Four patients (19%) remained ctDNA‐positive at 1–2 weeks after surgery, with all of them (100%) experiencing disease progression at later time points. In contrast, only 4 out of 12 ctDNA‐negative patients (33%) after surgery experienced relapse during follow‐up. Positive ctDNA in early postoperative plasma samples was associated with shorter progression‐free survival (P = 0.013) and overall survival (P = 0.004). Our findings suggest that, in early‐stage and locally advanced NSCLC, intraoperative plasma sampling results in high ctDNA detection rates and that ctDNA positivity early after resection identifies patients at risk for relapse.Circulating tumor DNA (ctDNA) has demonstrated great potential as a noninvasive biomarker to assess minimal residual disease (MRD) and profile tumor genotypes in patients with non‐small‐cell lung cancer (NSCLC). However, little is known about its dynamics during and after tumor resection, or its potential for predicting clinical outcomes. Here, we applied a targeted‐capture high‐throughput sequencing approach to profile ctDNA at various disease milestones and assessed its predictive value in patients with early‐stage and locally advanced NSCLC. We prospectively enrolled 33 consecutive patients with stage IA to IIIB NSCLC undergoing curative‐intent tumor resection (median follow‐up: 26.2 months). From 21 patients, we serially collected 96 plasma samples before surgery, during surgery, 1–2 weeks postsurgery, and during follow‐up. Deep next‐generation sequencing using unique molecular identifiers was performed to identify and quantify tumor‐specific mutations in ctDNA. Twelve patients (57%) had detectable mutations in ctDNA before tumor resection. Both ctDNA detection rates and ctDNA concentrations were significantly higher in plasma obtained during surgery compared with presurgical specimens (57% versus 19% ctDNA detection rate, and 12.47 versus 6.64 ng·mL ctDNA profiling in plasma revealed higher levels and detection rates of ctDNA during tumor resection as compared to pretreatment time points in patients with early‐stage or locally advanced NSCLC. Moreover, patients testing positive for ctDNA immediately after tumor resection had worse clinical outcomes than patients with undetectable ctDNA. Our research highlights the role of ctDNA assessment for guiding treatment in patients with respectable NSCLC. AFallele frequencyBWABurrows–Wheeler alignercfDNAcell‐free DNActDNAcirculating tumor DNAFFPEformalin‐fixed paraffin‐embeddedkbkilobasesMRDminimal residual diseaseNGSnext‐generation sequencingNSCLCnon‐small‐cell lung cancerOSoverall survivalPCRpolymerase chain reactionPDWBplasma‐depleted whole bloodPFSprogression‐free survivalqPCRquantitative polymerase chain reactionRECISTresponse evaluation criteria in solid tumorsSNPsingle nucleotide polymorphismSNVsingle nucleotide variant1Lung cancer is the leading cause of cancer‐associated death worldwide , with 80Profiling of circulating tumor DNA (ctDNA) in the blood plasma of cancer patients has shown great potential as a noninvasive biomarker for tumor genotyping and classification, monitoring of minimal residual disease (MRD), and detection of clonal heterogeneity over time , 12, 13.In general, pretreatment ctDNA detection rates in early‐stage and locally advanced NSCLC were substantially lower than in advanced metastasized stage IV lung cancer, mainly due to lower tumor sizes and minimal shedding of DNA molecules into the bloodstream , 17. ManHere, we present the results of a prospective study in resectable stage I‐III NSCLC. We profiled ctDNA before, during, and after tumor resection to evaluate the impact of intraoperative procedures on ctDNA characteristics and to assess its value as a biomarker for early outcome prediction.22.1Patients with resectable early‐stage or locally advanced NSCLC (stage I–III) qualified for inclusion into this trial (DRKS00009521). From October 2014 to June 2018, a total of 33 patients aged ≥ 18 years were enrolled at the University Medical Center Freiburg, Germany. From 21 patients, both tumor biopsies and serial blood plasma samples were available for further analyses Fig. . Additio2.2Blood samples from 21 NSCLC patients were collected presurgery at enrollment, during surgery from the jugular vein through a central venous catheter immediately after pulmonary vein clamping and before tumor resection , 1–2 weeks after surgery , and during follow‐up every 3 months  was stored at −80 °C.Blood plasma was separated from the cellular blood compartment by centrifugation at 800 Cell‐free DNA (cfDNA)‐isolation from blood plasma was performed using the QIAsymphony and the QIAmp Circulating Nucleic Acid Kit  according to the manufacturer´s instructions. Quality and quantity of the isolated cfDNA were assessed by Fragment Analyzer  and Qubit 2.0 fluorometer . Isolated cfDNA was then stored at −20 °C.n = 21) was isolated from formalin‐fixed paraffin‐embedded (FFPE) tissue specimens after microdissection of the tumor areas using the GeneRead DNA FFPE Kit . DNA quality and quantity of these samples were assessed as described above.DNA from primary tumor resection specimens (4Cl solution (8.4 g NH4Cl soluted in 1 L H2O) and incubated for 20 min at room temperature, followed by one centrifugation step for 10 min at 800 g. Lysed cells were then washed twice with PBS and lysis was completed by the addition of RLT+‐buffer  and homogenization with a syringe. Cell lysates were stored at −80 °C until further use. Cellular DNA from the lysates was isolated using the QIAsymphony and the ‘QIASymphony DSP DNA Mini Kit 192 version 1’ according to the manufacturer`s protocol and stored at −20 °C. In cases with no available PDWB, cellular DNA for SNP identification was obtained from segments of FFPE tissues with no evidence of histological tumor infiltration. Cellular DNA from these parts was isolated as described above, using the GeneRead DNA FFPE Kit .Cellular DNA from PDWB was used as germline control to identify single nucleotide polymorphisms (SNP) in all patients. To isolate cellular DNA from PDWB, cells were lysed by the addition of an NH2.3https://cancer.sanger.ac.uk/cosmic): BRAF , CDKN2A , CTNNB1 (exon 3), EGFR , HER2/ERBB (exon 20), HRAS , KEAP1 , KIT , KRAS , MET (exon 14), NFE2L2 (exon 2), NRAS , PDGFRA (exon 18), PIK3CA , PTEN , SDHA , SMAD4 , TP53   was sheared prior to library preparation by Covaris , using the following settings: 180 s with a duty factor of 10%, a peak incident power (W) of 175, and 200 cycles/burst. Next‐generation sequencing (NGS) libraries were generated from both cellular sheared DNA and cfDNA with the ThruPlex Tag‐Seq 48S Kit , using molecular barcodes  according to the manufacturer's instructions. In median, 17.2 ng (range: 6.2–59.4 ng) of cellular tumor DNA, 50 ng (range: 1.74–50 ng) of cellular DNA from PDWB, and 13.2 ng (range: 0.6–90 ng) of cfDNA were used for library preparation. After generating NGS libraries, target enrichment with complementary probes was performed using a customized targeted‐capture panel , according to the manufacturer's instructions. The following 18 commonly mutated genes in NSCLC were selected for the customized sequencing panel (17 kb), based on the COSMIC database (0) Table . Quantif2.4https://github.com/umich‐brcf‐bioinf/Connor), applying a consensus frequency threshold of 90% and a family size threshold of 10. Median deduplication rate was 91%. Variant calling of single nucleotide variants (SNVs) was performed using the software Sequence Pilot . SNPs were identified by sequencing each patient’s cellular DNA and discarded for further analyses.Paired‐end reads were mapped to the hg19 reference genome (GRCh37v28/hg19) with Burrows–Wheeler aligner (BWA) version 0.7.17 (default parameters), allowing soft‐clipping, and sorted and indexed with SAMtools. Deduplication was performed using Connor (n = 126 tests). At a threshold of 0.001% mean AF and a threshold of ≥ 18% for tumor genotyping, a false‐positive rate of 5% was observed  was defined for tumor genotyping based on the false‐positive rate in plasma samples from healthy controls (see below). SNVs passing this threshold were used for ctDNA monitoring analyses in blood plasma. Here, plasmas samples were considered ctDNA‐positive if the following rules applied: (a) detection of at least one predefined SNV from tumor genotyping; (b) a mean AF of at least 0.001% for the plasma sample (see ‘Statistics’ for definition of ‘mean AF’). This detection limit of 0.001% was established as follows: First, spike‐in experiments using artificial mutant DNA  revealed robust detection of SNVs above an AF of 0.1% Fig. A. We theved Fig. B.2.5Mutant allele frequencies were expressed as percentage (%) and set to the number of reads harboring a given variant divided by the total number of reads at the same genomic position. The AF of total ctDNA was calculated as the mean AFs of all tumor‐derived mutations detected by monitoring in blood plasma.graphpad version 8.0 software . To compare continuous data distributions, we used the nonparametric Mann–Whitney U‐test. Fisher’s exact test was applied to compare proportions of two nominal variables. For Kaplan–Meier analyses, log‐rank tests were used to statistically evaluate survival differences between two groups. For clinical outcome analyses, progression‐free survival (PFS) was considered the primary endpoint, defined as time from blood sample collection to any of the following: disease progression as defined by RECIST 1.1‐based radiographic assessment, death from any cause or last follow‐up visit. Overall survival (OS) was considered as secondary endpoint and was defined as the time from blood sample collection to death from any cause or last follow‐up. P‐values < 0.05 were considered as significant.Further statistical analyses were performed using 3n = 18/21, 85.7%). The two most frequent histopathology subtypes were squamous cell carcinoma (47.6%) and adenocarcinoma (23.8%). All patients underwent curative‐intent tumor resection that was followed by either adjuvant chemotherapy (n = 9), radiotherapy (n = 2), combined chemoradiation (n = 2), or no adjuvant treatment , followed by TP53 (67%), EGFR, and PDGFRA  with those obtained during surgery after manual lung and tumor palpation and immediately before tumor resection (n = 19). Samples collected during surgery had higher cfDNA concentrations , contralateral lung (n = 1), and liver (n = 1). Furthermore, MRD negativity was significantly associated with both favorable PFS  and KEAP1 (S338STOP) in one single plasma sample during follow‐up without any signs of radiological disease progression, indicating false‐positive ctDNA identification  and plasma samples (n = 96) from major disease landmarks to characterize the role of ctDNA as a noninvasive biomarker in this patient population. We demonstrate that ctDNA can be identified in 57% of patients, with higher detection rates in locally advanced stage III compared to early‐stage NSCLC (stage I‐II), in accordance with previous studies [The prognosis of stage I‐III NSCLC patients experiencing disease relapse after curative‐intent therapy is poor. Accurate biomarkers to identify patients at high risk for NSCLC recurrence are lacking. To address these challenges, we applied a custom‐targeted‐capture high‐throughput sequencing approach to prospectively collected tumor biopsies , almost two‐thirds of patients with negative MRD remained cancer‐free. Despite the rather small sample size of our study, these results, together with similar findings from other previously reported studies, suggest that ctDNA could potentially serve as a specific marker for relapse prediction and thus could guide adjuvant treatment decisions in the postoperative setting [et al. [Our second major finding was that ctDNA positivity 1–2 weeks after tumor resection potentially identified patients with a particularly high risk of future NSCLC relapse. While 100% of patients with detectable MRD early after surgery experienced disease progression  for MRD assessment . Howeveret al. (125 kb and 203 kb panels) [However, our study harbors some limitations. First, the sample size of our prospective cohort is rather small and larger studies are needed to validate the findings of this work. Furthermore, sensitivity of our sequencing approach could be increased by an alternative panel design. Our panel captures 18 NSCLC‐specific genomic regions and covers 17 kb of the genome. Yet, it has been shown that tracking a larger number of mutations per patient improves detection rates in solid cancers, especially in NSCLC , 28. Thi panels) , 29, 30.5In conclusion, our work highlights the advantages of ctDNA as a noninvasive biomarker in early‐stage and locally advanced NSCLC. Our findings suggest that intraoperative manual manipulation of the tumor leads to an increase of ctDNA in the blood and an increase of ctDNA detection rates. We further show that ctDNA detection early after tumor resection identifies patients at risk for relapse, indicating a potential future role as MRD marker for guiding adjuvant systemic treatment.F.S. received research funding from Roche Sequencing Solutions. S.L. received Advisory Board and/or scientific meeting/presentation sponsorship; Agilent, AstraZeneca, Illumina, Novartis, Roche, as well as research project sponsorship Bristol–Myers–Squibb. All other authors declare no conflict of interest.NvB, FS, and SWa involved in conceptualization; SWa, JaMi, JuMu, HB, JW, FS, and NvB involved in methodology; JaMi, SWa, GA, and MB involved in software preparation; SWa validated the data; SWa, JaMi, MR, GA, MD, MJ, UP, JW, and FS involved in formal analysis; SWa involved in investigation; SWi, A‐ML, DK, SS, LT, CZ, SL, PB, CG, JR, HB, LI, AM, SD, DP, BP, and JD contributed to resources; SWa and JM contributed to data curation; SWa, FS, and NvB. contributed to writing—original draft preparation; all authors contributed to writing—review and editing; SWa contributed to visualization; FS and NvB contributed to supervision; SWa contributed to project administration; FS and NvB acquired funding. All authors have read and agreed to the published version of the manuscript.Fig. S1. Flow chart of the prospective study with reasons for exclusion.Fig. S2. Spike‐in and specificity analyses.Fig. S3. ctDNA concentrations during surgery in stage I/II versus stage III NSCLC patients.Fig. S4. ctDNA as a biomarker during treatment.Click here for additional data file.Table S1. Patient characteristics shown for all individual patients of the cohort.Table S2. Target regions of the custom target capture NGS panel.Table S3. Overview of total cfDNA amount subjected to custom target capture NGS approach as well as the ctDNA status of the individual patients at all analyzed time points presurgery, during surgery, postsurgery, and follow‐up.Table S4. Overview of mutations detected by tumor genotyping.Click here for additional data file.


The coronavirus disease 2019 (COVID‐19) crisis and consequent changes in medical practice have engendered feelings of distress in diverse populations, potentially adversely affecting the psychological well‐being of cancer patients.The purpose of this observational longitudinal study was to evaluate psychosocial perspectives among patients with cancer on intravenous treatment during the COVID‐19 pandemic.The study recruited 164 cancer patients undergoing intravenous anti‐neoplastic therapy in a tertiary cancer center. Psychosocial indices were assessed at two points in time, corresponding with the beginning of the first wave of COVID‐19 pandemic in Israel (March 2020) and the time of easing of restrictions implemented to curtail spread of infection (May 2020). At Time 1 (T1), elevated COVID‐19 distress levels  were observed in 44% of patients, and associated with pre‐existing hypertension and lung disease in multivariate analyses but no demographic or cancer related factors. At Time 2 (T2), 10% had elevated anxiety and 24% depression as indicated by Hospital Anxiety and Depression Scale (HADS‐A/D). COVID‐19 distress at T1 was related to higher levels of HADS‐A at T2 (Spearman 0.33 p < .01), but not HADS‐D. Patients with breast cancer expressed greater COVID‐19 distress compared with other cancer types (p < .01), while both HADS‐A and HADS‐D were highest for patients with GI cancer. Patient report of loneliness and decreased support from relatives were factors associated with HADS‐A , while HADS‐D was not similarly related to the factors evaluated.Patients with cancer undergoing intravenous treatment may be vulnerable to acute adverse psychological ramifications of COVID‐19, specifically exhibiting high levels of anxiety. These appear unrelated to patient age or disease stage. Those with underlying comorbidities, breast cancer or reduced social support may be at higher risk. Unfavorable mental health has been observed among healthcare workers,The COVID‐19 pandemic reached Israel in February 2020, and enforcement of social distancing rules began in March 2020. On March 19, a state of national emergency was declared and a national lockdown prohibited citizens from leaving their homes unless for activities deemed essential. A gradual easing of restrictions began in early May. A second wave of COVID‐19 infection was announced during July. Located in the northern city of Haifa, Rambam Medical Center (RMC) is the tertiary referral center for 12 district hospitals, serving approximately 20% of the total nationwide population.The main objective of our study was to evaluate the psychosocial consequences of COVID‐19 on patients with cancer undergoing intravenous treatment in the oncology center during the peak of the crisis in Israel (March–May 2020). The cohort was longitudinally followed over the course of a month; first subjects completed a survey of demographics and general distress associated with COVID‐19, and ultimately, a validated assay of anxiety and depression. We evaluated indices of psychological well‐being and emotional and functional burden, considered the existence of more vulnerable subgroups of patients, and investigated a possible association between scores at different points in time.22.1The present study included patients with cancer receiving intravenous treatment administered at the ambulatory unit of the oncology center or inpatient service, within RMC. Inclusion criteria included patients of age 18 or more, with a cancer diagnosis within the past year, undergoing intravenous oncological treatment in RMC, and capable of reading and writing in the national language, Hebrew. Those with hematological malignancies were excluded, as treated in a separate department in our institution, as were patients who were not on active intravenous treatment . All participants signed a written informed consent. During the study period of March–May 2020, subjects completed two questionnaires for sociodemographic information as well as emotional and functional burden data. The study protocol was approved by the Institutional Ethics Committee.2.2Two questionnaires were administrated at baseline  and 1 month later  to evaluate participants' psychological state and psychosocial parameters.2.2.1The T1 questionnaire available in 2.2.2At T2, after the national lockdown and period of peak of infection, and with the results of T1, it was decided to distribute a validated questionnaire to assess and quantify patient psychological indices for comparison. The Hospital Anxiety and Depression Scale (HADS) questionnaire was chosen. HADS is a 14‐item validated screening tool with two subscales for anxiety (HADS‐A) and depression (HADS‐D). For each subscale, a cut‐off HADS score of equal to or greater than 8 is considered suggestive for anxiety or depression.2.2.3The T2 questionnaire included a series of questions designed to assess additional factors of emotional and functional burden. These were developed by the study team psychologist and social worker  was the dependent variable , lung (25%) and gastrointestinal cancer (25%), followed by genitourinary, head and neck, melanoma, gynecological and sarcoma, respectively. Treatment type included chemotherapy (74%), biological therapy (33%) and immunotherapy (25%), noting that some received more than one treatment modality. Patients were exposed to few people during the study period, suggestive of self‐isolation (p < .01). Current smoking was reported in 23% of patients, and frequent co‐morbidities included 22% hypertension and 17% diabetes mellitus. Participant characteristics are delineated in Table The study team requested participation from 164 patients, of these all 164 agreed and completed written consent. A full T1 questionnaire was provided by 161 patients and 99 patients went on to complete the T2 questionnaire Table .3.2As of June 10, 2020, there was no documented symptomatic case of COVID‐19 infection in any study participant.3.3The study group expressed moderate levels of distress related to COVID‐19 with mean value 3.1 (SD 1.62). However, 44% of patients had lower scores of 1 or 2, indicating greater distress Figure .3.4Mean HADS‐A was 4.3 (SD 3.65), while 17 patients (10%) manifested substantial symptoms of anxiety (HADS‐A ≥ 8). Mean HADS‐D was 6.7 (3.75), while 39 patients (24%) manifested substantial symptoms of depression (HADS‐D ≥ 8).Of groups with over 15 subjects, HADS‐A was highest among breast cancer patients, and HADS‐D in those with GI cancer, however HADS‐D was lowest among patients with breast cancer Figure . Despite3.5Significant association over time is observed for anxiety; patients with higher degree of COVID‐19 distress at T1 demonstrated higher HADS‐A scores at T2 (Spearman rank 0.33 p < .01) Figure . Regardi4The COVID‐19 crisis has posed a new and imminent threat to the continuity of care in cancer patients, possibly compounding any existing distress related to their cancer and future outlook. This has been previously assessed in different populations, points in time and locations worldwide. Cross‐sectional studies in China showed high anxiety and depression among the general population,The present study aimed to longitudinally evaluate psychosocial perspectives of patients with cancer undergoing intravenous treatment during the time of peak COVID‐19 infection, and found initially elevated COVID‐19 distress levels that predicted patient anxiety after 1 month, as well as loneliness and feelings of decreased social support. Nevertheless, similar correlations were not found for depression. Compared to the general population, depression is known to be relatively more common among cancer patients, especially those with metastatic disease. In the present study, depression levels were unequivocally higher than anxiety but homogenous and not correlated with COVID‐19 distress; anxiety levels, however, were heterogenous and significantly linked to COVID‐19 distress – thereby highlighting this distinct aspect of psychological well‐being as a potentially representative aspect of the experience of patients with cancer in the COVID‐19 era. As above, former studies evaluating non‐cancer patients did demonstrate COVID‐19‐associated depression. Taken together, these data suggest that anxiety may be an effective indicator of the mental state of cancer patients in relation to COVID‐19, while depression a more ingrained and enduring trait that is less affected by acute stress. To illustrate this, patients with breast cancer had high COVID‐distress and HADS‐A scores, but lower HADS‐D.This study identifies subgroups of patients who may be at greater risk for emotional distress and thereby benefit from early psychological support. The susceptibility of patients with underlying conditions or even poor perceived health is widely corroborated by other studiesIn international databases, a steep decline in oncology‐related procedures and visits has been observed.This study has several limitations. Firstly, a small sample size taken from a single institution was utilized. Patients chose to participate perhaps affecting degree of distress or willingness to partake in the study. The study cohort included mostly patients with metastatic disease, which may have influenced the results as a subpopulation more at risk and more distressed by COVID‐19. These reflect the patient distribution in the RMC oncology daycare. Importantly, COVID‐19 distress and HADs were not evaluated at both time points, rather one was compared to the other, limiting the strength of the data. At T1, a general survey was conducted to assess the degree of COVID‐19 distress, and as the epidemic progressed and the lockdown continued, the need arose for a validated quantification of patient distress, or the HADs survey. This does not represent change over time but rather a longitudinal comparison of separate factors, or initial COVID‐distress and eventual psychological state. In other words, those who were weary of COVID‐19 also eventually demonstrated high anxiety levels, which was not true for depression, thus isolating anxiety as a unique COVID‐19 related issue and providing further distinction between cancer‐anxiety and COVID‐19‐anxiety. It can be assumed that actual levels may in fact be higher as patients who did not arrive for treatment due to self‐isolation were not represented. Large interventional controlled trials are needed to support findings of patient distress and determine ramifications on patient treatment or outcome.While in some parts of the world the pandemic is abating, in other parts, peak levels are being experienced, and in still others, harbingers of additional waves are emerging — regardless, oncology teams should anticipate emotional distress among patients and prepare in advance. Availability of accurate information and the use of precautionary measures as hand‐washing and wearing a mask, have been shown to correlate with lower COVID‐19‐related anxiety and depression.Authors have no conflicts of interests to disclose.Conceptualization, I.T., T.G.L., K.K, L.G., I.B.A.; Methodology, I.T., T.G.L., I.B.A.; Investigation, I.T., T.G.L., I.B.A.; Formal analysis, I.T., T.G.L., I.W., A.R.B., R.A., I.B.A.; Resources, I.T., T.G.L.; Writing‐Original draft, I.T., T.G.L., I.W., I.B.A.; Writing‐Review & Editing, I.T., T.G.L., I.W., M.H., I.B.A.; Visualization, I.T., T.G.L., I.W., A.R.B., R.A., I.B.A.; Data Curation, I.T., T.G.L., I.B.A.; Validation, I.T., T.G.L., I.B.A.; Project Administration, M.H., I.B.A.All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. The study protocol was approved by the Institutional Ethics Committee . Patients provided written informed consent to participate in the study.Data S1. Supplementary materialClick here for additional data file.


Dipterocarpus turbinatus Gaertn. f. and Hopea hainanensis Merr. et Chun. Our whole‐genome duplication (WGD) analysis revealed that Dipterocarpaceae underwent a shared WGD event, which showed significant impacts on increased copy numbers of genes related to the biosynthesis of terpene, BAHD acyltransferases, fatty acid and benzenoid/phenylpropanoid, which probably confer to the formation of their characteristic fragrant oleoresin. Additionally, compared with common soft wood plants, the expansion of gene families was also found to be associated with wood formation, such as in CESA (cellulose synthase), CSLE (cellulose synthase‐like protein E), laccase and peroxidase in Dipterocarpaceae genomes, which might also contribute to the formation of harder, stronger and high‐density timbers. Finally, an integrative analysis on a combination of genomic, transcriptomic and metabolic data from different tissues provided further insights into the molecular basis of fragrant oleoresins biosynthesis and high‐quality wood formation of Dipterocarpaceae. Our study contributes the first two representative genomes for Dipterocarpaceae, which are valuable genetic resources for further researches on the fragrant oleoresins and superior‐quality timber, genome‐assisted breeding and improvement, and conservation biology of this family.Dipterocarpaceae are typical tropical plants (dipterocarp forests) that are famous for their high economic value because of their production of fragrant oleoresins, top‐quality timber and usage in traditional Chinese medicine. Currently, the lack of Dipterocarpaceae genomes has been a limiting factor to decipher the fragrant oleoresin biosynthesis and gain evolutionary insights into high‐quality wood formation in Dipterocarpaceae. We generated chromosome‐level genome assemblies for two representative Dipterocarpaceae species  To prova Figure . That ise Figure .et al., et al., D. turbinatus and H. hainanensis genomes, respectively, which were classified into five categories, that is, the WGD duplicates, tandem duplicates (TD), transposed duplicates (TRD), proximal duplicates (PD) and dispersed duplicates (DD)  have significant impacts on the increase of copy numbers in genes related to terpene and phenylpropanoid biosynthesis that are involved in the generation of aromatic scent, which may contribute to the characteristic fragrance (aroma component) in Dipterocarpaceae  Figure . WGD eveet al., et al., D. turbinatus and H. hainanensis showed that dozens of distinct chemical constituents, including the azulon, α‐gurjunene, α‐copaene, δ‐elemene and borneol, exist in these two species  accumulation of fragrant resins in D. turbinatus and H. hainanensis. The comparison of the expression level of TPS genes of D. turbinatus and H. hainanensis between wood and leaf tissues revealed that the expression levels of Diptu_12379.t1 (TPS‐a) and Diptu_12960.t1 (TPS‐b) were significantly higher in wood than leaves  plays a key role in yielding benzyl acetate in plants for floral scent, and we identified 9/6 BEAT homologous genes in D. turbinatus and H. hainanensis . D. turbinatus and H. hainanensis could encode 64/60 cellulose synthase (CESA) (including cellulose synthase‐like (CSL)), respectively, which is remarkably higher than the number of two representative softwood trees (Populus trichocarpa and A. sinensis) and other phylogenetic affiliation species  and further result in the formation of harder, stronger and highly dense wood compared with those softwood plants.Hardness, high strength and strong moisture resistance are the typical features of wood of Dipterocarpaceae plants  and phytohormones also participate in these complicated regulatory networks , jasmonic acid (JA) and abscisic acid (ABA) exhibited higher accumulation in the stem tissue of H. hainanensis than its leaf tissue, while almost no difference between leaf and stem tissues for D. turbinatus. Gibberellin accumulation in D. turbinatus stem tissue was over twofold higher than its leaf tissue; conversely, Gibberellin was preferred to be produced in H. hainanensis leaf rather than its stem  adenine  in e Figure . Howeverm Figure . Thus, tD. turbinatus and H. hainanensis at chromosome level by combining the long‐read sequences from Nanopore and Hi‐C data for super‐scaffolding. The first two Dipterocarpaceae genomes presented here provided a valuable opportunity to determine genome evolutionary signatures and elucidate the genetic basis of these high‐value metabolism products.Dipterocarpaceae are widely known trees (dipterocarp forests) in the tropics and predominate the international tropical timber market, and therefore play an important role in the economy of many Southeast Asian countries  genes , most displayed higher activity in stem tissue of D. turbinatus and H. hainanensis than their respective leaf tissues, further suggesting complicated fragrance composition in Dipterocarpaceae wood. Using genomic data, we classified various types of duplication‐induced gene family expansion. Functional enrichment of each type of duplication‐induced gene family expansion revealed that whole‐genome duplication and other types of duplication events have significant impacts on copy numbers of genes related to terpene, phenylpropanoid biosynthesis and lipid biosynthetic process  and 226 single‐copy nuclear genes from 14 representative species  indicated that the two Dipterocarpaceae species et al., et al., et al., P. trichocarpa and A. sinensis), Dipterocarpaceae own expanded CESA, CSLE, Laccase and peroxidase. Moreover, most of these genes exhibited higher expression in wood tissue than their expression status in leaves. CESA is involved in the primary cell wall formation and is thought to be the most important enzyme involved in the synthesis of cellulose microfibrils in plant cells , guaiacyl (G) and syringyl (S) lignin unit ’ in H. hainanensis genome compared with D. turbinatus  adenine showed higher accumulation in wood of both D. turbinatus woods displayed ~fourfold higher GA3 accumulation than its leaves. GA3‐treated Cannabis exhibited an augmented HMGR activity of MVA pathway that is primarily essential for the synthesis of sesquiterpene  and wood formation in these timber species at the genomic level, which will also be a valuable genetic resource for further research on the genome‐assisted breeding and improvement, and conservation biology of Dipterocarpaceae.In summary, we presented high‐quality assemblies of Dipterocarpus turbinatus Gaertn. f. (HCNGB_00001637) and Hopea hainanensis Merr. et Chun. (HCNGB_00001636) used in this study were collected from Ruili Botanical Garden . High‐quality DNA was extracted from fresh leaves by using QIAGEN® Genomic kits, and the DNA quantification was checked by Nanodrop and Qubit. PromethION Nanopore sequencer with the long‐read DNA sequencing type was used for genome sequencing. The SQK_LSK109 Ligation Sequencing Kit was used to prepare the sequencing libraries. Finally, a total of 27 and 26 Gb pass reads were generated for D. turbinatus and H. hainanensis, respectively.All plant materials of For short read Illumina sequencing, the genomic DNA was isolated from fresh leaves using a modified CTAB protocol   used Oxford Nanopore long reads to assemble the genome, including reads error correction with parameter ‘task=all (correct and assemble)’. We set read_cutoff = 1k, seed_cutfiles =10K while default parameters were used for other settings. At the genome polishing stage, NextPolish was used to correct the genome with three rounds of nanopore reads and thrice with Illumina sequencing reads.NextDenovo  with ‘eukaryota_odb9’ database . Among them, Piler detected repeat elements such as satellites and transposons, RepeatScout identified all repeat classes, MITE‐hunter discovered miniature inverted‐repeat transposable elements (MITEs) from the genomic sequence, while LTR‐FINDER predicted the location and structure of full‐length LTR retrotransposons. All results from ab initio prediction were merged as a homolog database to identify repetitive sequences by RepeatMasker (http://www.repeatmasker.org). We used LAI (LTR Assembly Index) to evaluate the assembly continuity by evaluating the assembly of repeat sequences. LTR‐RT candidates were obtained using LTRharvest with parameters ‘‐minlenltr 100 ‐maxlenltr 7000 ‐mintsd 4 ‐maxtsd 6 ‐motif TGCA ‐motifmis 1 ‐similar 85 ‐vic 10 ‐seed 20 ‐seqids yes’ and LTR_Finder (version 1.0.7) with ‘‐D 20000 ‐d 1000 ‐L 700 ‐l 100 ‐p 20 ‐C ‐M 0.9’  implemented in Phylogenetic Analysis by Maximum Likelihood (PAML)  was used to infer a homolog matrix of orthogroups (gene families) among these selected organisms ) and Circos .We use the MCscan pipeline , respectively. 4,539 gene pairs of D. turbinatus were distributed in 3,578 orthogroups, and 3,098 gene pairs of H. hainanensis were distributed in 2,550 orthogroups. Then, we identified a total of 1,631 orthogroups containing both gene pairs of D. turbinatus and H. hainanensis. Next, 125 out of 1,631 orthogroups were randomly selected for phylogenetic study by using the IQtree (version 1.6.12) with parameter of ‘‐bb 10000 ‐alrt 5000 ‐nt AUTO’. Next, we reconciled ancestral gene events  using a phylogenomic function in NOTUNG (version 2.9) by comparing the gene trees with the species tree. These trees were visualized with NOTUNG , tandem duplicates (TD), proximal duplicates (less than 10 gene distance on the same chromosome: PD), transposed duplicates (transposed gene duplications: TRD) and dispersed duplicates . The target species was A. thaliana, and the final gene number came from unique genes.To investigate http://www.timetree.org/), G. raimondii‐ D. zibethinus divergence time (60‐77 MYA), G. raimondii‐ T. cacao divergence time (62‐85 MYA).Divergence times in the phylogeny tree between each species were calculated using the MCMC tree program with ‐sampfreq 5000 ‐burnin 5000000 parameter. The sequential PHYLIP format nucleotide sequences and rooted phylogeny tree is derived from Figure Based on the following criteria, all candidate genes were screened: firstly, candidate gene sequences were identical to collect query gene sequences gathered from previous studies or public databases, by BLAST (< 1e‐5); and (2) The candidate genes feature should be similar with the online functional annotation or Swissprot functional annotation query genes.https://pfam.xfam.org/) was used to download HMMER domain structure models for each transcription factor when as per the role of TAPscan v.2 database for TFs (https://plantcode.online.uni‐marburg.de/tapscan/). Preliminary TF candidate genes were collected for each species (< 1e‐5) by searching the HMM profile. Then, parts of genes were filtered if they are not the homologs according to their functional annotation of SwissProt (< 1e‐5). In the end, we filtered genes that contained a wrong domain under the TAPscan v.2 transcription factor database domain rules.Regarding the identification of transcription factors (TFs), we used the HMMER search method for transcription factors. The Pfam website . The CYP450 genes were searched by both domains, PF00067 and BLAST, with the queries from rice and Atha.To identify genes involved in the terpenoid backbone biosynthesis pathway Figure , we collFinally, all searched candidate genes were used for phylogenetic analysis to distinguish the orthologs of corresponding functionally characterized genes. For each gene family, mafft‐7.310 used to align and Gblocks used to trim ambiguously aligned positions  packages were used to estimate gene expression levels from clean reads. The transcriptome reads were mapped to the assembled genome by bowtie2 with default settings. We continue to identify the differentially expressed genes by DEseq2  of gene expression in the leaf and stem (FPKM>1)  methanol. Next, samples with glass beads were put into the tissue grinder to grind for 90 s at 55 Hz. Following centrifugation at 12000 rpm at 4 °C for 10 min, take the supernatant, filter it through 0.22 μm membrane and transfer the filtrate into the detection bottle before LC‐MS analysis. Then, the sample extracts were analysed using an Ultra Performance Liquid Chromatography (UPLC) Vanquish (Thermo) and Q Exactive HF‐X system (Thermo). For the quantitative detection of phytohormones, stem and leaf tissue samples were used. The self‐construction database which is constructed by reference standards was used to perform qualitative analysis. Additionally, different concentrations of the standards were used to perform quantitative analysis.The authors declare that they have no competing interests.H.L. and S.K.S. conceived, designed and supervised the project. J.W., L.C., J.Y., C.H., J.L. and Y.Z. provided resources and materials. S.W., H. Liang., S.K.S. and H.W. analysed the data. S.K.S., S.W. and H. Liang. wrote the paper. H.L., Y.L., T.M., M.L., L.L., Y.X. and M.L. revised the manuscript. All the authors read, revised and approved the final version of the manuscript.Figure S1 Estimation of genome complexity of two Dipterocarpaceae trees.Click here for additional data file.Figure S2 Hi‐C contact matrix visualization for chromosomes of two Dipterocarpaceae reference genome assemblies.Click here for additional data file.Figure S3 Comparison of insertion dates of LTR‐RTs among D. turbinatus, H. hainanensis and A. thaliana.Click here for additional data file.Figure S4 The phylogenetic analyses of Malvales based on nuclear and chloroplast genes.Click here for additional data file.Figure S5 Phylogenetic evidence of shared WGD event between the D. turbinatus and H. hainanensis.Click here for additional data file.Figure S6 Syntenic blocks between genomes.Click here for additional data file.Figure S7 Phylogenetic tree of the TPS proteins.Click here for additional data file.Figure S8 Chromosomal distribution of the important fragrance related genes in D. turbinatus and H. hainanensis genomes, respectively.Click here for additional data file.Figure S9 Phylogenetic tree of the SABATH family, COMT and ICMT in the selected plants.Click here for additional data file.Table S1 Statistics of contig and chromosome level genome assemblies of D. turbinatus and H. hainanensis.Table S2 Benchmarking Universal Single‐Copy Orthologs (BUSCO) evaluation of D. turbinatus and H. hainanensis genomes.Table S3 Annotation of repetitive sequences in D. turbinatus and H. hainanensis genomes.Table S4 Functional annotation of the D. turbinatus and H. hainanensis protein‐coding genes.Table S5 KEGG enrichment of expanded gene families of selected evolutionary nodes.Table S6 KEGG enrichment of unique gene families in Dipterocarpaceae.Table S7 Statistics of expanded genes number induced by different types of gene duplication way.Table S8 Aroma components in Dipterocarpaceae.Table S9 Genes involved in the 2‐C‐methyl‐D‐erythritol 4‐phosphate (MVA) and mevalonate (MEP) metabolism in selected plants.Table S10 Comparison of TPS gene number in selected plants.Table S11 Comparison of BAHD gene number in selected plants.Table S12 The candidate genes involved in the benzenoid/phenylpropanoid synthesis pathways in D. turbinatus and H. hainanensis.Table S13 The expression level of candidate genes involved in the terpenoid volatile organic compounds (VOCs) synthesis in wood and leaf tissue of D.Table S14 Comparison of gene number of SABATH family and SAM‐binding methyltransferase superfamily in selected plants.Table S15 Comparsion of transcription factors of selected plants.Table S16 Comparison of various phytohormones content between wood and leaf.Table S17 Genes involved in the fatty acid metabolism.Table S18 Protein ENHANCED PSEUDOMONAS SUSCEPTIBILITY in selected plants.Table S19 Statistics of resistance (R) genes.Click here for additional data file.


Our data reveal that selection of enzymes for generating single cell suspensions from murine tissues influences detection of surface expression of antifungal CLRs. Using a method that most preserves receptor expression, we show that non‐myeloid expression of antifungal CLRs is limited to MelLec on endothelial cells in murine mucosal tissues. Candida, Aspergillus, and Cryptococcus [C‐type lectin receptors (CLRs) constitute a family of pattern recognition receptors (PRRs), many of which are involved in immunity . Of parttococcus , 2, 3. Ttococcus .Most of these “antifungal” CLRs are thought to be expressed by myeloid cells, but there is some evidence suggesting that these receptors are also expressed on nonmyeloid cells including endothelial and epithelial cells , 6, 7, 8In the literature, a range of proteolytic enzymes have been used for the generation of single cell suspensions of tissues. However, these treatments can affect the levels of surface molecules and have functional consequences . Little –CD31+ endothelial cells , club (Scgb1a1), and ciliated (Foxj1) epithelial cells. These data confirm that expression of MelLec is absent on all three major epithelial populations (Supporting Information Fig. Using the lung dissociation kit, which had the least effect on the ability to detect CLR surface expression, we next assessed the expression of these receptors on murine lung epithelial and endothelial cells ex vivo. As we have previously observed , expresslls Fig. . Howeverlls Fig. . To confAspergillus fumigatus and assessed receptor expression after 24 h. We determined that expression of MelLec was unaltered on lung CD45–CD31+ endothelial cells (Supporting Information Fig. –EpCAM+ cells (Supporting Information Fig. To determine if expression of MelLec could be modulated under inflammatory conditions, we intratracheally infected mice with Several reports have suggested that other anti‐fungal CLRs can be expressed by endothelial and/or epithelial cells , 6, 7, 8–CD31+ endothelial cells, and no CLRs were detected on CD45–EpCAM+ epithelial cells of the genital‐urinary tract (Fig. –CD31+ endothelial cells, and no CLRs were detected on CD45–EpCAM+ epithelial cells isolated from the gastrointestinal tract (Fig. Finally, we explored CLR expression on nonmyeloid cells at two other mucosal tissues frequently in contact with fungi, the genital‐urinary and gastrointestinal tract. Similar to the lung, only MelLec was detected on naïve CD45act Fig. . Unfortuact Fig. . The cavact Fig. . Thus, tIn summary, our data reveal that the selection of enzymes for generating single cell suspensions from murine tissues influences the detection of surface expression of antifungal CLRs, which may have consequences in subsequent in vitro analyses , 11. UsiThe authors declare no commercial or financial conflict of interest.https://publons.com/publon/10.1002/eji.202149192The peer review history for this article is available at Supporting InformationClick here for additional data file.


Anti‐leucine‐rich glioma‐inactivated 1 (LGI1) autoimmune encephalitis (AE) is characterized by complex manifestations of seizures. Here, we report a new seizure semiology, attempt to classify the disease by semiology type, and explore the metabolic pattern of each group.18F] fluoro‐2‐deoxyglucose (FDG) positron emission tomography (PET) scans. Voxel‐based metabolic analysis and z‐distribution analysis were carried out to determine the metabolic pattern.Anti‐LGI1 AE patients were retrospectively screened between May 2014 and September 2019 in our tertiary epilepsy center. All enrolled patients had seizures during long‐range video electroencephalogram (EEG) recordings, and all patients (except one) underwent [n = 17), faciobrachial dystonic seizures (FBDS)‐only (n = 6), FBDS‐plus (n = 8), and focal aware motor seizures (FAMS) (n = 2). No significant differences were found in the clinical manifestations or accessory tests except for the onset age (FIAS < FBDS‐plus) and seizure semiology. This was the first study to extensively describe the clinical manifestations and EEG of FAMS in anti‐LGI1 AE patients. In addition, we found that the patients with different semiologies all showed a wide range of abnormal metabolism, which is not limited to the temporal regions and basal ganglia, and extends far beyond our previous interpretation of FDG‐PET data.Thirty‐three patients were enrolled. According to the patients’ seizure semiology, we divided the patients into four groups: focal impaired awareness seizures . Furthermore, FDG‐PET scans showed a wide range of abnormal metabolic pattern of this patient and patients with other seizure semiologies, which is not limited to the temporal regions and basal ganglia and far beyond our previous interpretation (B). Previous studies found that FDG‐PET can significantly increase the sensitivity for detecting abnormalities in patients with AE compared with structural magnetic resonance imaging (MRI) or EEG.‐FDG (3.7 MBq/kg). Before injection, subjects fasted for at least 6 h, and their blood glucose level was <120 mg/dl. During image acquisition, subjects rested in a quiet environment with their eyes closed. A CT scan was recorded, followed by a 15‐min emission scan consisting of three 5‐min frames. Images were reconstructed using an ordered subset expectation maximization algorithm, and vendor supplied corrections for scatter and random corrections were applied. Normal controls underwent identical scans.Thirty‐two patients underwent FDG‐PET scans during initial clinical evaluations at diagnosis. Brain PET/computed tomography (CT) images were acquired with a hybrid PET/CT system  60 min after intravenous injection of [https://www.fil.ion.ucl.ac.uk/spm), implemented in MATLAB (MathWorks), was performed. FDG‐PET brain images were preprocessed, coregistered to individual T1‐weighted MRI, spatially normalized in Montreal Neurological Institute space, and normalized to global intensity with SPM12 software. Images were smoothed, and proportional scaling was used to adjust global values for metabolism. For statistical comparisons, healthy control subjects were divided into three groups  to match the number, sex ratio, and age of patients in the three different groups . Images from the three groups of patients were tested for relative hypo/hypermetabolism by comparison with the corresponding reference database on a voxel‐by‐voxel basis using the general linear model by means of the SPM12 two‐sample t‐test design with age included as a covariate. An uncorrected threshold of p < 0.005 was used to compare paired groups. Only clusters of more than 50 voxels were considered.Semiquantitative analysis using Statistical Parametric Mapping 12 (SPM12) (http://brainvisa.info) was used to extract areas with differences between the patient and normal controls. Clusters of >100 voxels (8 ml) and voxels with absolute values of >1.96 z‐score (p < 0.05) were considered to have significantly higher metabolism than healthy controls, and these areas were extracted before z‐score maps were displayed onto anatomical images. The details are provided in our previous literature.For the only patient in the FAMS group, z‐score mapping implemented in BrainVisa software (2.4p < 0.05 (two‐sided).Analyses were performed by using SPSS 13.0 software (SPSS). For categorical variables, Fisher's exact test was used for group comparison. For quantitative variables, the Shapiro–Wilk test indicated that the data did not conform to a normal distribution; thus, we adopted a nonparametric test  to make group comparisons. The onset age among groups was significantly different, and we made multiple post hoc comparisons by using the “all pairwise” method. Differences were considered significant at 33.1+<134 mmol/L); the positive rates of LGI1 antibody in blood and CSF were 100% and 93.5%, respectively; and 27/29 (93.1%) patients were LGI1‐positive in both the blood and CSF. Brain MRI scans were available for all patients before immunotherapy; specifically, 22/33 (66.7%) patients showed unilateral or bilateral abnormalities in the MTL, including hyperintensity in T2 images, swelling or atrophy, while only two (6.1%) patients showed basal ganglia hyperintensity. Notably, one patient in the FBDS‐plus group presented with abnormalities of the bilateral hippocampus and right lenticular nucleus simultaneously. Long‐range video EEG recording was also performed before immunotherapy, and patients exhibited clinical (93.9%) or subclinical (33.3%) attacks or both (27.3%). Except for one patient in the FAMS group, the remaining 32 patients all underwent FDG‐PET examination, and the average time to onset was 3.1 months; no patients had undergone immunotherapy at that time point. All of the 33 patients underwent immunotherapy: 11 received high‐dose corticosteroids, 15 received high‐dose corticosteroids combined with intravenous immunoglobulins, and seven received isolated intravenous immunoglobulins. There were 29 patients who took antiepileptic drugs; among them, 21 took one type of AED, six took two types and two took three types. All patients improved at discharge; specifically, the clinical seizures disappeared, and cognitive function improved significantly.As shown in Table p = 0.042), but this result needs to be interpreted cautiously due to the small sample size. In addition, compared with the FBDS‐only group, the MTL abnormalities and subclinical attacks of patients in both the FIAS and FBDS‐plus group showed trends of differences, but these differences did not reach statistical significance, probably due to the small sample size. The FAMS group was not included for comparison because there were only two patients in this group.Patients were divided into four groups according to seizure semiology, and then we made comparisons among the FIAS, FBDS‐only, and FBDS‐plus groups; however, except for the onset age, there was no significant difference in the clinical manifestations and accessory examinations. Specifically, post hoc comparisons suggest that the onset age of the FIAS group was lower than that of the FBDS‐plus group . Long‐range video EEG Figure  detectedBefore immunotherapy, the patient underwent a brain FDG‐PET scan. We calculated the z‐distribution compared with age‐matched healthy controls to understand the metabolic changes. As shown in Figure 3.3In comparison to the matched reference group, FDG‐PET scans of patients in the FIAS group displayed extensive hypermetabolism in the bilateral basal ganglia, paracentral lobule, precentral gyrus, postcentral gyrus, MTL, cerebellum, lingual gyrus, insula, right superior parietal lobule, right cuneus, and left superior frontal gyrus (69–12734 voxels; peak clusters in Brodmann areas 3, 4, 6, 7, 10, 13, 17, 18, 36, and 43; Table In the FBDS‐only group, we observed regionally limited hypermetabolism of the bilateral cerebellum and left medial globus pallidus (154–1163 voxels; peak clusters in Brodmann areas 25; Table Similar to the metabolic pattern of the FIAS group, patients in the FBDS‐plus group also presented a wide range of hypermetabolism, including the brain areas of bilateral basal ganglia, MTL, precuneus and cerebellum, left postcentral gyrus, insula and superior parietal lobule, right substantia nigra, middle occipital gyrus, and cuneus (58–6547 voxels; peak clusters in Brodmann areas 7, 13, 18, 19, 21, 38, 39, and 43; Table 4This study retrospectively analyzed 33 anti‐LGI1 AE patients in our tertiary epilepsy center. Seizures were observed in all patients during long‐range video EEG recordings. Then, we grouped patients according to the seizure semiology and acquired patients’ metabolic changes compared with the matched controls. This study was a larger sample study in which anti‐LGI1 patients had clinical and subclinical seizures. Importantly, we first described the clinical and EEG manifestations of two FAMS patients in detail, and we explored the metabolic pattern of each group divided by seizure semiology. All FDG‐PET scans were performed before immunotherapy due to its effect on metabolism.Involuntary movements have been described in up to 40%–68.8% of patients with anti‐LGI1 AE.Considerable research efforts have illustrated the limited and rigid metabolic pattern of anti‐LGI1 AE.Previously, we found that computer‐aided semiquantitative analysis of FDG‐PET scans could reduce the false negative rate of the MTL and basal ganglia up to 56% and 73%, respectively, compared with visual reading.We acknowledge a number of limitations in this study and future directions that should be taken. First, the sample size was too small, and the patients were from a single center. Second, we found specific metabolic patterns of different groups, but it is not a simple superposition relationship; for example, the metabolic pattern of the FBDS‐plus group was not the addition of the FIAS and FBDS‐only groups; this finding is difficult to explain and may be due to individual differences, small sample sizes or different onset ages. Third, it is difficult to explain the causes of some brain regions’ abnormal metabolism, and the results cannot currently be used in individual diagnosis. Fourth, although we have corrected for the number, sex ratio, and age of the patients, some patients had intracranial ischemic changes, which could cause metabolic changes. Considering the shortcomings of our research and the limitations in this field, multicenter collaboration to include more subjects is needed to verify the repeatability of the results. Furthermore, deep‐level mechanisms, such as genetic mutations5Our results show that FAMS can serve as a rare indicative symptom of anti‐LGI1 AE. Furthermore, we found that patients with different seizure semiologies all showed a wide range of abnormal metabolic patterns, which is far beyond our previous interpretation of FDG‐PET data, indicating that this type of AE is associated with widespread functional network alterations. Future larger sample studies and studies from other perspectivesOn behalf of all authors, the corresponding author confirms no conflict of interest. All authors agreed to the publication of the manuscript in its current form.RJ Lv and YD Zhang provided the clinical data. TR Li and RJ Lv acquired the literature data. Q Wang and XQ Shao polished the manuscript. TR Li drafted the manuscript. RJ Lv critically revised the manuscript for important intellectual content. All authors read and approved the final manuscript.Supplementary MaterialClick here for additional data file.


FBP1) gene, previously unreported, was identified. Diagnosis in adulthood is underreported in the literature, however, represents an important, albeit rare, cause of hypoglycaemia and lactic acidosis.Hepatic fructose‐1,6‐bisphosphatase (FBPase) deficiency commonly presents with acute crises during infancy when glycogen stores are depleted. In these patients, dependence on glycogenolysis means that the duration of normoglycaemia is related to liver glycogen stores. Clinical hallmarks of FBPase deficiency include hypoglycaemia and lactic acidosis with or without ketosis. Patients commonly present with hyperventilation, vomiting, tachycardia, reduced consciousness and glucagon‐resistant hypoglycaemia. Between crises, patients are usually well with normal growth and development; however significant ingestion of fructose, sucrose or glycerol during acute crises may be fatal, hence the importance of a prompt diagnosis. We present the case of a 30‐year‐old male who presented to our tertiary centre acutely unwell, shortly following a diagnosis of hepatitis C, which we speculate may have precipitated this severe presentation. He had similar, milder episodes throughout childhood. Furthermore, a pathological homozygous sequence variant in fructose‐1,6‐bisphosphatase ( FBP1 deficiency is an important, albeit rare, cause of hypoglycaemia and lactic acidosis in adults; we speculate that this severe presentation was precipitated by newly diagnosed hepatitis C.1FBP1 gene, which consists of seven coding exons and is located at chromosome 9q22.3.Deficiency of hepatic fructose‐1,6‐bisphosphatase (FBPase), a rate‐limiting gluconeogenic enzyme, is a rare autosomal recessive disorder that commonly presents with acute crises in neonates and infants.Acute crises are characterised by hyperventilation, vomiting, episodic irritability, tachycardia, somnolence and hypotonia, and may be fatal, especially in neonates and infants.Here, we report a case of an acute presentation associated with newly diagnosed hepatitis C, in a patient with suspected FBPase deficiency. Furthermore, a novel sequence variant, previously undescribed in the literature, was identified in this patient.2A 30‐year‐old male born to non‐consanguineous parents presented to our emergency unit with symptomatic hypoglycaemia . He reported 2 days of mild, intermittent abdominal pain and frequent food craving, not responsive to fruit, simple carbohydrates, or dairy products. Despite oral intake, he developed dysarthria, hyperhidrosis, somnolence, and asthenia, with subsequent onset of severe epigastric pain and vomiting immediately prior to presentation. He denied any blurred vision, dizziness, or seizures. There were no infective or episodic symptoms of note.© and Centrum©). He denied any use of prescription, recreational or over‐the‐counter medications, including psychotropic or anxiolytic medications.He had been recently diagnosed with hepatitis C, discovered incidentally during investigations for exertional chest pain with associated lactataemia. He denied recent alcohol intake although reported significant stress related to this diagnosis. His medication history was significant only for branded multivitamins  with a normal C‐reactive protein (4 mg/L [RR: <5 mg/L]). He was hyperkalaemic (6.5 mmoL/L [RR 3.9–5.3 mmol/L]), without concurrent acute kidney injury (serum creatinine 96 μmol/L [RR: 45–84 μmol/L]). Liver function testing demonstrated an acutely elevated ALT (163 unit/L [RR: 0–41unit/L]), with normal bilirubin and synthetic function. Of note, recent transient elastography (FibroScan©) performed during hepatitis workup showed only mild fibrosis (5.5 kPa). No infective focus was found on bacteriology specimens  or imaging (CT abdomen and pelvis) and toxicology screen was negative. Due to persistent acidosis and hyperkalaemia despite intravenous crystalloid and dextrose therapy, he required haemofiltration, following which he remained euglycaemic without intravenous dextrose.Emergency room investigations showed a severe lactic acidosis  and marked leucocytosis  and C‐peptide (50 pmol/L). He had significant ketosis (serum beta‐hydroxybutyrate 2.36 mmol/L), as well as elevated free fatty acids (3.16 mmol/L), urate (645 μmoL/L) and lactate (10.4 mmoL/L). His basal pituitary profile was normal.FBP1 gene was identified. In silico analysis predicted this to affect splicing, and clinical and biochemical findings were consistent with pathogenicity. Unfortunately, it was not possible to arrange genotyping of this individual's family members due to geographical constraints. Fructose‐1,6‐phosphatase activity in white cells was subsequently also found to be very low , thus confirming pathogenicity of the novel mutation. He was initiated on appropriate management, centred on avoidance of fasting, regular meals which include slowly absorbed carbohydrates, and uncooked cornstarch (UCCS) before bed as cover for overnight fast. Unfortunately, he was unable to tolerate the UCCS due to gastrointestinal discomfort. Therefore, he was encouraged to take a late‐night snack high in low glycaemic index (GI) carbohydrates and given an oral emergency regimen (ER) of dissolvable 25% glucose polymer (50 g/sachet to be dissolved in 200 mL of water) sachets to be used when unable to eat and drink normally. He was advised to have a low threshold for presenting to the emergency department for intravenous glucose if unable to tolerate, or not improving with, the oral emergency regimen.A clinical diagnosis of fructose‐1,6‐bisphosphatase deficiency was made and he was referred for genetic analysis and expert inherited metabolic disorders (IMD) advice. The Illumina HiSeq platform was used to sequence coding regions and splicing sites of 30 glycogen storage disorders/gluconeogenesis/glycogen synthesis genes using TruSight One Panel target enrichment system (Illumina). A homozygous previously unreported variant of unknown significance affecting a highly conserved nucleotide in the splice donor region of intron 1 of the ©). He achieved sustained virological response and had a normal liver ultrasound at 12‐month follow‐up. Following treatment completion, he experienced one further hypoglycaemic episode precipitated by a stressful personal event, which was easily terminated with one sachet of oral ER. He once again denied concurrent use of psychotropic or recreational drugs at this time. It was not possible to deduce whether fasting tolerance had increased, due to a change in the pattern of dietary intake following patient education.He completed a 12 week course of hepatitis eradication therapy consisting of elbasvir with grazoprevir  and around one‐third are missense variants (n = 35).FBP1 gene, c.170+4A>G,p.(?), a novel variant previously undescribed in the literatureFBP1 gene have been recorded, affecting introns 4, 5 and 7.All case reports identified in the literature pertain to non‐European patients, relevant due to differences in disease‐causing genetic variants of We speculate that this acute, severe presentation was precipitated by recently diagnosed hepatitis C. FBPase catalyses the hydrolysis of fructose 1,6‐bisphosphate to fructose 6‐phosphate, a common reaction to both the Calvin cycle and gluconeogenesis.Akt, an important protein kinase involved in glycogen synthase activation, another integral glycogenesis enzyme.Furthermore, maintenance of normoglycaemia during fasting in individuals with FBPase deficiency is closely related to liver glycogen availability.n = 23) reported a fasting tolerance of greater than 10 h when well.Guidelines published by British Inherited Metabolic Disease Group in 2008 suggest the avoidance of fructose and sucrose during acute illness.Despite the novelty of our case in European literature, and thus its benefit in advancing the evidence base, the authors recognise several important limitations. First, given that this is a single case report, it is not possible to draw any causal relationship between newly diagnosed hepatitis C and acute crisis. Furthermore, the avoidance of fasting as an important means to prevent hypoglycaemic episodes limits conclusions regarding the impact of hepatitis C eradication therapy on prolonging fasting interval.4FBPase deficiency, a key gluconeogenesis enzyme, is a rare autosomal recessive disorder that commonly presents acutely in early childhood when glycogen stores are limited or exhausted. The relationship between hepatic impairment, subsequent impact on glycogen stores, and precipitation of inborn errors of metabolism represents an important area of future study.Despite being underreported in the literature, up to 40% of patients under the care of Adult IMD services are diagnosed in adulthood.The authors declare no potential conflict of interests.All authors had direct involvement in the patient's clinical care, at resident or attending level, and thus acquisition of data. All authors were involved in the planning of the paper. Helena Fawdry wrote the first draft of the manuscript and all other authors provided extensive input into further drafts and all approved the final manuscript.Informed consent was obtained from this patient to be included in the case report.No experimentation or research was involved that required informed consent and no patient identifying data are included.


Adrenarche involves maturation of the hypothalamic‐pituitary‐adrenal axis and increased production of dehydroepiandrosterone and its sulfate ester, dehydroepiandrosterone‐sulfate (DHEA‐S). It occurs at ages 6 to 8 in industrialized populations, marking the transition from childhood to juvenility and cognitive development at middle childhood. Studies in subsistence level populations indicate a later age (8‐9) for adrenarche, but only two such studies currently exist for comparison.To investigate adrenarcheal age among Maya girls and its association with body composition and dietary variables. We hypothesized adrenarche would occur earlier given the current dual burden of nutrition in Mexico.25 Maya girls aged 7 to 9 from Merida, Mexico using ELISAs to measure salivary DHEA‐S, standard anthropometry for height, weight, and skinfolds, bioelectrical impedance for body composition variables, as well as a food frequency questionnaire for dietary information.Our hypothesis was rejected—adrenarche occurred close to 9 years. While no measures of body composition were significantly associated with adrenarcheal status, girls eating meat and dairy products more frequently had significantly higher DHEA‐S levels.Like other populations living in ecologically challenging environments, adrenarche occurred relatively late among Maya girls. Adrenarche has been linked to measures of body composition, particularly, the adiposity or body mass index rebound, but no relevant anthropometric measures were associated, possibly because of the small sample.Further studies are required to illuminate how adrenarcheal variation relates to developmental plasticity, body composition, pubertal progression, and animal product consumption in other transitional populations. Samples were kept on ice until transported to the laboratory at CINVESTAV‐Merida, and then stored at −80°C until assayed using a DHEA‐S ELISA . Samples were processed in a single batch according to manufacturer instructions. ELISA absorbance was read using a Biorad microtiter plate reader at 450 nm without correction. Reproducibility was assessed by two pooled, blinded quality control samples. Intra‐assay coefficient was 19.7%, detection limits 43 and 16 000 pg/mL, respectively, and lower limit of functional assay range, 188.9 pg/mL.Since DHEA‐S shows little diurnal variation, a single, morning (08:00‐12:30), stimulated saliva sample was collected in 5 mL polypropylene vials at least 1 hour after eating or drinking using flavorless gum base , and above the sample median z‐scores for body composition variables. We also tested for associations between adrenarcheal status and 5th percentile of z‐scores for height‐for‐age (short stature/stunting), 5th percentile weight‐for‐age (under‐weight), as well as 5th, 85th, 95th percentiles of BMI‐for‐age    CDC, .P < .05. Means are shown with standard deviations (SD).A Fisher's exact test was used to assess whether being above the sample median for absolute fat percentage, fat mass index, and fat free mass index was associated with being post‐adrenarcheal. Overfat was defined as >85th percentile of sex‐ and age‐specific reference curves for body fat derived from British children; no equivalent exists for Maya , and the Bioethics Committee of CINVESTAV.3Supporting Information Table P = .030), but not z‐score median‐for‐weight, presumably because this adjusted for age. Of ten stunted girls in the sample, only one (aged 9.12 years) was post‐adrenarcheal. Neither height, measured as a continuous variable, nor being stunted was significantly associated with adrenarcheal status. More overweight (>85th percentile of BMI z‐scores) and obese girls (>95th percentile of weight‐for‐height z‐scores) were post‐adrenarcheal, but no measure relating to body weight status was significantly associated with adrenarche  had lower birth weight than post‐adrenarcheal ones (n = 1).P = .007). The median DHEA‐S level in the lower consumption group was 59.49 pg/mL (IQR: 29.99‐104.25 pg/mL) compared to 302.90 pg/mL (IQR: 123.33‐430.50 pg/mL) for girls eating meat more frequently . BMI was neither correlated with meat  nor dairy consumption .Girls with greater weekly meat consumption frequencies had significantly higher DHEA‐S  consumption among Maya girls were associated with higher DHEA‐S levels.Campbell  suggestsThe results here push the boundaries of adrenarcheal age later in populations exposed to more challenging ecological conditions relative to girls in well‐nourished, affluent settings. How this later transition might affect the timing of adolescence and young adulthood requires further analyses of larger cross‐sectional and longitudinal datasets. The consumption of animal products in relation to adrenarche also deserves further investigation.GB, GC, LH designed the adrenarche study protocol. FD, HW, MV, and PG designed the initial study protocol that formed the core dual burden project. FD trained HW and the fieldwork team in anthropometrics, while MV trained them in bioelectrical impedance techniques. AV and LH collected the salivary samples, and AN conducted the hormonal assays in consultation with GC. SK performed the data analyses in consultation with GB and MV. AN, GB, and SK were the main authors while all others contributed ideas and suggestions to various drafts of the manuscript. We thank the girls who participated in the study and their families, as well as the field assistants who helped in various stages of this project. Special thanks goes to Jenice Tut and Hugo Azcorra who assisted in data collection. Finally we would like to acknowledge the late Dr. Omar Zapata, who kindly stored our saliva samples before analysis.Appendix S1: Supporting informationClick here for additional data file.


To compare the diagnostic efficacy among transvaginal sonography (TVS), transabdominal sonography (TAS), and 3.0 T magnetic resonance imaging (MRI) in early cesarean scar pregnancy (CSP).  The clinical data of 65 patients initially diagnosed with CSP in our hospital from November 2019 to November 2020 were selected for the retrospective analysis, and all patients received TVS, TAS, and 3.0 T MRI. Taking the pathological findings as the “gold standard”, the diagnostic efficacy of different diagnostic modalities in early CSP was compared. P < 0.05).  In terms of the AUC value, the result was 3.0 T MRI > TVS > TAS, and among the three diagnosis methods, 3.0 T MRI had the highest diagnostic specificity, and TVS and 3.0 T MRI had the highest sensitivity; other than intragestational hemorrhage, the detection rates of other signs of disease by TVS and TAS were significantly higher than 3.0 T MRI ( 3.0 T MRI has better diagnostic efficacy in early CSP than TVS and TAS, while TVS and TAS work better in diagnosing uterine bleeding, plumule, yolk sac, and fetal heartbeat than 3.0 T MRI. The results are more beneficial to the guidance on selecting treatment modalities. Cesarean scar pregnancy (CSP) is a type of heterotopic pregnancy at a special site in pregnant women who have undergone cesarean section (C-section) before, which is mainly manifested by embryos implanted at the lower uterine segment where the incision scar is located and is one of the long-term complications of C-section , 2. The β-HCG) value was found via blood examination; and ③ patients had completed clinical imaging data. Exclusion criteria: ① puerpera had other types of heterotopic pregnancy ; ② patients were complicated with other gynecological diseases that affected the diagnosis of CSP; ③ patients had an allergic constitution and could not accept MRI; and ④ patients were complicated with reproductive system malignancies or had mental diseases.The clinical data of 65 patients initially diagnosed with CSP in our hospital from November 2019 to November 2020 were selected for the retrospective analysis, and the study met the World Medical Association Declaration of Helsinki (2013) . InclusiThe examination instrument selected was the DU8-M2 color Doppler ultrasonic tester , and the probe frequency for TVS and TAS was 5–9 MHz and 1–5 MHz, respectively. First, the TAS was performed. Patients filled their bladder before examination and then lied in spine position, coupling agent was applied to their lower abdomen, and the probe was placed on their abdomen for exploration to carefully observe whether the size and shape of the uterus, the presence or absence of abnormal adnexal mass, gestation sac in the uterine cavity, abdominal or pelvic hydrops, and echogenicity inside internal uterine cavity were associate with the uterine scar. After that, patients emptied their urine and received TVS examination in lithotomy position, an ultrasonic probe wrapped with a sterile condom was put into their cavity via vagina for full-range scanning, routine examination of the uterus, pelvic cavity, and bilateral appendixes, and observation of size, location, and echogenicity of gestation sac, to find out whether they were related to C-section scar , 12. Mea3.0 T MRI: patients were in spine position and asked to control their breathing; the MAGNETOM Skyra 3.0 T MRI superconducting scanner (manufactured: Siemens AG) and body phased-array coil was used to perform routine scanning with the following scanning sequences. Turbo spin echo (TSE): T1WI , T2WI , slice thickness 5 mm, slice gap 1.0 mm, matrix 320 × 320, the field of view (FOV) 350 mm × 350 mm; fat-saturated T2WI sagittal sequence: TR 5,000 ms, TE 93 ms, slice thickness 5 mm, slice gap 1.0 mm, matrix 320 × 320, and FOV 260 mm × 260 mm. The images were analyzed and observed by 2 experienced radiologists.Taking the results of pathological diagnosis as the “gold standard,” the diagnostic efficacy of TAS, TVS, and 3.0 T MRI examinations was compared.2 test, and differences were considered statistically significant at P < 0.05.The data obtained in this study were analyzed by SPSS 26.0, the relevant accuracy rates of diagnosing CSP via 3 imaging modalities were examined by XThe results of pathological diagnosis showed that 54 cases were positive and 11 cases were negative for CSP. For comparison of results of the three imaging diagnosis modalities and pathological diagnosis; see Tables 1The AUC values of diagnosing CSP via TAS, TVS, and 3.0 T MRI were 0.727, 0.776, and 0.844, respectively, and among them, 3.0 T MRI had the highest diagnostic specificity, and TVS and 3.0 T MRI had the highest sensitivity .P < 0.05) Other than intragestational hemorrhage, the detection rates of other signs of disease by TVS and TAS were significantly higher than 3.0 T MRI (For analysis of ROC curves of the three imaging diagnosis modalities, see CSP is a special ectopic pregnancy manifested by the implantation of the gestational sac at the incision of a C-section. At the early stage of CSP, there are no specific clinical manifestations, which largely increase the difficulty of diagnosis, and with the progress of pregnancy, the CSP will easily trigger uterine rupture, major bleeding, and other symptoms, seriously threatening the life safety of patients . UltrasoThe study results showed that compared with 3.0 T MRI, the AUC value was lower in TVS and TAS in diagnosing the CSP, demonstrating that 3.0 T MRI had higher diagnostic efficacy in confirming early CSP. Further analysis found that the AUC value of TVS was higher than that of TAS, and the reasons might be as follows: although the TAS was able to display the relationship between CSP position and the lower uterine segment as a whole and was convenient to observe the uterine corpus and cervix comprehensively, it confirmed the diagnosis using the principle of large interface reflection , hence iIn conclusion, 3.0 T MRI has high diagnostic efficacy in early CSP diagnosis, and its effect is better than that of TVA and TAS, which is beneficial for physicians to carry out observation and develop treatment regimens, demonstrating its role in improving the prognostic effect. Shortcomings of this study: the included sample size was limited, and the findings still need more multicentered studies with large samples for further confirmation; in addition, the quantitative comparative analysis between the lesion and normal myometrium was not performed in this study, so it needs to be more deeply and meticulously explored in the future work.


Objectives: Although supporting preventative self-regulation and self-care activity for daily stress is important as dyadic coping, there seems a paucity of exploration of non-verbal interventions such as tactile communication. This preliminary experimental study assessed the efficacy of a short educational massage programme for healthy but stressed couples. The study aimed to investigate if the educational mutual massage (Positive Massage) programme has any acute and sustained effects on wellbeing, perceived stress and coping, and relationship satisfaction among couples.Design: A pseudo randomised two group design employing a delayed treatment element assessed the effects of the Positive Massage programme and subsequent at-home application. Thirty-eight participants completed a three-week massage course.Main Outcome Measures: Measurements of the Warwick-Edinburgh Mental Well-being Scale, the Rhode Island Stress and Coping Inventory, and the Positive Feelings Questionnaire were collected online using Qualtrics at three time points . Data were analysed with mixed ANOVAs.Results: Mental wellbeing, and perceived stress and coping significantly improved from before to after the Positive Massage programme. There was no significant decline after the cessation of the massage programme. Relationship satisfaction did not show significant changes from the initial assessment.Conclusions: The overall effects of the Positive Massage programme indicate the importance of developing further large scale studies of mutual massage as a safe and beneficial self-care activity. This innovative study has laid the groundwork for future studies into the possibility of mutual massage as a self-regulation dyadic coping strategy for home use to improve overall wellbeing. Decades of research has revealed that stress, especially chronic daily hassles stress, can be detrimental to health and wellbeing  due to its use as a therapeutic intervention for a diagnosed problem. However, a relatively small but significant number of studies exist showing positive effects of massage by lay people such as partners  has any effects on mental wellbeing, perceived stress and coping, and relationship satisfaction among couples;If there are any sustained effects after the completion of the massage programme at week 6; andIf healthy but stressed couples can learn and continue to apply massage with satisfaction at home in their daily lives.The aims of the study were therefore to investigate:massage designates PM rather than other forms of massage.To this end, a simple sequence of massage for home use, called Positive Massage (PM), was devised, and a short massage course (the PM programme) set up for people to learn and practice in close relationships  short massage (15 min) among adult couples rather than one-way (just receiving), aiming for a benefit to both parties’ wellbeing rather than a therapeutic effect for the receivers.Three distinctive elements in this preliminary experimental study were:www.qualtrics.com) in the participants’ homes at the same three time-points  for both groups  or B . There are both ‘between participants’ (groups A and B) and ‘within participants’ (repeated assessments) aspects of the data. The dataset was collected online using Qualtrics software (h groups . SPSS veh groups  was usedhttp://www.consort-statement.org/) (n = 2) and unexpected circumstances (n = 2). Not all the participants recorded the entire datasets at all time-points. Therefore, only 34 participants’ data were used for the analysis 1 and follow up frequency data 2&3, and 16 (i.e. group A) for analysis 2  = .05, p= .83, or relationship length F  = .15, p = .71.Recruitment was by voluntary response sampling via posters, flyers, email and social media. The study’s inclusion and exclusion criteria are shown in nt.org/)  shows, 4rg/) n =  and unexThe 14-item Warwick-Edinburgh Mental Well-being Scale (WEMWBS)   . The RISCI is a well-used inventory in stress and health related studies especially in health behaviours  A short programme for those in close relationships to learn together; 2) Its aim to promote wellbeing by stress reduction and empower connection via skills of caring touch; 3) Simple massage easily applied at home without hassles (i.e. no need to remove clothing and no oil); and 4) First-hand learning and practice in the supervised classes to equip couples with the confidence and skills to practice/exchange massages at home. The philosophical base of the programme shares the principles of positive psychology  at the first class; arm, neck and head massage (11 steps and 9 new components) at the second class; and all parts plus face massage (17 steps and 5 new components) at the third class. A guide for a 15 min sequence for each practice was given to the participants with an allowance to adjust the duration, and being flexible as to the part of the body to apply massage to according to the partner’s need/preference. The participants were encouraged to carry out the massage practice ideally three times a week for three weeks at home. Previous studies of lay massage suggest three times a week massage at home is feasible ;There would be a sustained effect of the PM programme manifest at follow up (T3) in group A ;Participants who undertake the PM programme would have practiced PM (give a massage and receive a massage) an average of at least once per week at home during the programme (frequency data 1);Participants who completed the PM programme in group A would go on to practice PM at least three times during three weeks after the programme finished (frequency data 2); andParticipants would indicate their intention to continue exchanging PM at T3 and also to recommend of PM to their friends and family (frequency data 3).Our hypotheses were:The effects of massage on mental wellbeing, stress and coping, and relationship satisfaction were compared with mixed ANOVAs. The between groups factor was treatment group (A and B) and the within subjects factor was time-point (T1 and T2). Bonferroni corrected paired-samples T-tests were employed to interrogate significant interaction effects further. See F  = 18.48, p < .001, F  = .58, p = .45, F  = 8.36, p = .007, t (16) = −4.10, p = .001, and that there was no significant difference in group B (Delayed Treatment) between T1  and T2 , t (16) = −1.47, p = .16.Mixed ANOVA revealed that there was a significant difference in the WEMWBS score between T1 and T2, F  = 6.68, p = .015, F  = .002, p = .96, F  = 8.38, p = .007, t (16) = 3.27, p = .005, but that there was no significant difference for group B between T1  and T2 , t (16) = −.29, p = . 78.There was a significant main effect of time-point on the RISCI Stress score, F  = 5.31, p = .028, F  = .999, p = .33, F  = 4.58, p = .04, t (16) = −3.2, p = .006, but that there was no significant difference for group B between T1  and T2 , t (16) = − .12, p = . 91.There was a significant main effect of time-point on the RISCI Coping score, F  = .36, p = .55, F  = 1.81, p = .19, F  = 3.80, p = .06, There was no significant main effect of time-point on the PFQ score, To test the potential lasting effects of the PM programme, data from the WEMWBS, the RISCI, and the PFQ at 3 time-points (including at week 6 follow up) were analysed by repeated measures ANOVA followed by Bonferroni corrected pairwise comparisons for group A only. Group B were not subject to follow up data collection. See F  = 8.05, p = .005, p = .003 but interestingly there was no significant difference between T2 and T3 , p = 1., or between T1 and T3, p = .10.Repeated measures ANOVA revealed that there was a significant effect of time-point on the WEMWBS score, F  = 6.62, p = .009, F  = 5.63, p = .016, p = .006, but no significant difference between T1 and T3  p = .16; or T2 and T3, p = .78. Similarly there was a significant improvement in Coping between baseline  and after massage practice  p = .01, but no significant difference between T1 and T3  p = .63; or T2 and T3, p = 1.There was a significant effect of time-point on the both RISCI Stress and Coping scores, Stress F  = .49, p = .62, p = .97, and no significant difference between T1 and T3  p = 1; or T2 and T3, p = 1.There was no significant effect of time-point on the PFQ score, Follow-up data from group A at T3 (week 6) showed, 14 (73.7%) out of 19 participants exchanged massage in both giving and receiving modes after the PM programme finished. The average frequency of massage practice was 5 during post three weeks (i.e. receiving a massage was 4.6 times and giving a massage was 5.4 times during three weeks). The frequency surpassed our hypothesised rate.After the PM programme, 25 (73.5%) participants out of 34 expressed their intention to continue exchanging massage at home. Fourteen participants (41%) indicated for 1–2 times a week; 9 (27%) for 1–3 times a month; and 2 (6%) for 3–4 times a week, while 9 (27%) expressed ‘not sure’ and none said ‘no’.Frequencies showed out of 34, 32 participants (94%) expressed that they would recommend the massage (PM) to their friends and families while 2 (6%) participants were not sure. Responding to the question on how they felt about exchanging massage, 18 participants (53%) indicated definitely positive and 14 (41%) probably positive, while 2 (6%) were not sure and none of the participants indicated negative. Our expectancy in hypothesis 5 was met with high rate of acceptance and willingness to continue PM.The analyses indicated that the PM programme had a significant positive impact on couples’ mental wellbeing, and perceived stress and coping. This result is consistent with the results previously published relating to emotional stress  appears to provide enough skills and confidence to lay couples such that they can execute massage and deliver significant positive effects on their wellbeing. The results suggest that such skills might be included as contributing to a dyadic coping strategy. A real-world dyadic coping strategy can be empowering for couples and may be particularly important for stressed couples, especially when considering the potential accumulation of chronic daily stress that may lead to infirmities, dysfunctional relationships, and even accelerating ageing  = 5.45, p = .026, t (16) = −2.64, p = .018, and that there was no significant difference in group B between T1  and T2 , t (16) = .46, p = .65. Therefore, further investigation of mutual massage effect on relational factors such as perceived closeness, being loved, connectedness and support would be worthy.This is not however, thoroughly reflected in the impact on relationship satisfaction, which at first sight might be disappointing. However, the near-significant interaction is associated with a medium to large sized effect . A previous study of fathers massage on their pregnant wives has also revealed a significant effect of massage on dyadic adjustment with a medium sized effect (Cohen’s d = 0.50)  = .071, p = .79, F  = .31, p = .58, F  = . 98, p = .33, F  = 4.89, p = .034, Additionally, although not a core aim of the study we went on to consider possible gender effects. Data from group A and B were collapsed and pre and post intervention data analysed with mixed ANOVAs. There were no significant gender differences on the WEMWBS score, t (18) = −1.9, p = .07, and that there was no significant difference for male between pre  and post , t (14) = 1.3, p = .23. This gender discrepancy in effects on relationship satisfaction may suggest the usefulness of future studies to explore the most likely population to receive the maximum benefits from such massage i.e. to compare the effect of one-way  massage and two-way (exchange within partner) massage.Paired-Samples T-tests revealed that there was a trend of differences in main effect for female between pre  and post , All the WEMWBS, the RISCI, and the PFQ scores from group A showed no significant differences between T2 (week 3) and T3 (week 6: follow-up survey). These follow-up data indicate the lasting effects of the PM programme on mental wellbeing, perceived stress and coping, and relationship satisfaction 3 weeks after the cessation of the programme. The combined results may suggest that the sustained effect is related to the participants’ continued practice of massage on each other after the cessation of the programme. If this is the case, it may reflect the self-regulatory aspect of PM based on the skills and confidence provided by the programme. An exploration between the compliance rate and the level of mental wellbeing in larger study, as well as a longitudinal study with longer follow-ups would add a deeper understanding regarding this proposal. Since the effects of PM seem to continue after the cessation of the programme, it is possible that the PM programme may provide a cost-effective intervention for mental wellbeing with stress buffering effects. A future cost-effect analysis of the massage programme with input from health economists would add to our understanding.Bodenmann’s  theory oThe simple easily accessible massage (PM) was well accepted by the sample, with 94% of participants indicating that they would recommend PM to their friends and families. This was mirrored by the fact that once the massage course had started, the attrition rate (9.5%) was very low, which is considerably better than for example, a 69% attritionAs a consequence of the pleasantness and popularity of the modality  and psychological factors  may be useful.Time commitment pressures in relation to the online data completion meant that not all participants provided data at all time-points. As a consequence sample sizes for analysis in this study became rather smaller over time. Replication with a larger sample is therefore recommended. A strength of this preliminary study was the study design using an innovative intervention with a delayed treatment component so that all participants were given the opportunity to learn useful massage skills as opposed to employing an inactive control group. However, in contrast, such a study cannot possibly have participants blinded, so demand characteristics are unavoidable. In order to further evaluate the effects of PM and the PM programme rigorously, randomised control trials using an active control group (e.g. relaxation) would be recommended. In addition, the sample in this study may not be representative of general population at large for a number of reasons. Those who responded to the recruitment adverts for the study may be characteristically specific, since they were self-selected volunteers, and as such may be more motivated to be compliant and to deliver positive effects. However, such an intervention would be applicable only to couples who are open and willing to undertake massage, and as such the whole population is not the target at this stage. Qualitative studies investigating how participants experience and perceive PM and the PM programme would be of value by adding a deeper understanding of such a health behaviour and its potential applicability to a wider group. Consideration of motivators and barriers to engagement that such a study could identify can provide important information to improve take up when developing possible health interventions  per week at home during the programme and continued exchanging massage thereafter. About three quarters of participants indicated their intentions to continue massage practice, and 94% participants expressed their willingness to recommend of PM to their friends and family. Therefore, the conclusions that can be drawn are that the PM programme delivered significant improvements in mental wellbeing as well as bringing significant reductions in perceived stress and significant improvement in coping among couples. There were sustained effects after the cessation of the massage programme. Healthy but stressed couples could learn and apply massage with satisfaction at home in their daily lives.massage is not only for therapy, but also for the healthy population to enhance mental wellbeing and provides coping skills and stress buffering effects. If supporting data can be gained from larger trials mutual self-regulation massage should perhaps be promoted as a positive health behaviour among healthy but stressed couples. This innovative study has laid the groundwork for future studies into the possibility of mutual massage as a preventative dyadic coping strategy for home use to improve overall wellbeing in stressed couples and possibly close relationships in a wider group.In addition to providing new insights, the present findings revealed this alternative aspect of massage function: The current preliminary study posits the effects of lay mutual massage in a position of an early stage feasibility trial; therefore further rigorous studies need to be done to confirm the findings. Suggestions for further studies include: larger randomised controlled trials of mutual massage effects on stress, coping and wellbeing, and relationship satisfaction compared to an active control group; longitudinal study over 12 months exploring the relationships between compliance, wellbeing and relationship satisfaction; explorations of other potential effects of PM on couples relational wellbeing that can be measured by perceived closeness, support, empathy, or self-efficacy; evaluation of the couples massage programme in terms of physiological parameters such as cortisol and oxytocin; qualitative studies to explore how couples experience home-based mutual massage such as PM and the PM programme, in particular regarding motivations and barriers to the use of PM; experimental studies to explore the most likely population to receive the maximum benefits from PM; and cost-effectiveness analysis of the PM programme for potential wider promotion.