Sc, low tissue infectivity and severe neuron losses in some special brain regions. Compared with other gPrDs, the positive reactions of cerebrospinal fluid (CSF) RT-QuIC of FFI patients were markedly low. However, the reactivities of RT-QuIC of the brain tissues, particularly different brain regions, of FFI cases are rarely described. In this study, three different brain regions from three FFI patients were subjected into two kinds of RT-QuIC assays using recombinant hamster PrP23-231 (rHaPrP23-231) and PrP90-231 (rHaPrP90-231) as the substrates, respectively. The results showed that the general RT-QuIC reactivities of the brains from FFI cases were significantly lower than that of sCJD. Analyses of the positive rates and the reactivities (lag time and rfu peak) of RT-QuIC identified that the homogenates of frontal lobe induced the most active reaction, followed by thalamus and callosum. The RT-QuIC reactivity in the tested brain sample was closely associated with the intensity of PK-resistant PrPSc. Moreover, we also verified that the sensitivity of the RT-QuIC of rHaPrP90-231 was much higher than that of rHaPrP23-231. Those data confirm that brain tissues of FFI patients are able to convert positive reactions in RT-QuIC and show regional-associated positive converting capacities.Fatal Familial Insomnia (FFI) is one of the most popular genetic prion disease (gPrD) in China. Unlike the other types of human prion diseases, FFI patients show distinctive neuropathological characteristics, such as less deposition of PrP PRNP is directly linked with the onset of FFI. Unlike sCJD and other genetic Creutzfeldt-Jakob disease (gCJD), spongiform degeneration and PrPSc deposits are not frequent in the brains of FFI, instead of severe neuron loss and active astrogliosis in some brain regions, especially thalamus [Fatal familial insomnia (FFI) is a special kind of inherited human prion disease with distinct clinical and neuropathological features compared with other human prion diseases, such as sporadic Creutzfeldt-Jacob disease (sCJD), Gerstmann-Sträussler-Scheinker diseases (GSS) –3. A spethalamus .FFI cases with D178 N mutation have been reported in different races of many countries and regions. Based on the surveillance data for human prion disease in China, D178 N FFI is most frequently detected genetic prion disease . PreviouSc in brain tissues of FFI. It may also reflect the different conformational structure of FFI prion. In this study, with the post-mortem brain tissues of three Chinese FFI cases, we tested the reactivities of three different brain regions in two kinds of RT-QuIC assays, using rHaPrP23-231 or recombinant hamster PrP from aa 90 to 231 (rHaPrP90-231) as the substrate. We found that the brain homogenates of FFI cases induced positive conversion in RT-QuIC. The positive converting ability of FFI brains was lower than that of sCJD brains. The lysates of frontal lobes of FFI and sCJD patients possessed apparently stronger converting abilities than that of thalamus and callosum. In addition, RT-QuIC assay of rHaPrP90-231 was markedly more sensitive than that of rHaPrP23-231.Compared with sCJD, the infectivity of the tissues of central nerve system (CNS) of FFI patients on the susceptible experimental animals is extremely low. It may link with the less amount of PrPThe present study was approved by the Ethical Committee of the National Institute for Viral Disease Control and Prevention  under the protocol 2009ZX10004-101, including the usages of the storage brain samples of FFI and sCJD patients from Chinese CJD surveillance network.Recombinant hamster PrP proteins  were prepared according to the method described previously ,7. BriefThe storage brain samples from three Chinese FFI patients were enrolled in this study, including the brain regions of frontal lobe, thalamus and callosum. The exact epidemiological, clinical, genetic and neuropathological data of these three cases were well documented previously .-5 dilution of each specimen was used as the seed for RT-QuIC.Ten per cent BHs of FFI and sCJD patients were prepared in lysis buffer  according to a previously described protocol ,10. To sTo detect the PK resistances of PrP in the brains of FFI patients, the BHs were exposed to different amounts of PK at the final concentrations of 5, 10, 15, 20 and 25 μg/ml at 37°C for 60 min prion to PrP-specific Western blot. Briefly, after separating in 12% SDS-PAGE, the proteins were transferred onto a nitrocellulose filter membrane . The membranes were incubated with 1:1000 diluted commercial PrP-specific monoclonal antibody (mAb) 6D11  at 4°C overnight, and subsequently, with a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody  at 37°C for 2 h with a dilution of 1:5,000.−5 diluted BH, 1X PBS, 170 mM NaCl, 1 mM EDTA, 0.01 mM ThT, 0.001% SDS and 10 µg of rHaPrP23-231 or rHaPrP90-231 in a final volume of 100 µl. Each sample was assayed in quadruplicated. The assay was conducted in a black 96-well, optical-bottomed plate  on a BMG FLUOstar plate reader (BMG LABTECH). The main working conditions were fixed as follows: temperature, 50°C; vibration speed, 900 rpm; vibration/incubation time, 90/30 sec; total reaction time, 90 h. ThT fluorescence  each reaction was automatically measured every 30 min and expressed as relative fluorescence units (rfu). The cut-off value was set as the mean value of the negative controls plus 3 times the standard deviation. A sample was considered to be positive when ≥2 wells revealed positive reaction curves.The details of the RT-QuIC assay was described previously . BrieflyQuantitative analyses of immunoblot images were carried out using software Image J. Data shown were means ± SD of triplicate samples from a single experiment and were representative of three independent experiments. Statistical analyses were performed using Student’s t-test.Sc in the prepared brain homogenates again, different concentrated PK from 5 to 25 μg/ml were introduced into the lysates of frontal lobe prior to Western blot. As shown in To access the reactivities of the brains of FFI patients in the RT-QuIC assays, the homogenates of thalamus, callosum and frontal lobe of three Chinese FFI patients were prepared. The epidemiological, clinical, genetic and neuropathological data were thoroughly described previously . To veri−5 diluted various brain homogenates were subjected into the RT-QuIC assays using rHaPrP23-231 and rHaPrP90-231 as the substrates, respectively, together with 10−5 diluted cortical homogenate of scrapie agent 263 K-infected hamster as the positive control for RT-QuIC. Meanwhile, same amounts of the brain lysates from an sCJD case were also administrated as the control. PK digested PrP-specific Western blots verified the presences of PrPSc in the homogenates of thalamus, callosum and frontal lobe. In the tests of thalamus, no positive reactions were observed in all three cases in the RT-QuIC of rHaPrP23-231, whereas a clear positive reaction was recorded in the test of sCJD (One microlitre of 10 of sCJD ). A slig of sCJD ). In the of sCJD ). In the of sCJD ). In the of sCJD ). In the of sCJD ,b). In lThe reactivities of three brain homogenates from three FFI cases and one sCJD case in two different RT-QuIC techniques are summarized in 50 of the tested specimens [Sc plaques in the brains of FFI cases are usually remarkably less than that of the patients of sCJD and other types of genetic prion diseases, such as E200 K gCJD [Sc, particularly the glycosylation profile, in the brains of FFI cases is different from that of most sCJD cases, revealing predominate-diglycosylated PrPSc [Sc but also related with the subtypes of PrPSc in the brain tissues [Sc show stronger reactivity in RT-QuIC than those with the type of MM2 PrPSc [Sc amount, the unknown-refined conformational diversity of PrPSc in the brains of FFI cases might also contribute to the low reactivity in the RT-QuIC assays.In this report, we have confirmed again that the brain tissues of FFI cases possess the apparent ability to induce the fibrillation in RT-QuIC. In line with the observations of other previous studies, the brain tissues from three tested Chinese FFI cases seem to be less capable to induce positive reactivity in RT-QuIC tests compared with those from sCJD cases at the same working dilution, although we do not titre the exact ECpecimens . Our recpecimens . Such ph0 K gCJD –15. In aed PrPSc ,15. It h tissues . The CSFM2 PrPSc . It is rSc amounts among various brain regions. Our previous neurological study on these three FFI cases has already showed relatively more PK-resistant PrPSc in the region of frontal lobe and remarkable less in callosum [Sc amount is also observable in the tests of the individual cases. The FFI-1 patient in this study containing very weak PK-resistant PrP signal in the frontal lobe in Western blot shows long lag time and low rfu value in RT-QuIC assays.Our data here also illustrate that the RT-QuIC reactivities of the different brain regions of FFI cases vary apparently, the frontal lobe with strongest RT-QuIC reactivity and the callosum with the weakest one. Such region-associated appearance has been observed not only in every individual case but also in the two different RT-QuIC assays. The region-dependent reactivity in RT-QuIC is also described in other studies . The cercallosum . The cloSc.It is not surprising that RT-QuIC technique using rHaPrP90-231 as the substrate shows markedly higher sensitivity in detecting the prions in the brain tissues of FFI and sCJD cases than that using rHaPrP23-231. As the second generation technique, RT-QuIC of rHaPrP90-231 has already shown reliable sensitivity and specificity in CSF RT-QuIC assays for sCJD . Many CS


In the pathogen infection and host defence equilibrium, plant viruses have evolved to efficiently replicate their genomes, to resist the attack from host defence responses and to avoid causing severe negative effect on growth and metabolism of the hosts. In this study, we generated chimeric tobacco mosaic virus (TMV) variants, in which the coat protein (CP) sequences were substituted with that of cucumber green mottle mosaic virus (CGMMV) or pepper mild mottle virus (PMMoV) to address the role of these in virus infection and host symptomology. The results showed that the chimeric viruses (TMV-CGCP or TMV-PMCP) induce stunting and necrotic symptoms in tobacco plants. We analyzed the transcriptomic changes in tobacco plants after infection of TMV and its chimeras using a high-throughput RNA sequencing approach and found that infection of the chimeric TMV induced significant up-regulation of host defence responsive genes together with salicylic (SA) or abscisic acid (ABA) responsive genes, but down-regulation of auxin (Aux) responsive genes. We further confirmed the increase in the levels of SA and ABA, together with the reduced levels of Aux after infection of chimeric TMV in tobacco plants. These data suggest novel roles of tobamovirus CP in induction of host symptoms and defence responses. Plant viruses are obligate intracellular parasites and rely on the host machinery for the establishment of their infection cycle . Virus-hTobamovirus with a positive single-strand RNA and the genome encodes at least four proteins (Tobacco mosaic virus (TMV) belongs to the genus proteins . Among tproteins . The 30 proteins . It was proteins . Furtherproteins .The researches of TMV and other tobamovirus species have greatly contributed to the development of virology and viral evolution . VirusesThe application of homologous recombination technology to construct chimeric viruses provide important tools to unravel the functions of specific viral RNA sequences or the coding proteins during infection . In a prNicotiana tabacum inoculated with the chimeric viruses showed significant stunting compared with those inoculated with wild-type TMV. We further investigated the global changes of gene expressions during TMV and the chimeric virus infection in N. tabacum using RNA-seq technology. The results showed that the host resistance genes were significantly up-regulated in leaves infected with TMV-CGCP or TMV-PMCP. Furthermore, infection of the chimeras also induced up-regulation of the genes involved in host defence responses and affected phytohormone biosynthesis or signaling. Specifically, the levels of salicylic acid (SA) and abscisic acid (ABA) were increased, whereas the levels of auxin was decreased by infection of the chimeric viruses.In this study, we constructed two chimera TMV-CGCP and TMV-PMCP, in which the CP of TMV was substituted with that of CGMMV and PMMoV using the homologous recombination technique. Intriguingly, To construct pCB-TMV-CGCP and pCB-TMV-PMCP that express chimeric TMV encoding heterologous coat protein sequences from CGMMV and PMMoV , the plaHindIII double digested pGD vector using ClonExpressTM MultiS One Step Cloning Kit , respectively, to construct pGD-TMCP-Myc, pGD-CGCP-Myc and pGD-PMCP-Myc.Plasmids of pCB-TMV-SY, pCB-CGMMV-LN and pCB-PMMoV-HLD were used as templates to generate 510 bp, 516 bp and 504 bp sized PCR products using primer pairs TMV-CP + /TMV-Myc- CP-, PMMoV-CP + /PMMoV-Myc-CP- and CGMMV-CP + /CGMMV-Myc- CP-, respectively. The three amplified DNA fragments were integrated to the BgIII and Agrobacterium tumefaciens GV3101. All of the PCR primers used in this study are listed in To construct plasmids of pCB-TMV-fsCP, pCB-TMV-CGfsCP and pCB-TMV-PMfsCP  that canagrobacterium infiltrated N. benthamiana leaves using Gooding’s method  cycles. A. tumefaciens cultures harboring pCB-TMV, pCB-TMV-CGCP or pCB-TMV-PMCP were infiltrated into N. benthamiana leaves at (5–7) leaf stage. The upper leaves of N. benthamiana infected with TMV, TMV-CGCP or TMV-PMCP at 7 days post inoculation (dpi) were harvested and mechanically inoculated on the leaves of N. tabacum variety NC89 for symptom observation and subsequent analysis.Plants of N. tabacum that were inoculated with wild-type or chimeric TMV using TRIzon Reagent . Three independent biological replicates were used for RNA-seq. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina  according to the manufacturer’s instructions and index codes were added to attribute sequences for each sample. The mRNA was purified from total RNA using poly-T oligo-attached magnetic beads and fragmented using divalent cations at elevated temperatures in NEBNext First Strand Synthesis Reaction Buffer (5X). The cDNA was synthesized and purified with AMPure XP system  before PCR, then the purified PCR products were submitted to sequence on an Illumina sequencing platform (HiSeqTM 4000) conducted by the Biomarker Technologies Corporation, Beijing, China.Total RNA was extracted from leaves of 1, with the SRA BioProject accession number . The clean reads were mapped to the reference genome sequence using Hisat2 tools soft.2 Differential expression analysis of TMV vs. TMV-CGCP and TMV vs. TMV-PMCP were performed using the DEseq.3 The differentially expressed genes (DEGs) were determined by adjusting P values to <0.01 using the Benjamini and Hochberg’s approach for controlling the false discovery rate <0.05. To further analysis of DEGs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway4 were implemented.The raw reads generated by Illumina sequencing were submitted to the Sequence Read Archive database at NCBI (SRA)N. tabacum inoculated with TMV, TMV-CGCP, TMV-PMCP or buffer control were ground into powder with liquid nitrogen. Approximately 50 mg sample powder were weighed and mixed with working internal standard solution. Then plant hormones were extracted with extraction solvent . The homogenate was incubated at 4°C on a shaker at a speed of 900 rpm for 30 min. Then trichloromethane (1 ml) was added to each sample and placed on the shaker at a speed of 900 rpm for an additional 30 min at 4°C. Two phases were formed by centrifuging at 14,000 rpm and 4°C for 5 min. Then the solvent from the lower phase (1.2 ml) was transferred to a new tube and concentrated using a nitrogen generator with nitrogen flow. The concentrated samples were resuspended in 0.1 ml methanol and centrifuged for 5 min at 14,000 rpm. Finally, the supernatants were filtered using a syringe and syringe filter and prepared for high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis.Plant salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), and indole-3-acetic acid (IAA) were determined as described with some modifications . The lea® qPCR Master Mix . Three biological replications were performed for each treatment. Actin was used as a reference for the analysis of gene expression. The primers used for RT-qPCR were shown in The RNA samples were reverse-transcribed to synthesize cDNA using a HiScript II Q RT SuperMix for qPCR kit . Real-time quantitative PCR (RT- qPCR) was performed using Universal ChamQTM Universal SYBRN. benthamiana infiltrated with pGD-TMCP-Myc, pGD-PMCP-Myc or pGD-CGCP-Myc were collected and grinded with plant protein extraction buffer . After centrifugation at 12,000 rpm for 10 min at 4°C, the supernatant was collected. The total proteins were separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and stained with Coomassie Brilliant Blue, thereafter transferred to polyvinylidene difluoride (PVDF) membranes . The membrane was treated with Myc-tag antibody  and alkaline phosphatase (AP)-conjugate goat anti-rabbit secondary antibody . The treated membrane was incubated with chemiluminescent substrate CDP-star  and visualized the target bands by using the Chemical luminous imaging system Tanon 5200 .The leaves of 3-week-old Nicotiana benthamiana is well applied as a model plant in the study of plant physiology or pathology including plant virology. Especially, N. benthamiana is extremely sensitive to virus infection and can be systemically infected by a wide range of viruses . Consequently, the leaves of N. benthamiana infected with the wild-type or the chimeras of TMV were extracted and mechanically inoculated onto the leaves of N. tabacum  ≥ 1.0 or ≤ −1.0 , auxin-responsive protein IAA14 (LOC107764405), heat shock protein 90 , WRKY transcription factor 40 , WRKY transcription factor 50 , serine/threonine-protein kinase RLK1 (LOC107794887), pathogenesis-related protein-1C  and TGACG-sequence-specific DNA-binding protein TGA-2.1 (LOC107782457). The results showed that the expression levels of the auxin responsive genes were significantly decreased, whereas expression of other genes were increased in chimeras infected plants compared with TMV, which were consistent with the results of RNA-seq , enhanced disease susceptibility 1 protein (EDS1) and heat shock proteins (Hsps) were significantly increased (MAPKKK). Four DEGs including RLK1, Hsp90, WRKY40 and WRKY50 involved in defence response were selected to verify their regulation using RT-qPCR. The results demonstrated that the expression levels of all the above genes were significantly up-regulated by the infection of chimeras of TMV (Plant virus infection often induces host-virus interactions with a strong plant immune response. Leucine-rich repeat LRR domain present in receptor-like kinases (RLKs) plays an important role in pathogen recognition through signaling pathways . In thisnfection . In addincreased . Infectis of TMV . The ress of TMV .NPR1), TGA Transcription Factor genes and pathogenesis-related protein-1 (PR1) were significantly increased upon chimeric TMV infection. By contrast, the expression of auxin signaling pathway-related genes including auxin transporter AUX1, AUX/IAA family and small auxin-up RNA (SAUR) were significantly decreased after infection of the chimeric viruses. In addition, expressions of ethylene (ET) related genes ethylene insensitive3 (EIN3) and ABA related genes including abscisic acid receptor PYR/PYL family and serine/threonine-protein kinase SRK2 (SNRK2) were induced in TMV-CGCP or TMV-PMCP infected plants. We selected PYL4, PR1, TGA2.1, and IAA14 to verify their regulation using RT-qPCR. The results demonstrated that the expression levels of PR1, TGA2.1, and PYL4 were significantly up-regulated and IAA14 was significantly down-regulated by the infection of chimeric viruses  when they trigger PTI or ETI responses. The SA is also widely mentioned in viral defence immunity . Exogenoker gene . Therefonduction . TherefoSA regulates plant basal defences via activating the host systemic acquired resistance (SAR) and hypersensitive responses. Induction of SAR also suppresses a serial of auxin-responsive genes , suggestN. benthamiana NbPCIP1 was found to interact with the CP of PVX, which affect infectivity, pathogenicity, and symptom expression of the virus (T42W) can restrict the accumulation of MP and result in strong resistance to TMV infection in plants containing CPT42W  can be found in the article/N. benthamiana plants and N. tabacum cv and NC89 and treated samples for transcriptome sequencing. MA, MY, and XB carried out the transcriptome analysis and analyzed the data. YW, MA, ZX, MY, and XB prepared the reagents and materials and analysis tools. MA and MY prepared the figures and tables. MY wrote the original draft. YW, MA, ZX, and XB reviewed the drafts of the manuscript. All authors reviewed and approved the manuscript.YW conceived the research project. MA, ZX, MY, and XB conceived and designed the experiments. MY, XB, and YH performed the main experiments. YC, JW, RZ, and YL cultivated 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.


Background: Anorexia nervosa (AN) is a psychiatric illness that without early effective treatment becomes chronic with high physical, psychological and social morbidity and high mortality. Pilates exercises can improve quality of life and increase body awareness in different clinical and healthy populations. The aim of this pilot study was to examine the acceptability of a Pilates program in a sample of female children and adolescents with AN by evaluating the psychopathological status, alterations in the perception of body image and health-related quality of life after 10 weeks.Methods: A total of 12 female patients (age: 14.6 ± 1.7 years) completed the 10-week Pilates program. Psychopathology (EDI-3), body image disturbance (CDRS) and quality of life (KIDSCREEN-27) were evaluated before and after the intervention. A satisfaction questionnaire was also provided.Results: Regarding psychopathology, although there were standardized reductions in seven parameters of those that form EDI-3, none of them reached significance. In relation to body image, significant, moderately standardized and substantial decreases were observed in the body dissatisfaction . There were significant, large standardized and substantial increases in physical well-being  and significant, moderately standardized and substantial decreases in autonomy and parent relation . Satisfaction data was positive.Conclusion: A Pilates program could help to improve perceived health outcomes by decreasing body dissatisfaction and increasing physical well-being in female children and adolescents with AN, so Pilates seems to be a beneficial complementary treatment in children and adolescents with AN. These findings from our pilot study are encouraging for future research with a substantially larger sample size, representing the first phase of a longer process. Anorexia nervosa (AN) is a psychiatric illness that includes in its description an intense fear of gaining weight, and a disturbance in the way in which one's body shape is experienced or a persistent lack of recognition of the seriousness of the low body weight . The lifBody image disturbance is a robust predictor of AN, illness relapse and often persists in otherwise recovered patients , 6. ThisPatients with AN report poorer health-related quality of life (HRQoL) compared to both the general population and other psychiatric/somatic diseases . PatientPhysical activity has been observed to have broad positive effects on children and adolescents, improving psychological functioning, body acceptance, quality of life, and physical and mental health in general –15. Liken = 23) were recruited from the Child-Mental Health Unit of the Vázquez Díaz Hospital . The clinical criteria according to the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5)  and the guidelines of the Declaration of Helsinki were followed, last modified in 2013. All patients were assessed by the same researcher to reduce interexaminer error. A code was assigned to each patient to blind the data in the statistical management.All female patients between 10 and 17 years old with a clinical AN diagnosis ( (DSM-5) . This stThis was a small uncontrolled pilot study. For each patient, a visit was scheduled where anthropometric data and questionnaires were collected (Pre). After the Pilates program, they were asked to complete the questionnaires again (Post). The time between admission to the hospital and the approval of the medical team to enter the Pilates program was 118.7 (± 11.6) days on average. During that time, patients received cognitive behavioral therapy and continued in it throughout the program. All patients were informed about the voluntary character of their participation and were assured that all the data were gathered anonymously.The Pilates program was conducted in group for an hour, three times a week for 10 consecutive weeks by a certified physiotherapist as Pilates Instructor. The exercises were performed on mats and each movement was repeated 10 times. Patients were instructed to perform the movements with control and precision, with attention to their breathing pattern  and to core control activation during execution . The detStanding height was measured to the nearest 0.1 cm following standard procedures using a balance with an incorporated stadiometer , while subjects were standing barefoot. Weight was determined to the nearest 0.05 kg through bioelectrical impedance analysis  with the subject in their underwear. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared.EDI-3 (Eating Disorder Inventory-3)  is a sel2 (very low weight) up to a BMI > 30 kg/m2 (obesity). The person is asked to indicate which figure represents their current body shape (perceived body shape) and what figure they would like to have . The discrepancy between these two classifications represents a measure of body dissatisfaction. A score of 0 was interpreted as satisfaction with body image, and a score different than 0 was interpreted as body dissatisfaction    is one oied = 1) . The valied = 1) , and it ied = 1) .To evaluate the HRQoL, the Spanish version of KIDSCREEN-27 was used. KIDSCREEN-27 has been previously used in adolescents with EDs as a measure of quality of life . It is aWe also provided a satisfaction questionnaire about the Pilates program performed at Post-assessment. It included the following 3 questions: “1. Do you think Pilates has improved your posture  and flexibility?; 2. Do you think Pilates has helped you into feeling better?; 3. Do you think Pilates has made you have less back pain?” Each question had five possible Likert-scale responses rated from 1 to 5 points: “strongly disagree” (1), “disagree” (2), “neither agree nor disagree” (3), “agree” (4) or “strongly agree” (5).p < 0.05. The Shapiro-Wilk test was used to test for normality of distribution. The Kruskal-Wallis test was used to compare demographic data among the patients who completed the program, those dropped out, and those who declined to participate in the study. The Wilcoxon test was used to evaluate the significant differences between the Pre and Post Pilates program. Data were also assessed for practical/clinical meaningfulness using an approach based on the magnitudes of change. Cohen's d statistic determined the effect size of the standardized differences in the selected variables, and Hopkins' scale . Significance was set at s' scale  and a cus' scale  were uses' scale .2, of which eleven were diagnosed with a restricting subtype and one with the atypical subtype. The demographic characteristics of the all patients are shown in Of the 15 patients included in the intervention, three of them were excluded in this pilot study for not completing at least 85% of the intervention. The program was completed by 12 female children and adolescents diagnosed with AN, with an age and BMI of 14.6 (SD = 1.7) years, and 19.6 (SD = 2.2) kg/mp = 0.046), forming nine patients of the total (n = 12) with body dissatisfaction in Pre and 5 in Post, and there was a small standardized decrease in the ideal body shape variable.The differences observed in the psychopathology analyzed by EDI-3 and body image disturbance are shown in p = 0.008), the difference exceeding the threshold to consider such clinically relevant changes. There were significant, moderately standardized and substantial decreases in autonomy and parent relation (p = 0.021). Small standardized and substantial decreases were found in peers and social support and a small standardized increase in psychological well-being.Differences in quality of life are shown in The correlations of the differences between Pre and Post of all the analyzed variables of the three questionnaires are shown in The descriptive statistics of the satisfaction questionnaire are shown in To the best of our knowledge, this is the first study that analyzes the acceptability of a Pilates program evaluating psychopathology, body image and quality of life in children and adolescents with AN. The main findings of this pilot study after the 10-week Pilates program were the significant and substantial increase in physical well-being, and the significant and substantial decrease in body dissatisfaction and autonomy and parent relation.In recent years, there are few studies that can be found in the scientific literature in which alternative treatments are used in patients with AN. Although we obtained standardized reductions in seven parameters of those that form EDI-3, none of them reached significance. Similar results were found in a study in which the effect of a Yoga program during 8 weeks in young people with EDs was studied, and although the overall scores of the psychopathology decreased, they were not significant after the intervention . HoweverThe biggest change and statistical consistency after our Pilates program is the decrease in body dissatisfaction. Pilates work with exercises focusing on the sensory increase of internal consciousness, and it has been suggested that this can activate multiple interoceptive channels, such as proprioception from the locomotor- and vestibular system, and also visceroception through controlled breathing . These fRegarding the quality of life, an increase in physical well-being was obtained among our patients, in line with other studies where self-perceived general health improved after attending Pilates in the clinical population  as in thThe clinical impacts that we can observe from this pilot study are that the inclusion of a Pilates program in hospital units for eating disorders in children and adolescents, together with conventional psychotherapy, has a good acceptance by these patients, even finding positive improvements that could help recovery from illness.There are several limitations to this pilot study. Firstly, as this is a single-arm study, time is the independent variable. The patients had been in psychotherapy for 17 weeks before entering the program and, although it is quite a long treatment time and they continued in it, without having a control group or an active control group, the improvements found may not be unequivocally attributed to the Pilates program. Secondly, the comparatively small sample size is certainly a major limitation of our study, being difficult to enroll a large number of affected children and adolescents for 10 weeks. There are known limitations of all nonprobability samples, including the unknown levels of sampling errors and their lower representativeness. Therefore, future research studying this issue within a control group design and large sample size is clearly necessary to validate and support the current results. Thirdly, although the responses to the satisfaction questionnaire were prepared according to the Likert-scale, the questions could be biased toward positive responses in retrospect. Therefore, open ended questions with several-alternatives would probably be a better way to evaluate the program. Fourthly, objective variables of physical or body changes were not evaluated in this study. Finally, specific questionnaires for associated symptoms such as depression or anxiety were not evaluated in the present pilot study. Thus, future studies should evaluate the depression and anxiety associated with AN after a Pilates program and new lines of research that analyze the possible physical changes that could be found in this population. Furthermore, it would be interesting to observe the possible differences stratified by age in studies with a large sample size. We are aware of the limitations mentioned above and the potentially confounding factors in this pilot study, and therefore the current results must be interpreted with care. However, it deserves to be pointed out that our study was intended as a pilot study and is, to the best of our knowledge, the first to analyze the acceptability of a Pilates program evaluating psychopathology, body image and quality of life in children and adolescents with AN.After attending the Pilates program three times weekly for 10 consecutive weeks, our patients reported high levels of satisfaction and a high positive overall response. This is certainly a good beginning for future qualitative studies in this field. Therefore, more research is required to prove that Pilates could help in psychopathology and improve the quality of life in this population. We can affirm that through this pilot study it is clearly described how a more definitive study might be conducted, so this represents the first phase of a longer process.A Pilates program could help to improve perceived health outcomes by decreasing body dissatisfaction and increasing physical well-being in female children and adolescents with AN, so Pilates seems to be a beneficial complementary treatment in children and adolescents with AN. These findings from our pilot study are encouraging for future research with a substantially larger sample size, representing the first phase of a longer process.All datasets generated for this study are included in the article/www.clinicaltrials.gov (Identifier: NCT03667183). The guidelines of the Declaration of Helsinki, last modified in 2013, were followed and, since all the patients were minors, they and their legal guardians signed written informed consent.This pilot study was part of a Clinical Research, which was approved by the Research Ethics Committee of the University Hospital Complex of Huelva (PI 005/16) and registered in SM-S, TM-G, and DM-I: conception and design of the study. SM-S, CM-G, TM-G, and DM-I: acquisition and analysis of data and approved final version. SM-S and CM-G: manuscript draft. 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.


To improve the delivery and reach of primary health care, a robust scientific foundation driven by research is needed. However, few family physicians conduct research, especially in sub-Saharan Africa. Early-career and trainee family physicians are a key part of the primary care research pipeline and have an expressed need for research training and mentorship.AfriWon Research Collaborative (ARC) was an online research training and mentorship pilot program whose objective was to increase research activity among participants from AfriWon Renaissance, the family physician young doctors’ movement of sub-Saharan Africa.ARC utilized a 10-module online curriculum, supported by peer and faculty e-mentorship, to guide participants through writing a research protocol. The feasibility, acceptability, and scalability of this program was evaluated via a mixed-methods RE-AIM-guided process evaluation using descriptive statistics and inductive/deductive thematic analysis.The pilot reached participants from Botswana, Democratic Republic of the Congo, Ghana, Nigeria and Sierra Leone and was adopted by mentors from 11 countries across three continents. Four of the 10 pilot participants completed a full research protocol by the end of the six-month core program. Seven out of the 10 participants, and nine out of the 15 mentors, planned to continue their mentorship relationships beyond the core program. The program helped instill a positive research culture in active participants. Some participants’ and mentors’ engagement with the ARC program was limited by confusion over mentorship structure and role, poor network connectivity, and personal life challenges.Online research training and mentorship for trainee and early-career family physicians in sub-Saharan Africa is feasible and acceptable to participants and mentors. Similar programs must pay careful attention to mentorship training and provide a flexible yet clearly organized structure for mentee-mentor engagement. Additional work is needed to determine optimal implementation strategies and ability to scale. For primary health care to achieve its promise of improving health, reducing cost, and decreasing inequity , primary3Around the world, primary health care research is limited by a lack of skilled researchers, training opportunities, and resources, with these challenges being further amplified in sub-Saharan Africa 5. Recomm3448Peer research mentorship has successfully filled gaps in mentorship within academic institutions where the small number of research faculty are over-burdened . Health The feasibility, acceptability, and scalability of an online research training program that provides peer and faculty e-mentorship to early-career FPs in sub-Saharan Africa, where internet connectivity and research mentorship experience tend to be limited, is unknown. In response to this need, members of this study partnered with AfriWon to develop and pilot test the AfriWon Research Collaborative (ARC) research training and mentorship program. This is the first of a series of papers on the ARC program. This paper describes the program components and reports on key implementation indicators.Figure 1), supported by an e-mentorship team . The six-month pilot ran from September 2019 to March 2020 and was limited to 10 participants. ARC’s program development was guided by two logic models that mapped the development of needed inputs, activities, outputs, and anticipated outcomes . Participants and mentors received a certificate of completion for meeting a priori criteria for engagement in the program .The ARC pilot program was designed to increase participants’ research activity through an online curriculum . Each module contained one or more recorded screencast lectures, supplemental reading materials, and short assignments or “deliverables” and was accessed via Google Classroom . All participants and peer mentors were provided with an e-textbook How to do Primary Care Research, which also informed the curriculum design  medical study.” (PPU1)The baseline prior research experience varied widely among participants. Some reported: Table 1)  back to the chapters that were not even part of the modules (…) so that my understanding of the modules could be broadened.” (PPH8)Engagement in ARC increased participants confidence, competence, and interest in primary health care research: [The participant] was unable to complete the program after his bereavement (Mentorship Summary 1); “he told me that he had no connection (…) and then after that [he] is still quiet.” (PRML1); “I guess that language is quite a handicap for the mentee.” ; “It has been a little [more] time consuming than expected. It was initially very difficult to create a balance between my routine residency work and the program.” However, other participants’ engagement was limited by personal challenges, network connection, language, and time: Figure 3 shows reported check-ins on each team. One team had high levels of communication between all members of the mentorship team. Other teams had varying levels of communication between participants and peer mentors, which typically mirrored participants’ deliverable completion rates. Significantly less than expected communication between peer mentors and faculty mentors was observed.Mentorship implementation by team was mixed. Figure 2) and three full team meetings. Another team implemented a tiered structure initially, but when the peer mentor had to stop at Module 8 for personal reasons, one of the faculty mentors became the primary mentor to the two participants. A third team reported using a more direct 1:1 mentoring relationship between each mentor and the participant. Two teams had little or no faculty mentor involvement in the mentorship structure.The team with highest mentorship communication rates also reported implementing the suggested tiered mentorship structure  While another peer mentor reported being guided in his mentorship approach by his faculty mentor:As the key themes shown in “[The faculty mentor] was able to answer any question I had. Like if you have two different participants, and one is behind, do you give more effort to the one who is behind or to both? I had a lot of questions like that (…) he gave me some really good advice.” (PRMH1)“Interaction with faculty mentors was too limited, positive/negative feedback would be appreciated with each deliverable.” ; “I commented on a few proposals of my mentee and phoned with him and the mentor in [country] and with the coordinator. But I feel very insufficient over my contribution.” The lack of involvement of the faculty mentors on some of the mentorship teams was noted by participants and faculty mentors alike: Across the entire program, participants completed an average of 5.5 of 10 curriculum deliverables and checked in with their peer mentors an average of 5.8 times over the 10 module time periods. Overall, four out of 10 participants, three out of five peer mentors, and three of 10 faculty mentors met the completion certificate requirements over the six months. Seven participants, four peer mentors, and eight faculty mentors expressed interest in continuing their mentorship relationships beyond the core program.There was limited cross-program engagement amongst participants or peer mentors despite setting up the dedicated participant and peer mentor WhatsApp groups and encouraging the use of the “comments” function in Google Classroom. During the program feedback group discussion, two peer mentors noted that they enjoyed hearing about others’ experiences and wished they had connected sooner. One participant commented that she was nervous to share her work and ask questions in the participant group forum:“One of the modules we had to write down our research questions [in the WhatsApp group]. I was very nervous (…) [about] the idea of having everybody reading and being like: ‘What was she thinking?’ (…) I think about three or so of us only posted our research questions, but I was very grateful because I was corrected (…) going forward, I think, as participants we do need to discuss and share ideas.” (PPH3)Figure 2). The initial anticipation was that the program would be completed in four months, with a mix of one and two-week modules. However, after module three, received feedback showed that many participants were struggling with the pace of the program. Therefore, “catch-up” periods seen in Figure 1 were added and all remaining modules were extended to two weeks, increasing the total program length to six months. It was initially a completion certificate requirement to participate in mentorship full team meetings. However, some teams experienced communication and scheduling difficulties since members came from numerous time-zones and some had inconsistent internet connectivity. Thus, this requirement was removed and teams were encouraged to meet in smaller groups or use email and/or WhatsApp for asynchronous communication as needed. Adaptability was highlighted as a strength of the pilot program: The program was adaptable which helped a lot; (it) was important to give all the participants leeway. (FRMH1)Adaptations were made during program implementation. Mentorship teams were originally designed to include two participants, peer mentors, and faculty mentors each, however, only five peer mentors joined the program and thus the mentorship structure was modified to include just one peer mentor per team A program completion celebration was held to virtually present ARC completion certificates and acknowledge the hard work by all, responsive to suggestions for building research culture by Mash, et al . A six-mI didn’t feel competent in my mentorship skills during the first period of the program.” A lack of confidence in mentoring others may have been another barrier to peer mentor recruitment. In addition, one of the mentors was mis-assigned as a peer mentor when she met the faculty mentor criteria by both training completion year and number of first-author publications. Also, two of the faculty mentors did not reach the first-author publication criteria. These oversights occurred because a method to systematically request and receive CVs from all mentors at the beginning of the program to cross-check self-report on application surveys was not in place. The resulting range of research experience among those assigned to the peer and faculty mentor role may have impacted team mentorship dynamics.It was difficult to recruit the intended number of peer mentors for an equal ratio of mentors and mentees; it was also a challenge to accurately assign mentors as a peer mentor or faculty mentor. One possible reason for low peer mentor adoption is the time-pressure experienced by early career FP researchers. A near-peer mentorship program in Uganda, in which masters students mentored undergraduate students, was limited by the near-peers’ heavy workload and conflicting priorities . One pee“There should be an indication for who calls the meeting between the peer mentors and faculty mentors.” .The tiered mentorship team structure was designed to capitalize on peer-mentorships, which can flatten hierarchies  and exteAdditionally, the participants were the target of the ARC program and thus the primary focus of peer mentor and participant relationship was clear: to check in each module and support the participant’s research protocol development. The primary focus of the peer mentor and faculty mentor relationship, however, was a broader goal of improving peer mentor’s mentorship skills. While peer mentors were encouraged at orientation to set learning goals and complete a mentorship reflection tool , it was Suggestions for improvement in the future include (…) having more defined guidelines for team functioning, when to reach out, how much to reach out, how much faculty mentor feedback is desired or “enough.” (FRMU2)okay to demonstrate that you don’t know something.” (FRMH1)Participants and mentors largely limited mentorship interactions to their teams and did not utilize platforms to engage horizontally across the program. An evaluation of an online learning platform for postgraduate trainees in Germany reported that a perceived lack of involvement by other users can become a negative feedback loop . NormaliThe pilot study suggests a number of strategies that may improve upon reach, adoption, and implementation of online research training and mentorship programs for early career physicians in low-resource settings. Directly linking such programs to doctoral training or junior faculty development programs may not only provide increased opportunities for peer mentor recruitment, but also enhance the pipeline effect of these programs. The mentees are provided with an example to follow if they decide to pursue further training, and the early-career peer mentor gains skills in mentorship, which is equally crucial to building the pipeline . The ARC7The small size of this pilot, which was restricted by available resources, limits interpretation of our findings. Our recruitment methods did not allow for analysis of non-participants and non-adopters, a key component of the reach and adoption domains . Our menIt is both feasible and acceptable to conduct online research training and mentorship for trainee and early career FPs in sub-Saharan Africa. Similar programs must pay careful attention to mentorship training and provide a flexible yet clearly organized structure for mentee-mentor engagement. Future work is needed to determine optimal implementation strategies and to scale up efforts to develop primary health care research capacity in sub-Saharan Africa.


Smoking cessation helps extend a healthy life span and reduces medical expenses. However, the standard 12-week smoking cessation program in Japan has several notable problems. First, only 30% of participants complete this program. Second, participants may choose not to participate unless they have a strong motivation to quit smoking, such as health problems. Third, the program does not provide enough support during the period between clinical visits and after 12 weeks.This study examined the efficacy of the 24-week ascure program to address the problems of accessibility and continuous support. The program combines online mentoring, over-the-counter pharmacotherapy, and a smartphone app.Using a retrospective study design, we investigated data for 177 adult smokers who were enrolled in the ascure smoking cessation program between August 2017 and August 2018. The primary outcomes were continuous abstinence rates (CARs) during weeks 9-12 and weeks 21-24. To confirm smoking status, we performed salivary cotinine testing at weeks 12 and 24. We also evaluated the program adherence rate. Finally, we performed exploratory analysis to determine the factors associated with continuous abstinence at weeks 21-24 to provide insights for assisting with long-term continuous abstinence.The CARs of all participants for weeks 9-12 and weeks 21-24 were 48.6% (95% CI 41.2-56.0) and 47.5% (95% CI 40.0-54.8), respectively. Program adherence rates were relatively high throughout (72% at week 12 and 60% at week 24). In the analysis of the factors related to the CAR at weeks 21-24, the number of entries in the app’s digital diary and number of educational videos watched during the first 12 weeks were significant factors.The ascure program achieved favorable CARs, and participants showed high adherence. Proactive usage of the smartphone app may help contribute to smoking cessation success in the long-term. Smoking is a preventable cause of many health problems, such as cardiovascular diseases, respiratory diseases, and malignant tumors ,2. ThereThis standard smoking cessation program, however, has 3 major problems. First, only about 30% of participants who start the program complete it, often because of the burden of the clinical visits . Second,As one way to address the shortcomings of the 12-week program, the “ascure” smoking cessation program was created by CureApp Inc. Its features address some of the concerns related to the standard 12-week program: It is conducted remotely; uses a smartphone app, which is familiar to young people, to provide follow-up assistance; and supports participants for 24 weeks. It also provides professional online mentoring and offers nicotine replacement therapy  delivered to the patient’s home. The ascure smartphone app was developed based on research with the CureApp Smoking Cessation app -12. App Smoking cessation programs based on smartphone apps are rapidly increasing globally ,14. DespThis study retrospectively examined the clinical efficacy of the ascure smoking cessation program and its impact on continuous smoking cessation. We also used exploratory analysis to determine the factors associated with continuous abstinence at weeks 21-24 to gain insights for supporting long-term continuous abstinence.This was a retrospective study evaluating the efficacy of the ascure smoking cessation program. We conducted this study in compliance with the Declaration of Helsinki, and all other applicable laws and guidelines in Japan. All study procedures were reviewed and approved by the Kanazawa University Institutional Review Board (2019-023 (3058)).We assessed 177 adult smokers participating in the ascure smoking cessation program in Japan from August 2017 to August 2018. All participants had their own iOS or Android smartphones, desired to quit smoking, and were members of one of 16 corporate health insurance societies. Participants were recruited mostly via self-selection. Some became aware of this program through individual recommendations from professionals  at medical checkups. Advertisements by email, post, or leaflets distributed at hospitals were used for recruitment. We included participants who smoked >10 cigarettes per day and who agreed to the study’s privacy policy by providing written informed consent for data to be used in our analysis. We excluded participants who had difficulty using their smartphones according to the instructions or who were diagnosed with a mental illness.The primary outcomes of this study were participants’ CARs during weeks 9-12 and weeks 21-24. During the online video sessions at weeks 12 and 24, mentors asked participants whether they had smoked during the month prior to the current session and asked them to perform salivary cotinine testing using iScreen . The results of the test were confirmed visually via video. This allowed supplementation of the self-reported continuous abstinence via salivary cotinine testing. We also assessed adherence to the program based on how many weeks participants took part in the online sessions . Furthermore, we used exploratory analysis to determine the variable factors associated with smoking cessation success at week 24  to provide potential insights for maintaining long-term continuous abstinence.The ascure smoking cessation program is a 24-week completely remote, online program including 6 online sessions with the exclusive ascure smoking cession smartphone app. The app was developed by CureApp Inc . As noted earlier, the app’s contents were developed based on the findings of clinical studies using the CureApp Smoking Cessation app for patients diagnosed with nicotine dependence . The ascThe app see  compriseParticipants were encouraged to watch 1-3–minute video tutorials on nicotine dependence that provided useful tips on smoking cessation based on behavioral therapy. A total of 24 video tutorials were provided during the 24-week smoking cessation program.After being exposed to the behavioral therapy through the tutorials, participants could make a personalized to-do list for quitting smoking. For example, they could list alternative behaviors such as brushing their teeth or stretching when they craved a cigarette. When participants carried out the registered alternative behavior, they could record it in the digital diary provided in the app.The app instructed participants to keep a digital diary on their smoking cessation status, physical condition, medication use, and any adverse events, either by selecting from prepopulated options or by writing entries in free form.Whenever participants experienced cravings or withdrawal symptoms, they could tap the “call” button to send a message to a personalized chatbot. The chatbot immediately replied and provided personalized advice on how to deal with the symptoms. The chatbot also provided encouraging messages on smoking cessation to app users to remind the participants of their motivation to quit smoking.Throughout the 24-week program, 6-8 interactive online sessions with experienced mentors  were provided. Participants met with mentors via a web-based counseling system at each planned visit . During the online sessions, the mentors asked participants about their smoking status and any difficulties they encountered. Participants were also provided with guidance on overcoming psychological dependence. Each online mentor in the ascure program had more than 3 years of experience as a nurse, public health nurse, dietitian, or pharmacist. All instructors joined the Japan Society of Tobacco Control or The Japanese Association of Smoking Control Science and were qualified by either society before their initial mentoring session. In-house training was conducted for 1 month to learn the system for smoking cessation.As part of the program, participants received over-the-counter nicotine patches or nicotine gum as a smoking cessation aid, which was delivered via parcel from a pharmacy  to their homes. This nicotine replacement treatment was typically used until week 8.We collected basic information on each participant via the app, including their age, gender, years of smoking, number of cigarettes smoked per day, and screening of tobacco/nicotine dependence using methods such as the Tobacco Dependence Screener (TDS) score , FagerstP values. P values <3.0 x 10-3 (0.05 divided by 15) were considered to be significant. There were 36 missing values for the TDS score and 40 missing values each for the Fagerström Test for Nicotine Dependence and KTSND. To address these missing data in the explanatory variables, we used a multiple imputation method. We used the Pearson Chi-square test for subpopulation analysis. All calculations and analyses were performed using R software version 3.5.2 ; packages used were ‘data.table,’ ‘stats,’ and ‘mice’.Continuous variables are presented as mean (SD) or median (interquartile range [IQR]), depending on their distribution. Categorical variables are presented as the number (percentage). For the exploratory factor analysis of the predictors of CAR at week 24, we conducted multivariable logistic regression analysis, selecting from among the standardized variables , while adjusting for age, gender, and TDS score. In multiple testing, the Bonferroni method was used to adjust the Program adherence was 71.8% (127/177) at week 12 and 59.9% (106/177) at week 24. Within the first 4 weeks of the program, 23 people did not book the subsequent online mentoring sessions, and 11 and 16 people, respectively, lost contact with their mentors during the week 4 and week 8 sessions. The biochemically validated CAR during weeks 9-12 was 48.6%  when counting dropout as a failure. Even within the group of 106 people who completed all 24 weeks, the subgroup who wrote more diary entries than the median number (62.5 times) had a higher success rate  compared with those who did not .Moreover, we calculated the CARs at weeks 21-24 separately by subgroups of the number of days that participants wrote in their diaries. Participants who wrote diary entries more than the median number  during the first half of the program achieved higher CARs  than those who did not . However, there was no significant difference between success or failure among people who completed all 24 weeks .Participants who watched educational videos more than the median number  also had significantly higher CARs  when counting dropout as a failure  of smoking cessation outpatients completed the standard 12-week treatment, ascure participants had a higher program adherence rate of 71.8% (127/177) at week 12 and 59.9% (106/177) even at week 24.Although the efficacy of the 2 programs cannot be compared directly, both programs measured efficacy with a 1-month CAR at weeks 9-12. In the case of the outpatient service, the self-reported 1-month continuous abstinence was confirmed by exhaled carbon monoxide concentration (<8 ppm) at the fifth clinical visit.Among the participants continuing the ascure program until week 12, the CAR at week 12 was 67.7% , whereas the CAR among individuals who completed the 12-week outpatient program was 82.1% (320/390) [This study evaluated the efficacy of the ascure smoking cessation program, which is characterized by online mentoring, a smartphone app, over-the-counter medications, and an extended follow-up period (24 weeks). First, we found that the CAR was 48.6% during weeks 9-12 and 47.5% during weeks 21-24. We also observed high adherence rates: 72% at week 12 and 60% at week 24. Unlike previous reports [This study has 3 important findings. First, the ascure smoking cessation program achieved moderately favorable CARs in comparison with the current standard Japanese outpatient program, mainly due to the adherence rate. While only about 30% of participants completed the outpatient program , the comSecond, proactive use of the smartphone app  during the first 12 weeks of the program was significantly associated with CARs during weeks 21-24. Keeping a smoking cessation diary is recommended by various Japanese academic societies  . This stThird, the ascure program seemed to attract more relatively young, female participants with low dependence, as represented by the Brinkman index. This may imply that, because of the completely remote aspect of the ascure program, it could recruit people who do not utilize the outpatient service voluntarily.This study was the first to examine the efficacy of a novel smoking cessation program for members of a Japanese health insurance society. We used a biochemically validated smoking status test to assess the efficacy of the program. However, this study has several limitations. First, the absence of a control group is a significant problem when assessing the efficacy of the program. Our finding that a predictor of smoking cessation was the intensive use of the program could merely reflect that motivated patients made more intensive use of the program. Future studies, such as crossover trials with a control group, are necessary to better evaluate the efficacy of the program. Second, several factors could limit the generalizability of the findings: participants’ level of motivation to engage in the program, participants’ sociodemographic status , and access to technology . Third, we evaluated 1-month continuous abstinence (as does the outpatient service in Japan). This limits the comparability with internet-based programs in other countries. Fourth, data on adherence to nicotine patches and the quality of video counseling, which might affect outcomes, were not collected. Finally, it may be necessary to evaluate smoking status even further into the future, such as at week 52 or beyond, to confirm the long-term efficacy of this program.The completely remote ascure smoking cessation program achieved favorable smoking cessation success rates while improving accessibility and adherence to a cessation program. Moreover, proactive use of the smartphone app may have contributed to successful smoking cessation over a relatively long period. The combination of internet-based counseling, a smartphone app, and over-the-counter medications might be a viable solution for long-term smoking cessation.


Mindfulness-based programs are a novel and promising approach for supporting teachers’ occupational health and well-being. Although rationales for mindfulness programs for teachers have been offered, the empirical research base evaluating approaches for educating teachers in mindfulness is still developing. This study reports the findings of a pilot study of a mindfulness-based program. This study is unique in that it is one of the only studies of the Mindfulness-Based Emotional Balance (MBEB) program to focus on early elementary teachers, to be implemented by a new instructor, and to recruit teachers via extrinsic motivators.A pre-post, uncontrolled pilot study of a 27.5-h mindfulness-based program for teachers was conducted with 21 pre-kindergarten–third-grade teachers from the Pacific Northwest of the USA. Program acceptability was assessed based on attendance and teacher reports of program benefits. Effect sizes for within-person changes (from pre- to post-program) in teachers’ skills and mindsets, well-being, occupational health, and teaching practices were calculated. Teachers also suggested improvements to the program.d| = 0.30 to 0.83, and ranged from small to medium for changes in teachers’ well-being |d| = 0.07 to 0.48, occupational health |d| = 0.14 to 0.39, and teaching practices |d| = 0.15 to 0.48. Teachers suggested shortening the program and linking it more closely to their work in the classroom.With regard to program attendance and acceptability, teachers attended 87% of sessions, with 58% of teachers reporting a personal benefit and 58% of teachers reporting a professional benefit of the program. Effect sizes for changes in teachers’ skills and mindsets ranged from small to large, |This study suggests that the MBEB program may be beneficial to early elementary teachers, even when implemented by someone other than the program developer, and when provided with extrinsic motivation to participate . Teachers’ suggestions regarding program length and structure are considered, along with useful avenues for future research on mindfulness-based programs for teachers. What uncertainties existed regarding feasibility? The feasibility of the Mindfulness-Based Emotional Balance (MBEB) program when implemented for early elementary educators was unknown. It was also unclear how extrinsic motivation and implementation by someone other than the program developer might impact feasibility.What are the key feasibility findings? Teachers attended 87% of sessions; 58% of teachers reported personal and professional benefits of the program. Effect sizes on program outcomes ranged from small to large |d| = 0.07 to 0.83, and teachers offered suggestions for adapting the program.What are the implications of the feasibility findings for the design of the main study? Findings suggest that the program can be offered to a wide array of teachers, that those trained by the program developer can competently implement the program, and that compensating participants may not have deleterious impacts on feasibility or program outcomes.Teachers’ occupational health and well-being are important for their overall quality of life, as well as for the well-being of their classes and students , 38. MinThe present study contributes to this growing body of literature by presenting the results from a pre-post, uncontrolled pilot study of the Mindfulness-Based Emotional Balance (MBEB) program for early elementary teachers. This study aimed to (1) assess the program’s acceptability; (2) assess changes in teachers’ skills and mindsets , well-being , occupational health , and classroom teaching practices  over the course of the program; and (3) explore teachers’ suggestions for improvements to the program.The logic model for the MBEB program is depicted in Fig. Previous studies of mindfulness-based programs for teachers have shown them to be efficacious in fostering change in  mindfulness skills and a self-compassionate mindset , 27, 45.Of particular interest in this study is teachers’ use of two emotion regulation strategies: cognitive reappraisal and suppression of emotional expression. Cognitive reappraisal involves a re-conceptualization of a potentially emotion-eliciting situation in a more positive or less emotional way. It is a healthy regulation strategy which is associated with higher levels of positive affect and lower levels of negative affect, depressive, and anxiety symptoms . ExpressWell-being has been conceptualized as the presence of pleasant  and the absence of unpleasant  emotional experiences . ThroughTeachers’ occupational health has been conceptualized as the presence of pleasant  and absence of unpleasant  emotional experiences in the work context , 30. It Mindfulness skills are theorized to improve teachers’ practices in part through the adoption of an attitude of kindness towards oneself and others , which mThe current study focuses on the 8-week, 9-session version of the MBEB program, which met after school for a total of 27.5 h . In compThe topics covered in each session of MBEB are detailed in Additional file One important note about the MBEB program in relation to other mindfulness-based programs is that the MBEB program does not aim to scaffold and transfer program-related skills specifically to the classroom context, but rather invites teachers to explore and use these skills in any area of their lives that they wish. This could include their work context, but it need not . In onlTo date, the MBEB program has been tested on parents and teachers of elementary-high school students with special needs, and elementary and secondary teachers. The program has demonstrated preliminary positive effects on participants’ skills and mindsets, well-being, and teachers’ occupational health , 45. ThePilot studies are a useful step in the research process which help to determine the feasibility and acceptability of a program and to explore possible population-specific adaptations , 8, 55. 1: To what extent did teachers attend and report benefitting from the program?RQ2: Did teachers’ skills and mindsets, well-being, occupational health, and teaching practices improve across the program?RQ3: How could the program be adapted to better suit the needs of early elementary teachers?RQWe hypothesized that teachers would experience improvements in their skills and mindsets, well-being, occupational health, and teaching practices over the course of the program. Due to the novelty of implementing this program with early elementary teachers, the investigation into teachers’ acceptability and suggested adaptations were exploratory and meant to inform future iterations of the program.The present study employed a pre-post, no-control group design , 39, 54.Data were collected at two time points: at the start of the school year (prior to the program) and at the end of the school year (after the program). Teachers completed an online survey containing questions regarding their demographics, skills and mindsets, well-being, and occupational health at each assessment. At each time point, teachers were videotaped in their classrooms on 3 days for 20 min. At post-program, teachers also answered questions regarding the MBEB program in an online survey.The study drew teachers from two elementary schools with linked preschool sites whose principals/leaders, with district support, were interested in introducing mindfulness and social-emotional learning to their teachers and students. Schools were designated as Title 1 and served primarily economically disadvantaged families and children. In recent years, the schools increasingly served immigrant families from Russia and Mexico, although all teachers were European-American. The teachers in this study were encouraged by their principal to participate but could opt in or out of the program. Participating teachers were paid for the time they devoted to the program. Thus, unlike other studies of MBEB in which all teachers were self-selected, the motivation for teachers to participate in this study included encouragement from school leadership and financial payment for time. Participating teachers provided their informed consent.M = 12 years, SD = 8); were 86% female, 100% Caucasian; and 71% had a post-baccalaureate diploma or graduate degree. In terms of grade, 10% were pre-kindergarten teachers (N = 2), 14% kindergarten teachers (N = 3), 28% first-grade teachers (N = 6), 10% second-grade teachers (N = 2), and 38% third-grade teachers (N = 8).As teachers’ experiences at pre-program did not differ by site, data were collapsed across the two sites/years of the study. Teachers had a range of teaching experience . Teachers were also asked “Would you recommend this program to your peers who are teachers?” Responses were Yes, No, and Unsure.The number of teachers who enrolled in the study and participated at each time point was recorded. Teachers self-reported on their mindfulness skills, occupational self-compassion, and emotion regulation skills.Mindfulness skills were measured using 38 items from the Five Factor Mindfulness Questionnaire [αpre = .92, αpost = .91). Items were scored on a 1 to 5 scale . Items included “When I’m walking, I deliberately notice the sensations of my body moving” (observing), “I’m good at finding words to describe my feelings” (describing), “When I do things, my mind wanders off and I’m easily distracted” (active awareness), “I tell myself I shouldn’t be feeling the way I’m feeling” (nonjudgement), and “In difficult situations, I can pause without immediately reacting” (nonreactivity). Items were scored so higher values indicate greater mindful skills.ionnaire , which aSelf-compassion was measured using Roeser et al.’s [αpre = .85, αpost = .78). The scale assesses teachers’ extension to compassion to themselves in the context of their teacher role. Items were scored on a 1 to 5 scale .et al.’s  adaptatiet al.’s  self-comCognitive reappraisal was measured using six items from the Emotion Regulation Questionnaire . Items were scored on a 1 to 5 scale .onnaire . Items were scored on a 1 to 5 scale .onnaire .Life satisfaction was assessed using a single item. “Taking all things together, how satisfied are you with your life as a whole these days?” Items were scored on 1 to 4 scale .Anxiety symptoms were measured using six items from the State/Trait Anxiety Inventory . Items were scored on a 1 to 4 scale .ventory . Teachers were prompted about a topic and selected the option (1 to 4) that most accurately described their current state. For example, one item prompted about sleep habits; options included the following: I can sleep as well as usual (1), I don’t sleep as well as I used to (2), I wake up 1-2 hours earlier than usual and find it hard to get back to sleep (3), and I wake up several hours earlier than usual and cannot get back to sleep (4). Higher values indicate greater depressive symptoms.ventory . Items were scored on a 1 to 7 scale ; higher values indicate stronger feelings of burnout.ventory . Items were scored on a 1 to 7 scale ; higher values indicate stronger feelings of burnout.ventory . Items were scored on a 1 to 7 scale ; higher values indicate stronger feelings of burnout.ventory . Items were scored on a 1 to 5 scale .e items . Items were scored on a 1 to 5 scale .g Scale : positive climate, negative climate, sensitivity, regard for student perspective, behavior management, productivity, and instructional learning formats. Approximately 35% of these videos were independently rated by two observers, inter-rater reliability > .85. Consistent with our logic model [Emotional support reflects teachers’ positive and negative classroom climate, their sensitivity to students, and regard for student perspective, including supporting student autonomy, ideas, and peer interactions. Classroom organization reflects teachers’ clear, proactive management of student behavior, productive use of time, and employment of a variety of learning formats that encourage student engagement. Observer scores for each time point were averaged such that each teacher received a score for observed emotional support and classroom organization at pre-program and at post-program.m . At eaic model , scales Much too long; 5 = Much too short). The second was an open-ended question, “What suggestions, if any, do you have for the instructor for making this program better in the future?”Teachers were asked two questions about the program. The first, “How do you feel about the length of the program?” was scored on a 1 to 5 scale . The proportion of teachers who would recommend the program to fellow teachers was also determined.d and correcting for dependence between means d and cored large , 49. ConThe average response regarding program length was calculated. Teachers’ free responses regarding their suggestions were coded using qualitative content analysis . Each reDuring the first year of the project, 15 teachers from site 1 enrolled in the program; two teachers withdrew after the initial wave of data collection, but prior to program participation Fig. . During Attendance for the 21 participating teachers was generally high, with 76% of participating teachers missing one or no sessions. On average, teachers attended 87% of sessions. Of the missed sessions, 31% were planned absences, 17% were sickness-related, and 52% were unannounced.Teachers reported on the personal benefit of the program, with 58% of teachers reporting that they benefitted “a moderate amount” or higher. Teachers also reported on the professional benefit of the program, with 58% of teachers reporting that they benefitted “a moderate amount” or higher. Half of the teachers reported that they would recommend the program to fellow teachers, 33% were unsure, and 16% reported that they would not recommend the program.d| = 0.30 to 0.83 and ranged from small to medium for changes in teachers’ well-being |d| = 0.07 to 0.48, occupational health |d| = 0.14 to 0.39, and teaching practices |d| = 0.15 to 0.48.Effect sizes for within-person change in each outcome between pre-program and post-program are displayed in Fig. Regarding program length, the average response was 2.33, indicating that most teachers reported that the program was “a little too long.” With regard to teachers’ suggestions, of the 12 teachers who responded to the post-program survey, half did not provide a response to this question. Responses for the six teachers who responded were coded thematically using qualitative content analysis. Three codes emerged: positive comments or responses suggesting no changes; suggestions to streamline the program either through shortening the sessions or limiting repetition of content; and suggestions for explicit instruction regarding the application of program content to teaching. Of the six respondents, three had positive comments or suggested no changes. Two teachers suggested streamlining the program, and two suggested explicit application of program content to teaching.Results from this pilot study demonstrate that the MBEB program can be implemented with, and have positive effects for, early elementary school teachers. This study adds three new directions to the existing research on the feasibility and success of the MBEB program: (1) the application of this program to early elementary teachers, (2) a test of program effects upon implementation by a new instructor, and (3) a test of program impacts when teachers were extrinsically motivated  to participate. Under these novel conditions for program implementation, teachers reported improvements over the course of the program in all domains assessed. These findings suggest that the program may continue to have positive benefits when it is offered to a wider array of teachers, that those trained by the program developer can competently implement the program, and that compensating participants may not have deleterious impacts on feasibility or program outcomes. These findings have important implications for the scale-up and dissemination of the MBEB program, together suggesting that the MBEB program holds promise for school-wide implementation. This study adds to the growing body of literature on mindfulness-based programs for teachers and the promise of such programming for promoting positive change for teachers , 25, 45.and result in greater improvements for all teachers’ occupational health and teaching practices.Teachers’ feedback suggested two specific directions in which to adapt the program. First, analyses of teachers’ free responses suggested that some teachers wanted more scaffolding with regard to transferring mindfulness skills to the classroom, despite the program being presented as non-instrumental. This feedback suggests that the program may not have met some teachers “where they were” in terms of their wanting to work on mindfulness primarily for classroom change. Balancing the capacity of the program to meet teachers’ personal and professional needs will be useful in developing future iterations of the program. For example, creating time at the end of each session for tying the content to everyday challenges and dilemmas of teaching practice may better suit the needs of certain teachers Teachers’ feedback also suggested that the program was a bit too long, which brings to light the complex interplay between dosage, duration, and culturally adapted programming, which the field of mindfulness-based programs has yet to fully explore. A key challenge of prevention science is balancing the development of evidence-based prevention programs that can be implemented with fidelity, while also catering to the needs and wants of those the program aims to support , 9. For and cultivate flourishing [A strength of this study was its assessment of both pleasant and unpleasant feelings. With most research on teachers' occupational health and well-being focused on the reduction of unpleasant emotions like stress and burnout e.g., , 48, 51), 5148, 5urishing .This pilot study has several limitations, including a reliance primarily on teacher self-report measures, and its focus on a small, racially homogenous sample. In addition, the processes through which these effects were inferred were not directly tested. This is a useful area of research that would contribute to our understanding of how effects are achieved and could inform program refinements that target such pathways. There is much still to be learned about the MBEB program for teachers and about the feasibility of mindfulness-based programs more generally. This pilot study brought to light several considerations that warrant further exploration, including the best approach to teaching mindfulness skills , finding the “correct” dosage, and whether such programs are suitable for a diverse group of teachers.The present study addresses calls to understand how teachers’ social and emotional skills, well-being, occupational health, and teaching practices may be improved through novel means like mindfulness training . ResultAdditional file 1. MBEB Program Sessions and Topics.


Arising from H. Mergental et al. Nature Communications 10.1038/s41467-020-16251-3 (2020)1. Of 31 initially discarded livers, 22 (71%) met the viability criteria during NMP and were transplanted with a 100% 90-day graft survival; however, three out of ten livers donated after circulatory death (DCD) developed biliary complications (30%) and required retransplantation. Because the trial was already initiated in 2016, more recent insights in viability testing should be considered when discussing the design and results of this clinical trial. Therefore, there are three issues that needs commenting to interpret the high rate of biliary complications reported: (1) the viability criteria used in this trial, (2) production of bile during NMP and (3) the use of end-ischemic NMP alone.The results from the VITTAL study, with back-to-base end-ischemic normothermic machine perfusion (NMP) for viability testing of high-risk donor livers, were eagerly awaited as in this field different techniques are currently being used1. The first reports on the value of bile composition as a tool to determine bile duct viability were published after the initiation of this study3. In our experience, a substantial number of especially high-risk DCD grafts do not meet the cholangiocellular viability criteria during NMP (Table 4. Transplanting high-risk (DCD) donor livers without monitoring bile composition during NMP poses a risk to the recipient, as other investigators have previously shown a high rate of post-transplant cholangiopathy after end-ischemic NMP of high-risk DCD livers5. Therefore, with the current knowledge it is not a complete surprise that Mergental et al. have noted a high incidence of post-transplant cholangiopathy (30% after DCD transplantation) and need for retransplantation in their series1.In contrast to hypothermic <12 °C) machine perfusion (HMP), livers are metabolically fully active during NMP. This leads to restoration of hepatobiliary metabolism, such as clearance of lactate, production of (coagulation) proteins, and, importantly, bile production. Two types of viability criteria for donor livers during NMP have been distinguished: hepatocellular (‘liver parenchyma’) and cholangiocellular (‘bile duct’) viability criteria , we have monitored bile composition 1. In our experience, we have always been able to explain low or absent bile flow during NMP by a technical issue with the biliary drain. Regularly, the tip of the biliary drain can be stuck against the bile duct wall, leading to obstruction of the lumen and limiting bile flow. We have tested biliary drains extensively in preclinical experiments, and observed that bile flow initiates more rapidly when a small diameter drain is used, as a result of increased capillary force. As a result, we have been using a small feeding tube catheter (8 French) with an open tip and side holes as a biliary drain9.In the study by Mergental et al., 23% of the livers did not appear to produce bile1. Since the VITTAL study was initiated in 2016, other centers shared similar experiences showing high percentages of biliary complications after NMP for high-risk (DCD) donor livers5. End-ischemic NMP exposes a donor organ to ischemia-reperfusion injury, to which the bile ducts are extremely sensible. It has been shown before that a short period of dual hypothermic oxygenated machine perfusion (DHOPE) reduces reperfusion injury of the liver and biliary tree10. Several studies have demonstrated a beneficial effect of performing DHOPE prior to either direct transplantation or NMP15. After combined DHOPE and NMP, hepatic adenosine triphosphate content and biliary bicarbonate and bilirubin were higher in comparison to livers that underwent NMP only15. As a result, in 2017, the DHOPE-COR-NMP trial was initiated with a combination of one-hour DHOPE, followed by one-hour controlled oxygenated rewarming, and subsequent NMP, with the aim to reduce ischemia-reperfusion injury at the start of NMP8. Interestingly, all livers in the DHOPE-COR-NMP trial met the hepatocellular viability criteria used in the VITTAL study, despite the fact that all livers were derived from high-risk DCD donors with a substantially higher median age, compared to the VITTAL study8. This could suggest that a short period of DHOPE prior to NMP is very effective in preventing additional reperfusion injury of the liver parenchyma (hepatocytes) at the start of NMP. Only livers that have suffered too much (warm) ischemic injury of the bile ducts during and after the DCD procedure may no longer be salvageable with DHOPE and will not meet the biliary viability criteria during the NMP phase.In the study by Mergental et al., it was chosen to perform end-ischemic NMP onlyIn conclusion, in a rapidly advancing field of liver machine perfusion, we are all still learning and it is through sharing of our experiences that we can collective determine the most optimal use of the new technology in the benefit or our patients. From the emerging evidence, it becomes clear that end-ischemic NMP is an important tool to assess and select donor livers that, based on traditional criteria, were initially considered not suitable for transplantation. Yet, end-ischemic NMP alone, as applied in the VITTAL study, does not protect the bile ducts against ischemia-reperfusion injury. Therefore, a short period of HMP prior the NMP, as well as the use of cholangiocellular viability criteria during machine perfusion, are important modifications that may help avoid post-transplant morbidity and graft loss due to cholangiopathy.