local anesthesia from the posterior iliac crest during the visit before treatment. All manufacturing and product‐testing procedures for the generation of clinical‐grade autologous MSCs were conducted in accordance with good manufacturing practice . Mononuclear cells were separated from BM by density gradient centrifugation  and washed with phosphate‐buffered saline (PBS). Cells were resuspended in Dulbecco's modified Eagle's medium‐low glucose  containing 10% fetal bovine serum (Gibco) and 100 U/mL penicillin/100 μg/mL streptomycin (Gibco). Cells were then plated at 2–3 × 106 cells/mL in 10 mL of saline solution. The clinical criteria used were: viability >80% and the absence of microbial contamination  when tested 3–4 days before administration and in the presence of CD73 and CD105 in >90% of cells and of CD14, CD34, and CD45 in less than 3% of cells by flow cytometry. Cells  were injected via the right hepatic artery using a coaxial angiographic catheter . BM‐MSCs were injected using a coaxial angiographic catheter (Boston Scientific) placed in the hepatic artery. All procedures following the first and second MSC administrations were performed on an outpatient basis. Routine biochemical parameters, symptoms and signs, diuretic requirements, and overall CP score were assessed retrospectively.On the day of injection, MSCs were harvested using trypsin/EDTA, washed twice with PBS and once with saline solution, and then resuspended at a final concentration of 5 × 10After first and second infusions, aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total bilirubin, international normalized ratio (INR), creatinine, alpha fetoprotein, and quantitative HBV DNA polymerase chain reaction (PCR) values were improved compared to the laboratory results at initial presentation. Platelet count fell from 93.1 (1000/μL) at the initial presentation to 78.0 (1000/μL) at the last follow‐up. The CP score improved after the second infusion and was 5 (class A) at week 80  . In our patient, liver volume increased from 14 870 to 20 270 mm2 in cross‐sectional view and from 8780 to 14 640 mm2 in coronal view at week 80 after completing MSC infusions.Several studies have reported positive outcomes for stem cell therapy based on biochemical and pathologic results in LC patients. Most previous studies have not investigated the effect of MSC transplantation on liver volume. However, Zekri et al.Many authors have suggested that hepatocyte‐like cell differentiation and MSC to hepatocyte differentiation are promising sources of liver regeneration and the main therapeutic mechanisms responsible for the beneficial effects of MSCs in LC patients.et al.s and diuretics. The authors reported improvements in serum albumin, CP score, and alpha fetoprotein. Salama et al.Causes of LC also require consideration. Many studies have targeted alcoholic LC, and because alcohol is not permitted in these patients, few factors meaningfully affect study results and introduce bias. However, when studying the therapeutic effects of MSCs in patients with LC of viral origin, patients who do not take antiviral agents are preferred, but these patients are far more difficult to enroll than patients with alcoholic LC. For this reason, the number of studies on LC of viral origin is smaller than studies on alcoholic LC. Terai The major limitation of this case report is that this patient received an antiviral agent concomitantly. However, we consider our experiences meaningful because CT after two administrations of MSCs showed an increase in hepatic volume, reduced nodularity, and loss of ascites. In addition, after two MSC infusions, the clinical symptoms and biochemical test results improved. Our observations suggest that MSCs might meaningfully increase liver volume and improve liver function in a background of LC. The majority of studies undertaken to assess the effectiveness of MSC implantation in LC patients have used biochemical tests and pathologic reports to assess outcomes rather than radiologic findings to assess liver volume. We hope that the present case study encourages the additional use of radiological findings to clarify the effects of MSCs on liver volume.


Microstructural changes associated with degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNc) in Parkinson's disease (PD) have been studied using Diffusion Tensor Imaging (DTI). However, these studies show inconsistent results, mainly due to methodological variations in delineation of SNc. To mitigate this, our work aims to construct a probabilistic atlas of SNc based on a 3D Neuromelanin Sensitive MRI (NMS‐MRI) sequence and demonstrate its applicability to investigate microstructural changes on a large dataset of PD. Using manual segmentation and deformable registration we created a novel SNc atlas in the MNI space using NMS‐MRI sequences of 27 healthy controls (HC). We first quantitatively evaluated this atlas and then employed it to investigate the micro‐structural abnormalities in SNc using diffusion MRI from 133 patients with PD and 99 HCs. Our results demonstrated significant increase in diffusivity with no changes in anisotropy. In addition, we also observed an asymmetry of the diffusion metrics with a higher diffusivity and lower anisotropy in the left SNc than the right. Finally, a multivariate classifier based on SNc diffusion features could delineate patients with PD with an average accuracy of 71.7%. Overall, from this work we establish a normative baseline for the SNc region of interest using NMS‐MRI while the application on PD data emphasizes on the contribution of diffusivity measures rather than anisotropy of white matter in PD. These dopaminergic neurons contain neuromelanin, loss of which manifests as depigmentation of the SNc. This has been well established as an early histological feature of PD . Typically, these studies initially delineate the SNc and then perform analysis on the computed ROI. Table Taken together, the findings have been widely heterogeneous and perhaps could be implicated to inconsistencies in stages of disease severity as well as to variable sample sizes. More importantly, the techniques employed for delineating the SNc may have a significant bearing on the variability in the reported results. For example, studies have employed T2 weighted, proton density weighted spin echo and inversion recovery based contrasts to precisely localize the SNc ROIs , Bangalore, India. The diagnosis of idiopathic PD was based on the UK Parkinson's Disease Society Brain Bank criteria , and Unified Parkinson's Disease Rating Scale (UPDRS‐III) OFF‐state scores, and levodopa equivalent daily dose (LEDD) were recorded. The OFF state was defined as 12 hr after the last dose of levodopa, and 48 hr after the last dose of a dopamine agonist. Age and gender matched healthy controls with no family history of Parkinsonism or other movement disorders were recruited.Another group of 27 healthy controls  whose NMS‐MRI sequence was acquired as part of a different study  for these subjects were acquired using a single‐shot spin‐echo EPI sequence with repetition time (TR) = 8,583–9,070 ms, echo time (TE) = 60‐62 ms, field of view (FOV) = 128 × 128 × 70m, voxel size = 1.75 × 1.75 × 2mm, slice thickness = 2 mm. Diffusion gradient was applied in 15 directions, with For creating a probabilistic atlas of SNc, a different set of controls were scanned on the same scanner. T1‐weighted images were acquired using the above‐mentioned protocol. Neuromelanin contrast sensitive sequences were acquired using a fast spin echo 3D T1 acquisition with TE: 2.2 ms, TR: 26 ms; flip angle: 20°; reconstructed matrix size: 512 × 512; field of view: 180 × 180 × 50mm; voxel size: 0.9 × 0.9 × 1mm; number of slices: 50; slice thickness: 1 mm; and acquisition time: 4 min 12.9 s. These images covered only the areas between the posterior commissure and inferior region of pons.2.3Probabilistic atlas was built from NMS‐MRI images of 27 subjects as shown in Figure 2.4Two additional sets of snROI markings were generated, one was by manual segmentation (by rater 2 [R2]‐author A.S) and the other was generated by a fully automated deep learning model known as U‐Net  and combined dice scores of atlas with raters R2 and R3 using a Student's 2.5https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FMRIB58_FA) using SyN algorithm and mutual information similarity index in ANTS. The transformation matrix of this registration was used to transform the MD, RD, and AD maps to MNI space. Diffusion measures of bilateral SNc were extracted for all subjects using MNI registered diffusion maps and the atlas described in earlier, which was thresholded at a 0.5 probability.DWI images of patients with PD and healthy controls were manually visualized for quality assessment. All preprocessing steps were done using FSL5.0.9  threshold of 0.05.Statistical analysis on diffusion measures between PD and HC was performed using multivariate analysis of covariance (MANCOVA) model, with FA, MD, RD, AD of left, and right SNc as dependent variables, PD and HC as grouping variables and age and gender as covariates. In addition, an independent To evaluate associations between the microstructural changes to the severity of the disease, the residuals from the diffusion measures after regressing out age and gender were correlated to the UPDRS‐III OFF scores (where available), the age of onset of disease (AoO), duration of illness (DoI) and LEDD of patients with PD.2.7Further, to understand the discriminative power of these diffusion measures, a multivariate random forest (RF) was implemented to delineate PD from the HC. RF is a popular decision tree based machine‐learning algorithm Breiman, . At eachIn this study, RF model was implemented on a total dataset of 232 subjects, out of which 75% was used for training and 25% for testing. A total of eight normalized diffusion measures comprising AD, FA, MD, RD for left and right SNc were used as feature set for the classifier.Number of predictors or features sampled for splitting at each node and the number of trees in the forest were the two primary tuning parameters in the model  was found between overlap of rater's SNc marking and overlap of atlas and rater's markings, thus providing additional support to the reproducibility and validity of the atlas.The probabilistic and thresholded (0.5) atlas of the SNc computed from NMS‐MRI images of 27 controls is shown in Figure 3.3f = 17.28, p = .00004; right snROI: f = 6.470, p = .011), MD , RD  in SNc of PD patients as compared to controls. As shown is Figure f = 0.621, p = .431: right snROI: f = .470, p = .493). The mean and SD of all diffusion measures in HC and PD groups along with their t‐stats and FDR corrected p‐values are shown in Table MANCOVA results showed significantly higher AD , MD , RD  as compared to right SNc. FA of the left SNc was found to be significantly lower as compared to right FA in the PD group  .Asymmetry of all diffusion measures of bilateral SNc was observed for both HC and PD group, with PD group showing a higher significance as shown in Table 3.5L, RDR and RDL were found to be the three topmost ranked features as shown in Table The average classification accuracy, sensitivity and specificity for basic RF model were 73.4%, 0.736 ± 0.01, and 0.731 ± 0.01, respectively, whereas that for RF‐RFE model was 71.7%, 0.736 ± 0.01, and 0.686 ± 0.05, respectively. Average feature ranking was consistent for basic RF and RF‐RFE model wherein MD4We created a probabilistic atlas of the SNc by precisely extracting the SNc ROIs using NM rich MR sequence and employed it to accurately delineate the SNc to create a normative atlas that can be used in future PD studies. We applied this atlas to a large cohort of PD patients to gain understanding of the microstructural abnormalities. Our results not only endorsed earlier findings but also facilitated fresh evidence supporting presence of micro‐structural changes in PD substantia nigra compacta using diffusion MRI analysis. We demonstrated higher diffusivity values in the SNc in PD, with no changes in anisotropy and significant asymmetry of the diffusivity values.The SN is anatomically divided into the SNc and the SN pars reticulata (SNr), where SNc is further subdivided into nigrosomes and the nigral matrix. Nigrosomes are the primary sub regions of the SNc where dopaminergic neurons are degenerated in PD Click here for additional data file.


It is unclear whether the mnemonic advantage of social media extends to a population less familiar with these platforms and whether knowing the content is from social media sources influences memory. This study examined how the presentation of news-like content in social media affected both item and source memory across two age groups. Younger adults (For item memory, both age groups correctly recognized tweets more than headlines. Source identification was more accurate when format and content were congruent than incongruent. Signal detection analyses indicated that the source advantage for congruent items was largely driven by a bias to select the format that matched the content’s original source and that this tendency was stronger in older adults.These results replicate previous literature on the mnemonic advantage of social media content. Although both younger and older adults remembered the content of social media better than the content of news sources, older adults were more sensitive than younger adults to congruency effects in source memory. These findings suggest that older adults rely more on their prior knowledge of conventional language and style in traditional and social media. We examined how younger and older adults remember the content and source of items from social media and from traditional news sources. Participants studied news headlines and tweets that were photoshopped to look like CNN-online posts or tweets. Social media content was remembered better than news content, suggesting that the social nature of the material is well-remembered by both younger and older adults. When asked whether studied items had been presented in Twitter format or CNN format, errors revealed that remembering source information is difficult when content and format do not match. Older adults in particular were more likely to assume that a news headline had been studied as a CNN post, even if it had been studied as a Twitter post (and vice versa). In other words, it is easy to misremember where one encountered a piece of information - reading the news on social media might make it harder to remember where the information was actually found. These findings suggest that the erosion of boundaries between news sources and social media has potential implications for remembering the source of information.Social media have become a platform for sharing news, memes, and other content in addition to personal information. Sharing news stories on one’s social media page is easy and common. In addition to remembering A majority of American adults who use the Internet have social media accounts  and news headlines selected from CNN online that were formatted to appear as items on a Twitter feed or news items on CNN. Item and source memory tests were administered to examine how content and perceived source were remembered. Given the blending of news and social media platforms and the ease with which information can be shared across platforms  it is worthwhile to investigate memory differences for social media and news across ages. In this study, we were particularly interested in situations when the content does not match the format of the platform  and how source memory may be affected if readers are accessing news across multiple sites.Source memory involves the recollection of details and context surrounding the encoding of an event . Stereotypes are one form of schematic knowledge. For example, older adults have been shown to be more sensitive to stereotype-consistent information than younger adults, such that the two age groups were equally accurate at attributing stereotype consistent information to the proper source, but older adults were less accurate on stereotype inconsistent content  does not align with the content . If older adults have a richer knowledge of what items are “news” items, seeing these items in the context of a tweet might be particularly disruptive. In other words, when the source does not align with the content, it may become more difficult for older adults to identify the correct source, as they can no longer rely on the integrative nature of a congruent manipulation .Credibility is another factor that may impact source memory. Older adults who rate social media posts as less credible or reliable may not devote the processing resources required during the encoding phase to compensate for the age-related deficits. Mutter, Lindsey, and Pliske  examinedNevertheless, Mickes et al.  found thHowever, Mickes et al.  did not In the present study, we extended Mickes et al.’s  findingsIf the content, specifically the socially relevant nature of social media, is driving memory, tweets should be remembered better than headlines, regardless of format, consistent with Mickes et al. . AccordiRegarding source identification processes, due to older adults’ deficits in source memory coupled with lower familiarity with the platform and possibly the content of the tweets, we expected younger adults to identify the source accurately more often than older adults  from Colby College and healthy older adults  from the surrounding area participated in the study.t (34.17) = 4.92, p < .001 . Younger adults were compensated with candy and an opportunity to earn a $5 gift card through a raffle draw. Older adults were compensated at a rate of $10 per hour. The study was approved by the Institutional Review Board at Colby College. The minimum sample size was determined based on the observed effect size calculated from experiment 1 in Mickes et al.  more than younger adults , t (72) = 3.96, p < .001, 95% CI , d = 0.94, whereas younger adults used social media  more frequently than older adults  = 6.06, p < .001, 95% CI , d = 1.78). Thus, older adults were less likely to use social media than younger adults and those who did use it tended to do so less often. Conversely, younger adults read the news less often than older adults.Based on responses to a questionnaire, described In the Materials, all younger adults reported using social media  compared to 53% of older adults. Of those who reported using social media, 74% of younger adults used social media 1–5 h a day, whereas 53% of older adults used social media 0–2 h per day. About 19% of younger adults reported reading the news daily compared to 78% of older adults. Frequency of social media and news use  were converted to numerical values, with higher values reflecting more use. Frequency of reading the news was on a 5-point scale  and frequency of social media use was on a 5-point scale .SE = 1.44) and tweets averaging 61.03 characters in length (SE = 2.13), with minimal to no hashtags or links. Tweets were slightly longer than headlines (p = .064). All identifying content  were removed, and replaced with names from an online random name generator (http://random-name-generator.info/) and headshots selected online from public sources. The same names as CNN bylines and as Twitter user names were used. The format (CNN and Twitter background) and content (headline and tweet) were factorially crossed to create four conditions: congruent  or incongruent  and study status (studied versus non-studied), yielding four different scripts. An approximately equal number of participants were tested in each experimental script. The full set of stimuli is available in the Additional file We also created a brief questionnaire on news and social media use. Participants indicated what type of social media they used, how often they checked social media, the source of their news, and how often they checked the news.Participants were tested individually. The experiment was programmed using the Qualtrics platform . Analyses on raw accuracy are reported in Additional file To analyze item recognition, we conducted analyses in line with Mickes et al. . We calcd’ scores. Age was a between-subjects variable and content and format were within-subject factors. Tweets , regardless of format, were recognized better than headlines , F = 6.52, p = .013, partial η2 = 0.08. No other effects were reliable, all F values < 1.81, all p values > .18. In summary, we replicated Mickes et al.’s (We conducted 2 (age) × 2 (content: headline and tweet) × 2 (format: CNN and Twitter) mixed analysis of variance (ANOVA) on the et al.’s  finding M = 0.58, SE = 0.02, 95% CI ) performed worse overall than younger adults , F = 32.02, p < .001, partial η2 = 0.31. The source of tweets  was correctly identified more than the source of headlines , F = 12.43, p = .001, partial η2 = .015. Three higher order effects were reliable: the content by age interaction, F = 10.09, p = .002, partial η2 = 0.12, the content by format interaction, F = 124.46, p < .001, partial η2 = 0.63, and the three-way interaction between age, content, and format, F = 39.02, p < .001, partial η2 = 0.35. No other effects were significant, all F values < 0.76, all p values > .39.We defined source identification as correctly selecting the format in which an item was studied. Thus, correct source identification required retrieving specific details about the encoding event, such as recollecting that a statement was associated with the Twitter logo or with CNN’s logo. To analyze source identification, 2 (age) × 2 (content) × 2 (format) mixed ANOVA was conducted on the proportion of correct source responses . Older adults  = 20.96, p < .001, partial η2 = 0.34. The effect of format was not significant, F = 0.32, p = .57, partial η2 = 0.01, but there was a significant interaction between content and format, F = 15.95, p < .001, partial η2 = 0.28. The source of headlines was better recognized when presented in the CNN format than in the Twitter format, t = 3.69, p = .001, 95% CI , d = 0.55, and the source of tweets was better recognized when presented in the Twitter format than in the CNN format, t = − 2.78, p = .008, 95% CI , d = 0.44. In summary, younger adults showed a source identification advantage for social media content and a large congruency effect.For the sake of brevity, we focus on the three-way interaction  = 114.30, p < .001, partial η2 = 0.79. Older adults better identified the source when the format and content were congruent; this occurred for both headlines, t = 6.88, p < .001, 95% CI , d = 1.23, and for tweets, t(31) = − 7.88, p < .001, 95% CI , d = 1.38. As is evident in Table p values > .24). However, for incongruent items, older adults showed a significantly marked deficit in accuracy . Thus, the three-way interaction indicates that older adults were more affected by the incongruency than younger adults were - they had similar performance on congruent items but markedly decreased performance on incongruent items.Older adults showed no effect of content or format, d’ scores d’ for source identification by holding source response constant to determine whether the effect was driven by a biasTo further explore the factors driving the large age effect as a function of congruency we calculated d’ scores. Overall, younger adults  had better source memory than older adults , F = 23.08, p < .001, partial η2 = 0.24. The source of tweets  was accurately identified more than the source of headlines , F = 12.67, p = .001, partial η2 = 0.15. These main effects were qualified by a content-by-age interaction, F = 8.52, p = .005, partial η2 = 0.11. Older adults showed no difference between source memory for tweets and headlines, t(31) = − 0.43, p = .67, 95% CI , d = 0.08. Conversely, younger adults had better memory for the source of tweets , than for the source of headlines , t(41) = − 4.89, p < .001, 95% CI , d = 0.75. These analyses suggest that in older adults the congruency effect was partially driven not by better source memory, but by a strong bias to respond with a given format on the source test based on the content of the item rather than item-specific memory for the study event . When we accounted for response bias , older adults showed worse source memory than younger adults. In sum, analyses on d’ were consistent with accuracy analyses and highlight the fact that content appears to bias a source decision.We conducted a 2 (age) × 2 (content) × 2 (format) mixed ANOVA  was identified more accurately than that of non-studied headlines  = 5.58, p = .02, partial η2 = 0.10. This suggests that some linguistic structure or content makes tweets more easily identifiable as coming from social media, whereas the source of headlines might be less obvious. Interestingly, older adults were more likely to correctly identify the source of tweets and headlines  than younger adults  = 13.99, p < .001, partial η2 = 0.23. There was no interaction of content and age, F < 1.0.Thus, the congruency effect for source decisions suggests such decisions were made using the content and not via an episodic retrieval process. For example, if an item was judged more likely to be a tweet because of content or stylistic elements, participants might have indicated it was from Twitter via a plausibility judgment or educated guess. To examine this, we examined source judgments for false alarms to foils , for which a source judgment would reflect a guessing strategy. We conducted 2 × 2 ANOVA with content (new headlines versus new tweets) and age as factors. Data from 27 younger and 23 older adults were included in the analyses, due to missing data from some participants. The source of non-studied tweets  was identified more accurately than the source of headlines , t(156) = 2.04, p = .04, d = 0.35. Thus, both experimental and naïve participants were able to identify the source of tweets more accurately, likely due to content and linguistic cues present in the tweets.To further determine whether the tweets and headlines differed in the extent to which source could be inferred solely from content, we examined whether naïve participants were successful at recognizing the source of the items when they were presented with no identifying (or contradictory) formatting information. Forty-one participants recruited on Amazon’s Mechanical Turk platform were presented the stimuli one at a time  and asked to indicate whether the items were news headlines or social media posts . Additional details on this study are reported in Additional file r(57) = −.62, p < .001, news reading, r(71) = .49, p < .001, and education, r(71) = .57, p < .001, we controlled for age in the tests of correlation. None of the correlation values were reliable, all p values > .07, suggesting that none of the measures predicted item or source memory performance.Finally, to examine whether pre-experimental experience with social media or news sites and education affected how well participants recognized the items or their source, we tested correlation between measures of social media and news use and memory performance. Because age was significantly correlated with social media use, To explore the factors that affect source and item memory for social media and news items, younger and older participants studied tweets and headlines that were formatted to look like items found on a Twitter feed or on CNN’s online platform. No age-related declines in item recognition performance were seen; however, consistent with Mickes et al. , item meRegarding item memory, formatting information that indicated source of the material had no effect on performance. One hypothesis was that source information might have affected the encoding process, if social media posts were perceived as less relevant or important and afforded fewer attentional resources. User-generated sources are rated as less credible than other sources  to 7 (very gossipy). Participants read the following definition of gossipy: “Gossip refers to the general sharing of details of other people’s lives, such as casual or unconstrained conversation or reports about other people, typically involving details which are not confirmed as true (according to the Oxford Dictionaries).” Stimuli were presented in random order and participants responded at their own pace. As noted above, ratings for two headlines were missing. On average, tweets were rated as more gossipy  than headlines , t(156) = 2.06, p = .04, d = 0.33. Overall, perceived level of gossipy content appeared to be low; however, the higher ratings for social media posts than news headlines does suggest that this specific aspect of the content might be influencing retention.The fact that a similar memory enhancement effect was found for tweets as for Facebook posts and comments from news and entertainment sections  and its origin , this information must be bound during the encoding phase (Raj & Bell, Older adults’ sensitivity to the effects of congruency suggests they might have relied on mechanisms such as plausibility judgments, including factors such as content or language, that might have served as cues for the source. Additional experience with language might support older adults in making such judgments or recognizing particular stylistic differences between social media and traditional news. Such a finding is consistent with evidence that older adults have richer semantic schemata that they can rely on (Umanath & Marsh, d’, their memory for source was significantly lower than younger adults. Older adults seem to be relying more on the content, rather than the format, as their incorrect attribution of source for incongruent items was much higher than in younger adults.However, this reliance on knowledge or sophisticated guessing of the source could have significant costs if individuals make determinations about the source of information based solely on content, because source is a key indicator of information validity or credibility. Although older adults appeared to have similar source memory to younger adults for congruent items and the age effect in the analyses on correct source identification was relatively small, when response bias was taken into account in the analyses on We also acknowledge that there may be differences in how one decides whether an item is “old” or “new,” which could subsequently affect the accuracy of source judgments for items declared to be old. Starns, Hicks, Brown, and Martin  propose We acknowledge three important limitations in the present study: the use of different presentation rates during encoding, the timing of the social media questionnaire, and the confound between stimulus type and font size. The different presentation rate was intended to equalize performance across ages due to generalized slowing in older adults (e.g., Salthouse, A second limitation concerns the administration of the social media and news-use questionnaire. Participants completed this measure as a filler task before the recognition test. The items on this questionnaire might have biased participants to attend differentially to the different items on the test. Although this is a valid concern, it is worth noting that the formatting of the items during the encoding phase made the manipulation of item type quite explicit. Because of pre-existing associations and differential experience with the types of content and the two different sources, re-directing participants’ attention to the fact that the stimuli came from social media or news sources might have generated some bias in responses. Although we cannot rule this out, there are two lines of evidence that make such a concern less likely. First, if the questionnaire in some way biased participants to differentially attend to information of one type or another, such effects might have emerged in item memory, such that older adults, who preferentially attend to news over social media, would show a reversal of the mnemonic advantage of social media content. As noted above, this did not occur. Such a bias might manifest on the source memory test, such that older adults would preferentially attribute content to the news source and younger adults to the social media source. However, this did not seem to be the case. A second line of evidence suggesting that the timing of the questionnaire had a limited impact comes from research on false memories. The popular Deese-Roediger-McDermott paradigm for eliciting false memories involves presenting lists of words that all converge on one, non-studied critical lure (Roediger & McDermott, Confounding by font size, as noted in “Materials”, was intended to maintain a more naturalistic presentation. It is possible that different stimulus sizes might have affected performance; specifically, older adults might have had more difficulty with the smaller font size of the tweets due to deficits in perceptual processes (Baltes & Lindenberger, Remembering where a piece of information was seen can be difficult. The negative effects of a mismatch between content and format might be particularly relevant as traditional and social media sources become more integrated. Because up to 67% of individuals report obtaining news from social media sources (Shearer & Gottfried, Additional file 1. Supplemental Materials.