Detecting dysthyroid optic neuropathy (DON) in the early stages is vital for clinical decision-making. The aim of this study was to determine the feasibility of using an optic-nerve-based radiomics nomogram on water-fat imaging for detecting DON.This study included 104 orbits (83 in the training cohort) from 59 DON patients and 131 orbits (80 in the training cohort) from 69 thyroid-associated ophthalmopathy (TAO) without DON patients. Radiomic features were extracted from the optic-nerve T2-weighted water-fat images for each patient. Selected radiomics features were retrained to construct the radiomic signature model and calculate the radiomic score (Rad-score). The conventional MRI evaluation model was constructed based on apical crowding sign, optic-nerve stretching sign and muscle index. The radiomics nomogram model combining the Rad-score and conventional MRI evaluation factors was then developed. Predictive performance of the three models was assessed using ROC curves.Eight radiomics features from water-fat imaging were selected to build the radiomics signature. The radiomics nomogram  had superior diagnostic performance than did the conventional MRI evaluation model . Decision curve analysis confirmed the clinical usefulness of the radiomics nomogram.This optic-nerve-based radiomics nomogram showed better diagnostic performance than conventional MRI evaluation for differentiating DON from TAO without DON. The changes of the optic-nerve itself may deserve more consideration in the clinical decision-making process.The online version contains supplementary material available at 10.1186/s13244-022-01292-7. Radiomics analysis of the optic-nerve on the water-fat imaging has the ability to predict DON patients.Radiomics nomogram of the optic-nerve performed better than conventional MRI evaluation.The features of the optic-nerve may deserve more consideration in predicting DON in the future.Dysthyroid optic neuropathy (DON) is the most feared complication of thyroid-associated ophthalmopathy (TAO) from optic dysfunction. DON affects approximately 5%-8% of TAO patients , 2. DON Inflammation, ischemia of the optic nerve and mechanical compression at the orbital apex are the most widely accepted mechanisms of DON , 6. PrevWater-fat imaging can provide uniform and reliable fat suppression and is widely available for clinical application –14. AlexRadiomics can extract and analyze innumerable quantitative image details that cannot be detected with visual inspection by human experts, which has been widely applied in a clinical-decision support system to improve diagnostic accuracy , 18. TheThe main objective of our study was to build an optic nerve-based radiomics model on water-fat imaging and to compare its diagnostic performance with that of conventional radiological evaluation for differentiating between DON and TAO without DON.This single-center retrospective study was approved by our Institutional Review Board (IRB ChiECRCT-20170087). A total of 104 orbits from 59 DON patients were included from Tongji Hospital of Huazhong University of Science and Technology  between August 2017 and 2020 December. One hundred thirty-one orbits from 69 TAO without DON patients who were treated at Tongji Hospital were also included. They were age-matched with patients with DON. The inclusion and criteria of participants are as follow: (1) age > 18 years, (2) clear refractive media that allowed sufficient image quality, and (3) no treatment with systemic glucocorticoids for at least 3 months before the study. The exclusion criteria of participants are as follow: (1) poor MR image quality; (2) patients with other neurological or ophthalmologic diseases that could explain the vision loss; (3) patients with signs of severe corneal exposure; and (4) patients who had undergone steroid therapy, radiotherapy, or surgical decompression; (5) TAO without DON patients whose age did not match with DON patients.Thyroid-associated ophthalmopathy (TAO) was diagnosed based on the criteria, established in 1995 by Bartley et al. Dysthyroid optic neuropathy (DON) was made on the basis of the presence of two or more of the following clinical findings: relative afferent pupillary defect when unilaterally affected, color visual defect, reduced visual acuity, papilloedema, visual field defect, and abnormal pattern visual evoked potential test.Participants were randomly divided into the development and validation sets in an 8:2 ratio. Eighty-three orbits from 44 DON patients and 80 orbits from 40 TAO without DON patients were allocated to the development sets, and the subsequent orbits from participants were allocated to the validation sets. Table All participants underwent MRI within one week of the clinical diagnosis of DON or TAO. All MRI scans were conducted on a 3.0 T MR machine  using a 32-channel head coil. The patients were placed in the head-first supine position with their eyes closed during the MRI scanning . CoronalApical crowding: Crowding of the optic nerve at the orbital apex by enlarged extraocular muscles. It was evaluated on coronal water-IDEAL-T2WI imaging as described by Nugent et al. . Additi: Fig. S1Optic nerve stretching: Globe protrusion exceeds the interzygomatic line at least 21 mm. It was evaluated on axial water-IDEAL-T2WI images as described by Rutkowska-Hinc et al. .Muscle index (MI): Assessing the ratio of the extraocular muscle meridian to orbital meridian. Using the method described by Lynn Barret et al. . Additi: Fig. S2The conventional MRI assessments were independently conducted by two radiologists (two trained neuroradiologists with more than 5 years’ experience in diagnosing DON). They were blinded to the diagnoses (TAO with DON or TAO without DON). The conventional MRI assessment included:www.itksnap.org) on the coronal water-IDEAL-T2WI images. VOI was drawn on each image slice that contained the optic nerve tissue by two radiologists with more than 5 years of experience in diagnosing DON independently. The radiologists were unaware of the results of diagnoses (TAO with or without DON). The details of radiomics feature extraction are shown in the Supplementary Material  of the optic nerve tissue was manually segmented with ITK-SNAP software  algorithm was applied to assess the features. The least absolute shrinkage and selection operator (LASSO) logistic regression with tenfold cross-validation was applied to identify the optimal predictive DON features in the development set. The radiomics score (Rad-score) was built using the selected features, weighted by LASSO logistic regression model.The conventional MRI prediction model was developed using logistic regression. Then, a multivariate logistic regression analysis included the independent conventional MRI factors. The Rad-score was selected to build the radiomics nomogram model. The calibration curve and Hosmer–Lemeshow test were applied to investigate the performance characteristics of the radiomics nomogram. Receiver operating characteristic (ROC) curves were constructed to compare the performances of each model for differentiating DON from TAO without DON. The specificity, sensitivity, accuracy, and area under the curve (AUC) of each model were calculated. The AUCs were compared by using Delong’s test. Decision curve analysis (DCA) was applied to determine the clinical usefulness of the radiomics nomogram and the conventional MRI evaluation model by quantifying the net benefits for different threshold probabilities in the validation set.p < 0.05 were considered as statistical significance level. The packages of R3.6.1 that were used are shown in the Supplementary Material . p = 0.019 in development cohort, p < 0.001 in validation cohort). Apical crowding sign occurred in DON patients statistically more often than in TAO without DON patients . And the optic nerve stretching sign occurred in DON patients statistically more often than in TAO without DON patients   and the optic nerve stretching sign (p < 0.05) remained as independent factors in the conventional MRI model.Significant differences existed between groups with regard to the MI, apical crowding sign and optic nerve stretching sign. Multiple logistic regression analyses showed the apical crowding sign .Table The DCA for the radiomics nomogram and conventional MRI evaluation model are shown in Fig. In this study, we developed and validated an optic nerve-based radiomics nomogram on water-fat imaging to provide a noninvasive tool for differentiating DON from TAO without DON. We compared the radiomics nomogram model, radiomics signature model and conventional MRI evaluation model. We summarize our main findings as follows: (1) the radiomics nomogram demonstrated good diagnostic performance in the training and the test cohorts; (2) the radiomics nomogram showed superior predictive performance than using the conventional MRI evaluation model alone; (3) microstructure changes of the optic nerve itself may deserve more consideration for detecting DON, compared to using indirect orbital signs.We chose the MI, apical crowding sign and optic nerve stretching sign as conventional MRI evaluation methods because the overwhelming majority of imaging studies demonstrated their importance in distinguishing DON from TAO without DON. Based on our results, the conventional MRI evaluation model did not achieve a high AUC  for differentiating DON from TAO without DON. Moreover, the evaluation of the apical crowding sign and optic nerve stretching sign could be influenced by the reader’s experience. Barrett et al. demonstrated MI < 50% could almost certainly rule out DON. However, MI was not found to significantly contribute to the conventional MRI evaluation model. MI represents a way to quantitatively approximate the orbital apex crowding. The diagnostic performance of MI was not excellent in our study .http://pyradiomics.readthedocs.io/en/latest/features.html). The least axis length feature yields the smallest axis length of the ROI-enclosing ellipsoid, which is consistent with the orbital apical crowding pathological mechanism in DON. The LAHGLE feature is used to measure the proportion in the image of the joint distribution of large-size zones with higher gray-level values. The SRLGLE feature is used to measure the joint distribution of shorter run lengths with lower gray-level values. Our results revealed the heterogeneity among the gray levels in the optic nerve images between DON and TAO without DON. This finding indicated that microstructural changes of the optic nerve in DON could provide additional information for detecting DON.To our knowledge, this study is the first application of radiomics analysis based on water-fat imaging in detecting DON. Given the potential value of the optic nerve, we drew the optic nerve tissue on the coronal water-IDEAL-T2WI images. LASSO logistic regression analysis showed that least axis length, large area high gray-level emphasis (LAHGLE) and short run low gray-level emphasis (SRLGLE) were the main three predictive radiomics signature features (The definitions were adapted from In the current study, radiomics nomogram model showed better predictive performance than did the conventional MRI evaluation model, and the Rad-score was retrained as the most important predictive factor in the radiomics nomogram. The DCA indicated the radiomics nomogram was more beneficial compared with the conventional MRI evaluation model. Thus, the radiomics nomogram may assist radiologists in detecting DON and making clinical decisions in a timely manner.DON is a severe complication of TAO caused by optic nerve dysfunction, and a previous review indicated that no specific features were found in the optic nerve imaging . HoweverOur study had several limitations. First, this study was a retrospective single-center study. Second, the sample size was relatively small. The potential selection bias may exist and may have affected the robustness of the prediction model. Prospective multicenter studies with large patient numbers are needed to generate the nomograms from bench to bedside. In addition, our study has only focused on water-fat imaging. Combining the analysis of functional MRI may provide a more comprehensive understanding of the potential value of assessing the optic nerve itself to predict DON.We demonstrated the feasibility of developing an optic nerve-based radiomics nomogram for detecting DON in patients with TAO. The radiomics nomogram of the optic nerve could serve as a noninvasive clinical tool in the clinical-decision making process. We believe that the changes of the optic nerve itself deserve more consideration in the task of differentiating DON and TAO without DON in the future.Additional file 1. Additional information of Radiomics feature extraction and Radiomics nomogram calculation formula.


Comorbid depression in knee osteoarthritis (OA) is associated with declines in physical activity, but how persistent depressive symptoms impact physical activity over time remains unclear. We aimed to determine how the persistence of depressive symptoms affects physical activity in knee OA. Participants  from the Osteoarthritis Initiative had radiographic disease (Kellgren-Lawrence grade ≥ 2) in at least one knee. The Center for Epidemiologic Studies Depression Scale  assessed depressive symptoms from baseline through the first three annual follow-up visits, and persistence was operationalized using the cumulative average severity of symptoms over time. Self-reported physical activity was measured from the first to fourth annual follow-up visit using the Physical Activity Scale for the Elderly . The primary method of analysis utilized marginal structural models and included exposure by time interactions in the structural outcome model. Baseline depressive symptoms negatively impacted physical activity at the first follow-up , but at later time points, effect estimates were closer to the null and not statistically significant. The association between time-averaged CES-D scores from baseline through the first follow-up and physical activity at year two was -0.1410 ; and 0.2578  for average CES-D scores through follow-up visit two and physical activity at year three. Thus, the negative influence of persistent depressive symptoms on physical activity decreased over time. Physical activity may not consistently decline with persistent depressive symptoms in adults with knee OA.


Background: Mismatch repair-proficient (pMMR) microsatellite stability (MSS) in colorectal cancer (CRC) indicates an unfavorable therapeutic response to immunotherapy with immune checkpoint inhibitors (ICIs). However, the molecular characteristics of CRC patients with pMMR MSS remain largely unknown.Methods: Heterogeneities between mismatch repair-deficient (dMMR) microsatellite instability (MSI) and pMMR MSS CRC patients were investigated at the single-cell level. Next, an MSS-related risk score was constructed by single-sample gene set enrichment analysis (ssGSEA). The differences in immune and functional characteristics between the high- and low-score groups were systematically analyzed.Results: Based on the single-cell RNA (scRNA) atlas, an MSS-specific cancer cell subpopulation was identified. By taking the intersection of the significant differentially expressed genes (DEGs) between different cancer cell subtypes of the single-cell training and validation cohorts, 29 MSS-specific cancer cell marker genes were screened out for the construction of the MSS-related risk score. This risk score signature could efficiently separate pMMR MSS CRC patients into two subtypes with significantly different immune characteristics. The interactions among the different cell types were stronger in the MSS group than in the MSI group, especially for the outgoing signals of the cancer cells. In addition, functional differences between the high- and low-score groups were preliminarily investigated.Conclusion: In this study, we constructed an effective risk model to classify pMMR MSS CRC patients into two completely different groups based on the specific genes identified by single-cell analysis to identify potential CRC patients sensitive to immunotherapy and screen effective synergistic targets. Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. The treImmunotherapy represented by immune checkpoint inhibitors (ICIs) has shown good curative effects in many malignant tumors, but its application in CRC is still limited to patients with mismatch repair-deficient (dMMR) microsatellite instability (MSI) status. The lacThe differences in immune landscapes between dMMR MSI and pMMR MSS CRC patients have been preliminarily explored at the single-cell RNA (scRNA) level. HoweverIn this study, the heterogeneities between dMMR MSI and pMMR MSS CRC patients were systematically elucidated by the scRNA atlas. A specific cancer cell cluster was identified in MSS CRC patients. Furthermore, a specific MSS-related gene list was assembled by integrated analysis of the training and validation scRNA cohorts, which were subsequently used for constructing the MSS-related risk score. This MSS-related risk score could efficiently separate CRC patients into two subgroups with completely different immune characteristics in the external transcriptome cohorts. CRC patients in the low-score group were identified as “hot” tumors with high abundances of immune cells and an inhibitory immune microenvironment, which means they are potentially sensitive to immunotherapy.https://www.ncbi.nlm.nih.gov/geo/), and the clinical information of these CRC samples is shown in https://portal.gdc.cancer.gov/) as a transcriptome training cohort (TCGA-CRC cohort), while the microarray data of the other 527 CRC patients (73 patients with dMMR status and 454 patients with pMMR status) were downloaded from the GEO database (GSE39582) as a transcriptome validation cohort (GEO-CRC cohort) with MSI-H status (MSI group) and seven CRC patients  with MSS status (MSS group) were used as a single-cell training cohort (GSE132465 and GSE144735), while tC cohort).All of the scRNA data were analyzed by the “Seurat” R package in this study. SpecifiThe risk score of each CRC sample was calculated by the single-sample gene set enrichment analysis (ssGSEA) algorithm based on transcriptome data according to the specific single-cell marker genes. CRC patients in the transcriptome training and validation cohorts were divided into high- and low-score groups according to the median value of the risk score. Survival analysis between the two groups was conducted by the “survMisc” and “survminer” R packages.Abundances of 28 immune cells in each CRC sample were calculated by the ssGSEA method according to the specific markers as previously reported . CorrelaCellMiner is a web tool that predicts the sensitivity of target genes to drugs based on the NCI-60 cell line set. In thisp-value <0.05 was considered statistically significant.All of the data were analyzed by R 4.1.0 in this study. Continuous quantitative data between two groups were compared by Student’s t-test or the Wilcoxon test. Correlation analyses were conducted by the Spearman method. The survival data were analyzed by the log-rank test. A A total of 9366 single cells from CRC patients with MSI status and 9603 cells with MSS status were used to construct the single-cell atlas as a training cohort. The expression profiles of the CRC patients with MSI and MSS status, as well as the correlation between the numbers of features and counts of the single cell, are visualized in It has been reported that the enrichment of CD8 T cells in CRC patients with MSI status is an important reason for their relatively higher sensitivity to immunotherapy with ICIs. Our anaTo investigate the differences in the integrated role among different cell types between the MSI and MSS groups, cell-cell communication analysis was conducted separately. The number and strength of the cell-cell interactions in the MSI group are shown in To validate the specific cancer cell subtypes in the MSS group, a single-cell validation cohort including 11,220 cells from CRC patients (5555 cells in the MSI group and 5665 cells in the MSS group) was analyzed. The expression profiles of the CRC cells, as well as the correlation between the numbers of features and counts, are visualized in Next, DEGs between specific cancer cell Cluster 14 and other cancer cell clusters were identified . By takiFirst, the expression status of the 29 single-cell marker genes was analyzed in the TCGA-CRC cohort. The results showed that most of these genes were significantly differentially expressed between the MSS and MSI groups . Next, tUnder this condition, how to reverse the inhibitory immune microenvironments of the CRC patients in the low-score groups becomes the most pressing question. The correlations between the immune cells and single-cell marker genes were then analyzed. The results showed that these genes have a strong correlation with immune cells, especially CD55 with Th2 cells and LCN2 with Th17 cells . These rIn the GEO-CRC cohort, these 29 single-cell marker genes were also significantly differentially expressed between the dMMR and pMMR groups . The risImmunotherapy represented by ICIs has been approved as the second-line treatment for metastatic CRC with dMMR MSI status, but only a few CRC patients with pMMR MSS status can benefit from immunotherapy due to the lack of immune infiltration and low TMB status. HeterogSome studies have aimed to divide pMMR MSS CRC patients into different subgroups to screen potential patients who are sensitive to immunotherapy or a combination therapy scheme. HoweverIn addition, it is noteworthy that some of these 29 specific single-cell marker genes have been studied in tumor immunotherapy, but theThere are still some limitations of the present study. First, whether this MSS-related risk score can efficiently divide pMMR MSS CRC patients into “cold” or “hot” subtypes needs to be verified in a large number of samples from multiple centers. Second, whether the targeted intervention of these 29 single-cell marker genes can reverse the inhibitory immune microenvironment of “hot” CRC patients and thus enhance their sensitivity to immunotherapy needs to be confirmed by basic and clinical studies.Taken together, the heterogeneities between the dMMR MSI and pMMR MSS CRC patients were systematically elucidated at the single-cell level for the first time. As a result, a specific MSS cancer cell cluster and a specific MSS cancer cell gene list was identified. Next, an MSS-related risk score was constructed based on the 29 specific single-cell marker genes. This risk score model is useful to categorize CRC samples into “cold” or “hot” tumors, which will help us to judge the potential sensitivity of pMMR MSS CRC patients to immunotherapy. Moreover, this model will also provide fundamental knowledge to screen synergistic targets to sensitize pMMR MSS CRC patients to immunotherapy and improve the overall prognosis of CRC patients.


Countries of the Greater Mekong Sub-region aim to achieve malaria elimination by 2030. In the region, malaria is concentrated in high-risk areas and populations such as forest-going mobile and migrant populations (MMPs). However, routine protective measures such as long-lasting insecticidal nets do not prevent all infectious bites in these high-risk populations. Evidence for the effectiveness of a personal protection package tailored to forest-going MMPs which is acceptable, feasible, and cost-effective for reducing malaria transmission is required to inform the malaria elimination toolkit in the region.Plasmodium spp. infection diagnosed by rapid diagnostic test. Difference in prevalence of malaria infection will be estimated across intervention and control periods using generalized linear mixed modelling. Nested within the stepped-wedge cluster-randomised controlled trial is a mixed-methods study to explore the acceptability of the personal protection package, feasibility of implementing a personal protection package as a vector control intervention, and knowledge, attitude and practice of MMPs regarding malaria prevention; and cost-analysis to determine the cost-effectiveness of implementing a personal protection package.A personal protection package consisting of long-lasting insecticidal hammock net, insect repellent and health communication pamphlet was developed in consultation with relevant implementing partners from Cambodia and Lao PDR. An open stepped-wedge cluster-randomised controlled trial will be conducted over a period of 12 months in a minimum of 488 villages (~ 428 in Lao PDR and ~ 60 in Cambodia) to evaluate the effectiveness of the personal protection package. Villages will be randomised into 11 blocks, with blocks transitioned in random order from control to intervention states at monthly intervals, following a 1-month baseline period. The primary outcome of the trial is the prevalence of This study, using a rigorous design and mixed-methods methodology, will evaluate whether a personal protection package can reduce residual malaria transmission among forest-going MMPs in Cambodia and Lao PDR. It will also measure implementation research outcomes such as effectiveness of the intervention package, cost-effectiveness, acceptability, and feasibility, in order to inform potential national and regional policy.Trial registration This trial was prospectively registered on ClinicalTrials.gov (NCT05117567) on 11th November 2021The online version contains supplementary material available at 10.1186/s12879-022-07724-5. Plasmodium spp. parasites transmitted by Anopheles spp. mosquitoes [In the Asia–Pacific region, more than two billion people are at risk of malaria, a vector-borne disease caused by squitoes . The Gresquitoes , largelysquitoes . In recesquitoes , 4. Thessquitoes .Plasmodium spp. infected mosquitos [However, there are several challenges impeding progress towards malaria elimination in the GMS. One of the main challenges is the considerable heterogeneity of malaria transmission in the region. In the GMS, malaria is highly concentrated in hard-to-reach areas along international borders, and in forests and forest fringe communities including forest-going mobile and migrant populations (MMPs) –9. MMPs osquitos . Targetiosquitos . Howeverosquitos  togetherosquitos , 13—do nosquitos . TherefoP. falciparum infections [Few studies have examined the effectiveness of personal protection in reducing malaria in the region , particufections . Furtherfections . Howeverfections —to maximPlasmodium spp. infection in forest-going MMPs and individuals in their residing villages; a mixed-methods study to explore the acceptability of a personal protection package, feasibility of implementing a personal protection package as a vector control intervention, and knowledge, attitude and practice of MMPs regarding malaria prevention; and cost-analysis to determine the cost-effectiveness of implementing a personal protection package. The main text of this paper focuses on the trial with other study components and data collection tools detailed in Additional file This study has three components—an open stepped-wedge cluster-randomised controlled trial to evaluate the effectiveness of personal protection package in reducing the prevalence of Plasmodium spp. infection. The package was designed in consultation with relevant implementing partners: National Malaria Programmes: Cambodia National Center for Parasitology Entomology and Malaria Control and Laos Center of Malariology Parasitology and Entomology; and non-governmental organizations: Health Poverty Action (HPA), PEDA and CHIAs in Cambodia and Lao PDR.The intervention in the trial is a personal protection package to be used by the forest-going MMPs for prevention of The intervention package includes three items—WHO pre-qualified long-lasting insecticidal hammock net (LLIHN) (high-density polyester monofilament yarn blended with 2% (w/w) permethrin), two insect repellent bottles (1-piperidinecarboxylic acid 2-(2-hydroxyethyl)-1-methylpropylester, also known as Icaridin), and an MMP-tailored health communication pamphlet. In addition to the pamphlet, behavioural change communication will also be delivered in the forms of health communication sessions in groups and health communication posters posted at or around the MMP’s workplaces. The intervention package will be distributed through the malaria volunteers operated by implementing partners.Plasmodium spp. infection diagnosed by rapid diagnostic test (RDT). Secondary outcomes of this trial include symptomatic malaria infection diagnosed by RDT, Plasmodium spp. infection as determined by polymerase chain reaction (PCR), Plasmodium spp. infections with drug resistance mutations, prevalence and levels of antibodies to Plasmodium spp. and mosquito salivary antigens  and Lao PDR  will be included in this trial Fig. . The parThe provinces included in the trial have been selected based on the presence of malaria volunteer network in the province, capacity of implementing partners for field implementation, high malaria burden and high MMP activities.Villages/worksites managed by the implementing partner in the selected provinces will be screened against the following exclusion criteria by investigators and implementing partner staff. A village/worksite will be excluded from the study if it: (1) has no malaria cases or annual parasite incidence less than 1 in any of the past three years (2018–2020), (2) has no MMPs, (3) has no malaria volunteer actively working in the village/worksite, (4) has a government health facility for malaria services, or (5) has another malaria volunteer program operated by any organizations other than implementing partner.After the village/worksite has been selected, the MMPs with the following criteria will be included in the study for receiving the intervention : (1) currently living in the selected villages/worksites, and (2) being any of the following types of workers—traditional slash-and-burn and paddy field farming communities visiting their forest farms (commonly ethnic minority groups), seasonal agricultural laborers, forest workers in the informal sector , transient or mobile camp residents associated with commercial projects , and formal and informal cross-border migrant workers.The study design for the trial component of this study is an open stepped-wedge cluster-randomised controlled trial, randomised at the village/worksite level and conducted over a 12-month period, following a one-month baseline period , yielding an approximate total of 64,416 RDT tests over 12 months. Given this sample size, the trial will be able to detect a minimum relative reduction of 34% in the odds (OR = 0.66) of RDT-detectable malaria infection attributable to the intervention  with time-varying fixed factors for intervention status and time, and crossed random effects for village and time implemented to account for the dependencies inherent in the data given the stepped-wedge design. We will also explore any village-specific heterogeneity in effect by specifying a random effect for the intervention and the extent to which effectiveness of intervention is time-dependent. GLMM will be extended to include model terms for country (main and interaction effects), and these will used to assess the extent of country-specific heterogeneity in intervention effect. Analyses of secondary outcomes will also involve multi-level modelling  and GLMM). Statistical analysis will be performed using Stata version 17.Targeting residual malaria in high risk areas and populations in the GMS will be critical to achieve regional elimination goals . This steligible to receive the intervention and are to be recruited in the same manner if this intervention was adopted as part of malaria services provided by malaria volunteers. The study setting of the trial and the organisation and flexibility of intervention delivery in the trial through malaria volunteers is similar to the usual care setting. The intensity of follow-up has the potential to be greater than what would be observed during usual care as malaria volunteers have testing targets to meet to ensure that high study power is achieved. The primary outcome of RDT detectable Plasmodium spp. infection is the detection method used in routine malaria testing so will be of high relevance to normal malaria control practice. The primary analysis will be intention-to-treat and all data will be used for primary outcome analysis regardless of village data completeness across time or level of intervention fidelity.A strength of the study is that it is a pragmatic randomised trial undertaken in the “real world” and with usual care  and therefore will provide evidence to support whether to deliver personal protection packages to MMPs through established malaria volunteer networks as part of national malaria programs. We considered our trial design against the nine domains of the PRECIS-2 (PRagmatic-Explanatory Continuum Indicator Summary tool version 2)  to assesTo pragmatically evaluate personal protection packages to reduce malaria in MMPs we chose a stepped-wedge cluster-randomised trial design. There are several advantages to using a stepped-wedge cluster-randomised trial design over a parallel group design (where each cluster is randomised to either an intervention or control condition for the entire duration of the study). In the stepped-wedge design clusters act as their own controls as they experience both the control and intervention conditions during the study period and all clusters ultimately receive the intervention which is important where the intervention is thought to have a largely positive effect and exclusion of study subjects from such benefit might be considered unethical . HoweverThe proposed study will provide an evidence base for the distribution of tailored personal protection packages for high-risk MMPs in malaria elimination programs in the GMS through established malaria volunteer networks. The pragmatic trial design representing “real-world” distribution of the intervention, together with the nested mixed-methods study which measures essential outcomes for policy adoption of a new tool or strategy will ensure trial findings are translated into National Malaria Guidelines of GMS countries in order to meet national and regional malaria elimination goals.Additional file 1. Supplementary methods for the nested mixed-methods study, quantitative and qualitative data collection tools, and participant information and consent forms.


Adolescence is a critical age where steep declines in physical activity and increases in sedentary time occur. Promoting physical activity should therefore be a priority for short- and long-term health benefits. Wearable activity trackers in combination with supportive resources have the potential to influence adolescents’ physical activity levels and sedentary behavior. Examining the pathways through which such interventions work can inform which mediators to target in future studies.The aim of this paper is to examine the impact of the Raising Awareness of Physical Activity (RAW-PA) intervention on potential mediators of behavior change after intervention, and whether these mediated the intervention effects on physical activity and sedentary time at 6-month follow-up.RAW-PA was a 12-week intervention, grounded in social cognitive theory and behavioral choice theory, aimed at increasing physical activity among inactive adolescents through combining a wearable activity tracker with digital resources delivered via a private Facebook group (n=159 complete cases). The targeted potential mediators were identified from previous studies conducted in adolescents and included self-efficacy, peer support, family support, teacher support, self-regulation strategies, barriers, and enjoyment. Outcomes included sedentary time as well as light- and moderate-to-vigorous–intensity physical activity. A series of mixed linear models were used to estimate intervention effects on physical activity and sedentary behavior at follow-up and on potential mediators after intervention and to test whether there were indirect effects of the intervention on physical activity and sedentary behavior via mediators.P=.03). However, indirect effects for each outcome were not statistically significant, indicating that perceived barriers to physical activity did not mediate intervention effects for physical activity or sedentary time.Adolescents in the intervention group (n=75) engaged in higher sedentary time and lower light intensity at 6-month follow-up compared to the wait-list controls (n=84). There were no intervention effects for moderate-to-vigorous–intensity physical activity. The intervention group perceived more barriers to physical activity than the wait-list control group at 6-month follow-up (mean adjusted difference=1.77; 95% CI 0.19-3.34; RAW-PA did not beneficially impact hypothesized mediators in these inactive adolescents, despite strategies being designed to target them. This suggests that the lack of overall intervention effects on physical activity and sedentary time observed in the RAW-PA study could be due to the limited impact of the intervention on the targeted mediators. Future studies should consider different strategies to target theoretically informed potential mediators and identify intervention strategies that effectively target key mediators to improve physical activity among inactive adolescents. Finally, intervention effects according to level of wearable tracker use or level of engagement with the intervention should be explored. This may provide important insights for designing successful wearable activity tracker interventions.Australian New Zealand Clinical Trials Registry ACTRN12616000899448; https://anzctr.org.au/Trial/Registration/TrialReview.aspx?id=370716&isReview=trueRR2-10.1186/s12889-016-3945-5 Most adolescents worldwide do not engage in sufficient physical activity to benefit health, including cardiometabolic health, and cognitive development ,2. This The past decade has seen advances in the development of commercially available wearable activity trackers. Such devices can measure physical activity, enable self-monitoring, and provide real-time feedback. Wearable activity trackers are increasingly being used in physical activity research as both a measurement and intervention tool, while some devices have the capability to provide feedback and alerts for periods of sedentary time ,9. EmergPrevious technology-based physical activity interventions using activity trackers have shown that multicomponent interventions may be more effective than using wearables as a stand-alone strategy ,11. ThatThe Raising Awareness of Physical Activity (RAW-PA) study was a multicomponent intervention for adolescents living in areas of socioeconomic disadvantage that aimed to integrate more physical activity into their day by combining an activity tracker with digital resources that specifically targeted evidence-based behavior-change techniques. Grounded in social cognitive theory  and behaEthical approval was obtained from the Deakin University Human Research Ethics Committee (2016–179) and the Victorian Department of Education and Training.The RAW-PA study was evaluated using a cluster-randomized controlled trial design, with schools being the unit of randomization . The triThe eligibility criteria for participants included the following: (1) being ≥13 years old; (2) having access to the internet outside of school; (3) not engaging in regular organized physical activity or sport outside of school; (4) not meeting physical activity guidelines of ≥60 minutes of moderate to vigorous physical activity (MVPA) every day; and (5) not a current or past owner of an activity tracker. All eligible students n=280) who returned informed written parental consent and student assent were recruited into the study ) of the analytic sample were calculated and descriptively compared with excluded participants. As per the Consolidated Standards of Reporting Trials recommendations , baselinsuppression role), for example, because the intervention failed to impact important intermediate factors (a path) or that the intermediate factors did not impact the outcome as hypothesized (b path). Accordingly, modern approaches to mediation emphasize the importance of testing the a and b paths irrespective of the total intervention effect on the outcome. A visual representation of the hypothesized mediator model, including the several pathways assessed, is depicted in a path), while adjusting for baseline levels of the mediator. For potential mediators on which there was a statistically significant intervention effect at T1, single-mediator analyses were conducted to determine the indirect effect of the intervention on physical activity and sedentary time at 6-month follow-up (T2) via the mediator (assessed after intervention [T1]). This involved fitting a model while simultaneously estimating the effects of the intervention  and the potential mediator (b path) on each outcome variables at 6-month follow-up (T2). The mediated, or indirect, effect  was calculated as a × b, following the “product of coefficients” method, and this quantity was bootstrapped with 1000 resamples to produce percentile-based 95% CIs [c path). All models in this stage were adjusted for baseline levels of both the mediator and total physical activity or sedentary time (depending on outcome of interest).Mediation analysis of randomized controlled trials is important both when an intervention effect on the outcome is observed and when it is not . In the  95% CIs . The modBaseline (T0) participant characteristics and accelerometry variables at baseline and 6-month follow-up (T2) are shown in a path). There was evidence of an intervention effect on the perceived barriers to physical activity score after intervention, with adolescents in the intervention group perceiving more barriers to physical activity compared to those in the wait-list control group . There was little evidence of intervention effects for the remaining potential mediators.The mean scores for each mediator at baseline (T0) and after intervention (T1) are shown in c path) and direct  effects of the intervention on physical activity and sedentary behavior at 6-month follow-up . The total effects indicated that adolescents in the intervention group engaged in higher sedentary time and lower LPA at 6-month follow-up (T2) compared to the wait-list controls. These differences were still observed while adjusting for the perceived barriers to physical activity score .As the perceived barriers to physical activity were the only potential mediators for which there was a statistically significant intervention effect, formal mediation analyses were only conducted for this factor. a path), with adolescents in the intervention group perceiving more barriers than those in the wait-list control group, this did not mediate the intervention effects on physical activity or sedentary time at 6-month follow-up. No intervention effects were observed on any of the remaining factors identified as potential mediators, despite the intervention aiming to change target variables  hypothesized to be causally related to youth physical activity and sedentary time [This study aimed to examine the impact of the RAW-PA intervention on potential mediators of behavior change after intervention, and whether these mediated the intervention effects on physical activity and sedentary time at 6-month follow-up. While the intervention had a significant adverse effect on the perceived barriers to physical activity after intervention (ary time ,20.While there has been some variability of intervention effects on the barriers assessed in previous studies conducted in youth ,36,37, tWhile the analyses found evidence of an intervention effect on the perceived barriers to physical activity, there was no evidence of an indirect mediating effect. That is, the perceived barriers to physical activity after intervention did not mediate physical activity and sedentary time at 6-month follow-up. This suggests that the change in perceived barriers may not be on the causal pathway , which iTo date, few activity tracker interventions, regardless of population group, have examined mediators of physical activity and sedentary time. A recent review highlighted that activity tracker studies have instead focused on mediators of outcomes such as task motivation in adult populations, with intervention effects observed for mediators including self-efficacy and self-awareness . Despitemissions [This is the first study, to the authors’ knowledge, to examine potential mediators of change in physical activity and sedentary time during an activity tracker intervention conducted in adolescents. The mediators were targeted within weekly missions  and weremissions . Howevermissions . Second,In conclusion, an intervention effect was only observed for barriers to physical activity, with adolescent’s perceiving more barriers than those in the wait-list control immediately after intervention. However, this did not mediate changes in physical activity and sedentary time at 6-month follow-up. This suggests that the lack of overall intervention effects on physical activity and sedentary time observed in the RAW-PA study could be due to the limited impact of the intervention on the targeted mediators. Future studies should identify intervention strategies that effectively target key mediators to improve physical activity among inactive adolescents. They should also explore additional potential mediators that may explain changes in the use of activity trackers and digital resources over time. Finally, intervention effects according to the level of wearable tracker use or the level of engagement with the intervention should be explored. This information is critical for the design of future successful interventions to increase physical activity in adolescents.


Vaccine-associated hypermetabolic lymphadenopathy (VAHL) after a COVID 19 vaccination is a common adverse event and also a diagnostic challenge especially in patients with a history of a malignancy.d-glucose positron emission tomography (FDG-PET) performed 3 weeks later revealed an FDG uptake in those swollen lymph nodes and in the novel lymphadenopathy in the left axilla and left subclavicular regions. Both biopsy specimens from the right supraclavicular and hilar lymph nodes revealed only multiple small granulomas with multinucleated giant cells without malignancy, consistent with sarcoidosis. The left axilla and subclavicular lymphadenopathy detected by the FDG-PET subsequently spontaneously regressed.A 47-year-old woman presented with enlarged lymph nodes in the right hilar, subcarinal, and right supraclavicular regions detected by computed tomography as a postoperative follow-up study of thyroid cancer. Fluorine-18 fluoro-2-deoxy-The coincidental occurrence of VAHL and lymphadenopathy in sarcoidosis patients could cause diagnostic confusion especially in those with breast cancer.Sufficient attention should be paid both to the injection site and the time interval between the vaccination and imaging test in the era of nationwide mass vaccinations against COVID 19. •Vaccine-associated hypermetabolic lymphadenopathy (VAHL) after a COVID 19 vaccination is a common adverse event.•Sarcoidosis is a multisystem granulomatous disorder of an unknown etiology and often involves the lymph nodes.•The coincidence of VAHL and sarcoidosis poses a diagnostic challenge in patients with a history of breast cancer. Attention should be paid to both the injection site and time interval between the vaccination and oncological imaging test in the era of widespread COVID-19 vaccinations. It often involves the lymph nodes, and a bilateral hilar lymphadenopathy (BHL) is a characteristic imaging finding We report a case of a COVID19 vaccine-associated hyper metabolic lymphadenopathy (VAHL) concomitant with sarcoidosis in a patient with breast cancer.This work has been reported in line with the SCARE criteria 2A 47-year-old woman presented with enlarged lymph nodes in the right hilar, subcarinal, and right supraclavicular regions detected by computed tomography (CT) as a follow-up study of thyroid cancer . She hadd-glucose positron emission tomography (FDG-PET) performed 3 weeks later revealed an FDG uptake in those swollen lymph nodes and in the novel lymphadenopathy in the left axilla and left subclavicular regions  was performed on the right hilar lymph node through a 21-gauge needle and the histology showed an epithelioid granuloma along with lymphoid tissue . WhereasAn additional echocardiography and ophthalmologic examination revealed no ocular or cardiac involvement, suggesting no therapeutic requirements. The left axillary and subclavicular lymphadenopathy regressed spontaneously and continued to be undetectable by ultrasonography at 7 months after the PET imaging.3Sarcoidosis is a multisystem granulomatous disorder of unknown etiology that could involve any organ such as the lymph nodes, lungs, eyes, or skin VAHL after a COVID19 vaccination (BNT162b2/1273) is a common adverse event and the most frequently involved site is the axilla, followed by the supraclavicular, neck, and pectoral regions 4A coincidental occurrence of a VAHL and lymphadenopathy in patients with sarcoidosis could cause diagnostic confusion especially in those with breast cancer. An EBUS-TBNA is a feasible option for a histological confirmation of a hilar lymph node. Sufficient attention should be paid to both the injection site and time interval between the vaccination and oncological imaging test in the era of nationwide mass vaccinations against COVID 19.Not applicable.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Not applicable.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.AK wrote this paper. YO reviewed the pathological findings. All authors read and approved the final manuscript.Not applicable.Masayuki Yoshida.Not applicable.Not commissioned, externally peer-reviewed.The authors declare that they have no competing interests.


F-actin dynamics (polymerization and depolymerization) are associated with nucleotide exchange, providing the driving forces for dynamic cellular activities. As an important residue in the nucleotide state-sensing region in actin, His73 is often found to be methylated in natural actin and directly participates in F-actin dynamics by regulating nucleotide exchange. The interaction between His73 and its neighboring residue, Gly158, has significance for F-actin dynamics. However, this weak chemical interaction is difficult to characterize using classic molecular modeling methods. In this study, ab initio modeling was employed to explore the binding energy between His73 and Gly158. The results confirm that the methyl group on the His73 side chain contributes to the structural stability of atomistic networks in the nucleotide state-sensing region of actin monomers and confines the material exchange (Pi release) pathway within F-actin dynamics. Further binding energy analyses of actin structures under different nucleotide states showed that the potential model of His73/Gly158 hydrogen bond breaking in the material exchange mechanism is not obligatory within F-actin dynamics. F-actin is polymerized from G-actin in living cells and acts as the structural basis for the mechanical performance and dynamic behaviors of microfilament networks . F-actinIn addition to conformational changes, the His73 residue on the H-loop also directly participates in nucleotide exchanges during F-actin polymerization. On natural actin monomers, His73 is the only residue that is often modified with an extra methyl, for which the biological function is still cryptic. According to in vivo experiments, mutations of His73 would significantly change the ATP hydrolysis rate . The metIn this study, we evaluate the stability of the nucleotide state-sensing region by analyzing the binding energy between His73 and Gly158 via ab initio modeling. Actin structures with different methylation and nucleotide states are studied to understand the structural significance of His73 conformational changes in nucleotide exchanges. The analysis provides insights into an improved understanding of the critical role of His73 in regulating nucleotide exchanges in F-actin dynamics.bsseE) is comparable to the binding energy (bE) characterization, confirming that the basis set superposition error is obligatory for the calculation of weak chemical interactions in biological structures.To theoretically validate the structural significance of His73 methylation in F-actin dynamics, six actin structures with different methylation states were considered in the present ab initio study, which were all in the ATP binding state. Using the method elaborated in The ab initio calculation results suggest that actin structures with side chain methyl groups all present higher structural stability. Nyman, et al. proposed that the side chain methylation of His73 would affect the interaction between His73 and its neighboring residues . A typicAccording to the ab initio characterization of the selected actin structures, the aforementioned H-bond (N-H···O) between His73 and Gly158 can be located on the actin structures 1ATN and 2A3Z refer to . HoweverThe sequential conformational changes in actin monomers can trigger ATP hydrolysis and create a pathway for material exchanges in F-actin polymerization and depolymerization. In these conformational changes, the rearrangement of the H-loop after ATP hydrolysis putatively creates a pathway for Pi to release from the actin monomer after ATP hydrolysis ,8. To vaThe binding energies between His73 and Gly158 on ATP-actin can be either higher or lower than ADP-actin based on the calculation of different actin structures. The conformations of His73/Gly158 are shown in It can be found that the ATP-actin structures do not necessarily experience an H-bond interaction between His73 and Gly158. ADP-actin can also present a highly stabilized His73/Gly158 interaction, which confines the material exchange pathway during F-actin polymerization and depolymerization. The molecular mechanisms for actin structure conformational changes in material exchange are still cryptic in biophysics analyses. More ab initio modeling of the interactions between other keynote residues concerning nucleotide-binding states should be developed to obtain more convincing evidence on the prominent conformation changing mechanisms which directly regulate nucleotide exchange processes and affect F-actin dynamics in cellular processes.The interaction between the residues in the abovementioned actin structures is difficult to characterize using classic molecular dynamics simulation. Ab initio quantum chemistry methods can provide more information about the variation of weak interaction change in different states . FreedmabE) between the His73 and Gly158 is defined as:HisE73, GlyE158, and combE represent the energies of the isolated His73, Gly158, and the combined super-molecule, respectively, and bsseE denotes the basis set superposition error. The calculated binding energies between His73 and Gly158 were used to evaluate the stability of the atomistic networks of actin monomers with different nucleotide and His73 side chain methylation states. The molecular visualizations were carried out using VMD [All the ab initio calculations were performed using the Gaussian09 package . A Densising VMD  and Avogsing VMD  softwareTo understand the role of His73 side chain methylation in nucleotide exchange in F-actin dynamics, six Oryctolagus skeletal muscle actin with different methylation states were first selected. Three of these crystal structures were of the methylated His73, including 1ATN , 1Y64 2, and 2A3An H-bond between His73 and Gly158 was reported to be important in stabilizing the molecular structures of actin monomers before ATP hydrolysis . To valiIn the present study, we employed the ab initio method to investigate the structural significance of His73 in F-actin dynamics. The ab initio characterization of the binding energy between His73 and Gly158 in actin monomers suggests that the methyl group on the His73 side chain contributes to the structural stability of atomistic networks in the nucleotide state-sensing region of actin monomers and confines the pathway of material exchange in F-actin dynamics. However, the potential His73/Gly158 H-bond breaking mechanism for the formation of the Pi release pathway is not obligatory during F-actin depolymerization. As a quantum mechanics-level method, ab initio modeling is the first method ever employed to understand the structural significance of His73 in F-actin dynamics and shows great potential to understand various structural basis findings associated with dynamic cellular processes.


Since the beginning of the COVID-19 pandemic and development of new vaccines, the issue of post-vaccination exacerbation or manifestation of demyelinating central nervous system (CNS) disorders has gained increasing attention.We present a case of a 68-year-old woman previously diagnosed with multiple sclerosis (MS) since the 1980s who suffered a rapidly progressive severe sensorimotor paraparesis with loss of bladder and bowel control due to an acute longitudinal extensive transverse myelitis (LETM) after immunization with the mRNA Pfizer–BioNTech COVID-19 vaccine. Detection of Aquaporin-4-antibodies (AQP4) in both serum and CSF led to diagnosis of AQP4-antibody positive neuromyelitis optica spectrum disorder (NMOSD). Treatment with intravenous corticosteroids and plasmapheresis led to a slight improvement of the patient’s symptoms.Pathogenic mechanisms of post-vaccination occurrence of NMOSD are still unknown. However, cases like this should make aware of rare neurological disorders manifesting after vaccination and potentially contribute to improvement of management of vaccinating patients with inflammatory CNS disorders in the future. So far two cases of AQP4-antibody positive NMOSD have been reported in association with viral vector COVID-19 vaccines. To our knowledge, we report the first case of AQP4-antibody positive NMOSD after immunization with an mRNA COVID-19-vaccine. Post-vaccination exacerbation or manifestation of inflammatory central nervous system (CNS) disorders is a rare phenomenon that has been reported in association with few types of vaccinations (e.g. vaccination against human papilloma virus (HPV), influenza, tetanus) . HoweverSince the beginning of vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the global COVID-19 pandemic, few cases of adverse events associated with occurrence or deterioration of demyelinating CNS diseases such as multiple sclerosis (MS) have been published . RegardiHerein, we present a case of AQP4-antibody positive NMOSD presenting as severe LETM following first vaccination with the mRNA Pfizer–BioNTech COVID-19 vaccine (BNT162b2) in a female patient with suspected MS for over 40 years.A 68-year-old Caucasian woman presented with a rapidly progressive severe sensorimotor paraparesis a few days after having received the first mRNA COVID-19 vaccination (BNT162b2) in May 2021. The patient’s history showed the suspected diagnosis of a secondary progressive MS for over 40 years with unknown date of transition. After having suffered a severe relapse in the early 1980s the patient had lived with a residual mild paraparesis. The patient had never received a disease-modifying therapy (DMT). Relapses had been treated with intravenous corticosteroids. Regarding her vaccination history, the patient had regularly received the vaccines recommended by the German Standing Committee on Vaccination without any relevant side effects prior to the following events.In April 2021, the patient first suffered a deterioration of the paraparesis shortly after having been vaccinated against tetanus and pneumococci. She was admitted to an external hospital. Spinal cord MRI showed signs of atrophy of the cervical and thoracic spinal cord without Gadolinium (GD)-enhancement. Cerebrospinal fluid (CSF) analysis did not show any abnormalities. Due to a suspected MS relapse the patient was treated with intravenous corticosteroids. Subsequently the symptoms receded and the patient reached prior level of disability.On the 5th of May 2021 the patient received the first mRNA COVID-19 vaccination and 23 days later developed a severe exacerbation of the paraparesis with sensory level of T8 and inability to walk as well as loss of bladder and bowel control (EDSS 7.0). A new spinal cord MRI now showed T2-signal alteration ranging from C4 to T10 with GD-enhancement from C3 to C5 as sign of an acute LETM . Considering the highly active disease progression, DMT with eculizumab was initiated promptly. Since the patient showed deterioration of symptoms both times she was immunized, required meningococcal vaccination was not applied for safety reasons and prophylactic antibiotic therapy was initiated in the induction phase of eculizumab. The patient later decided not to receive the vaccination at all, hence therapy was changed to satralizumab. In the follow-up period of six months no new relapses have occurred. The patient also declined the second COVID-19 vaccination.AQP4-antibody positive NMO represents a rare chronic inflammatory disorder of the CNS as a result of an autoantibody- and complement-mediated astrocytopathy due to high expression of the antigen AQP4 at the astrocytic endfeet, which is followed by secondary demyelination and often extensive axonal and neuronal damage, which primarily affects the spinal cord, optic nerves and brain stem areas . Due to In this case the patient’s clinical presentation had been classified as MS in the early 1980s and not re-evaluated since, presumably as she had not suffered from a severe relapse until recently. Furthermore, the patient has never been treated with a DMT. Interestingly she already had suffered a mild exacerbation without any correlate in spinal cord imaging after having been vaccinated against tetanus and pneumococci, possibly indicating slight activation of the immune system. Following immunization with an mRNA COVID-19 vaccine the patient suffered from a severe disease exacerbation presenting as LETM which led to clinical re-evaluation and ultimately diagnosis of NMO due to detection of AQP4-autoantibodies in both serum and CSF.The timeline of these events is highly suggestive for a potential triggering of so far self-contained autoimmune-mediated processes increased by the COVID-19 vaccination. Of note, causality between vaccination and relapse or manifestation of NMOSD have not been verified yet. Also, specific testing for serum autoantibodies against SARS-CoV-2 as a potential marker for the immune response to the COVID-19 vaccine has not been performed. Furthermore, it has to be taken into account that the patient received three types of vaccinations in a short period of time, so possible delayed effects of the first vaccinations or combined effects of an unspecific “bystander immune activation” as a consequence of these different vaccinations could be associated with the disease exacerbation observed in this particular case , 11. SinTaken together, this case adds evidence to the few reported cases of NMOSD manifestation upon COVID-19 vaccination and warrants to potentially avoid repetitive vaccinations against COVID-19 combined with other vaccinations with only a short in-between interval, if possible. Furthermore, effective treatment must be ensured in patients suffering from inflammatory CNS diseases before immunization. Finally, regarding the advances in diagnostic possibilities for neurologic disorders re-evaluation of diagnoses from the past should always be considered.


Two-armed FabscFv-Fc is a favoured bispecific antibody (BsAb) format due to its advantages of the conventional IgG structure. Production of FabscFv-Fc requires expression of three polypeptide chains, one light chain (LC), one heavy chain (HC) and a scFv fused to the Fc (scFvFc) at optimal ratios.We designed a set of internal ribosome entry site (IRES)-mediated multi-cistronic vectors tailoring to various expression ratios of the three polypeptides to study how the chain ratios affect the FabscFv-Fc production.Expression of HC and scFvFc chains at 1:1 ratio and excess LC gave the highest yield of correctly assembled product. Compared to the use of IRES and multiple promoters, using 2A peptides for co-expression of the three polypeptides gave the highest titre and correctly assembled product.The results obtained in this work provide insights to the impacts of hetero-chain ratios on the BsAb production. Statement of Significance: Two-armed FabscFv-Fc is a favoured bispecific antibody formats but challenging to produce. This paper presents a general strategy to customize the expression ratios of polypeptide chains to produce correctly assembled FabscFv-Fc at high titres. Similar approach can also be applied to optimize the production of other formats of bispecific antibody classes. Bispecific antibodies (BsAbs) have emerged as a promising class of next-generation antibody therapeutics. In contrast to the standard monoclonal antibodies, BsAbs can recognize two different epitopes simultaneously, thereby allowing them to perform a wide range of applications that cannot be achieved with their monospecific counterparts, such as blocking multiple pathogenic mediators simultaneously and redirecting effector cells to the proximity of tumour cells. More than one hundred formats of BsAbs have been developed, which can be broadly categorized into two groups, fragment-based (no Fc) and IgG-like asymmetric BsAbs . The IgGet al. found out that expressing the common LC, HC1 and HC2 at 3:1:1 ratio was optimal for efficient assembly of the correct product [et al. [IgG-like asymmetric BsAbs usually consist of three to four polypeptide chains, which are often engineered to promote cognate chain pairing through framework engineering. For instance, correct LC/HC pairing can be achieved by using orthogonal Fab interface or CrossMab (one Fab with a CH1 domain switched with the partner CL domain) . Alterna product . In anot [et al.  revealedChinese hamster ovary (CHO) cells are the dominant host cells for producing therapeutic antibodies due to their advantages of robust cell growth and capability of performing proper post-translational modifications . StudiesBesides integration sites, the expression level of genes are also determined by the DNA regulatory elements applied on them. Co-expression of multiple genes from a single vector can be achieved by using multiple promoters (MP), internal ribosome entry site (IRES) or 2A peptides. Arrangement of MP in one vector can result in transcriptional interference, where an active transcriptional unit suppresses the expression of other units . As suchTwo-armed FabscFv-Fc is one of the most favoured asymmetric IgG-like BsAb formats due to its advantages of the conventional IgG structure. Currently 18 molecules that are developed using the FabscFv-Fc format are in clinical trials . ProductThe protocols for construction of landing pad and targeting vectors were described in The stably transfected CHO cell pools expressing different targeting vectors were generated via RMCE . They weCulture supernatants from fed-batch cultures were collected at day 14. They were purified by protein A followed by size-exclusion chromatography (SEC) analysis. The percentage of high molecular weight (HMW) and low molecular weight (LMW) species in each sample was quantified based on the total peak areas that were eluted before and after the monomeric peak, respectively. The detailed protocols were described in The purified samples were subjected to SDS-PAGE, followed by Coomassie Blue staining to assess the presence of different species in a sample. 2–3 μg of each sample were resolved on the NuPAGE™ 4–12% Bis-Tris Protein Gels (Thermo Fisher Scientific) in 3-(N-Morpholino)-Propanesulfonic Acid (MOPS) buffer following manufacturer’s protocol under both reducing and non-reducing conditions. The gels were then incubated in the fixing buffer (50% Methanol and 10% Acetic acid) for 10 min, and then followed by 10-min staining in 0.1% Coomassie blue staining solution on an orbital shaker. De-staining was performed using 30% ethanol until the background is clear. Gel photos were taken with the ChemiDoc Imaging System (Biorad).Western blot was performed to detect the intracellular polypeptides in stably transfected pools. Cell lysates (5 μg each) were separated in SDS-PAGE followed by dry transfer onto a Hydrophilic polyvinylidene fluoride (PVDF) member. Membranes were blocked for 1-h followed by overnight incubation with corresponding primary antibodies. The detailed protocol was described in Genomic DNA (gDNA) was extracted and subjected to two junction PCRs to confirm the successful cassette exchange after recombinase-medidated-cassette-exchange (RMCE) in each stable cell pool. The detailed protocols and a list of primers used for junction polymerase chain reactions (PCRs)  via RMCE . The MCLTo express the three chains constituting KIH-based antibodies at different ratios, we designed one set of targeting vectors to carry the trastuzumab LC, engineered trastuzumab HC with a “knob” formed at CH3 domain (HCK) and an empty Fc chain (FcH) or a Fc chain fused with an anti-CD3 scFv (scFvFcH). The CH3 domain in FcH or scFvFcH was engineered to form a “hole”. The three chains were linked through multiple IRES elements . After iSince knob/hole heterodimerization is critical for proper assembly of KIH-based bispecific antibodies, we set out to study the impact of varying the knob to hole chain ratios on HC heterodimerization. To this end, one-armed (OA) KIH antibodies consisting of three chains, LC, HCK and FcH, were used as model antibodies . The asyWestern blot analysis of the intracellular polypeptides in these stable cell pools were then performed using antibodies against the LC and Fc regions. The relative abundance of different chains in each sample was quantified by measuring the band intensity . A contrThe stably transfected pools were characterized in 14-day fed-batch cultures to examine the effects of different LC:HCK:FcH ratios on the expression level and assembly of one-armed KIH antibodies. Varying the ratios of the three polypeptide chains by reducing expression of LC, HCK or FcH had no impact on cell growth as indicated by the similar viable cell density (VCD) over the 14-day fed-batch culture period across different stable pools . HoweverThe culture supernatants harvested from different stable cell pools were purified by Protein A to obtain materials for chain assembly analysis. SEC was used to examine the product purity profile by distinguishing the size difference of species resulted from correct and incorrect chain assembly. When expression of HCK and FcH polypeptides were roughly at 1:1 ratio, more than 90% of the product was correctly assembled with minimal aggregate or incompletely unassembled fragments . ReducinUpon understanding how different ratios of subunits influenced the assembly and expression of HC heterodimerization, we further studied the optimal expression ratios for producing the 2-armed FabscFv-Fc BsAb. In contrast to the OA KIH antibodies, FabscFv-Fc had the FcH chain fused with an anti-CD3 scFv . Such fuChain assembly efficiency was studied using SEC and SDS-PAGE under reducing and non-reducing conditions. The products expressed from the three control vectors, which contained genes encoding two of the three chains, had high levels of aggregates and fragments. In contrast, the vectors expressing all three chains and HCK and scFvFcH at 1:1 ratio produced products with similar SEC profiles , containThe LC/HCK/scFvFcH stable pools, which expressed the HCK and scFv-FcH at 1:1 ratio and LC in excess by using multiple IRES, gave the highest titre and most efficient assembly of FabscFv-Fc . ReducinUnder reducing condition, the purified products expressed from IRES and 2A targeting vectors showed three distinct bands corresponding to the correct MW of the three peptides LC, HCK and scFv-FcH, respectively . MajoritMany antibody framework engineering strategies, such as KIH technology, are effective to solve the multiple chains-mispairing problems. However, efficient production of correctly assembled BsAb still requires expression of their constitute chains at optimal ratios. Being able to design vectors that can customize the relative expression levels of multiple chains is advantageous for robust production of complex BsAb molecules. To study how the chain ratios affect the FabscFv-Fc production, we designed a set of IRES-mediated multi-cistronic vectors tailoring to a range of various expression ratios of their constitute chains and stably expressed them in CHO cells via targeted integration. We found out that expression of HCK and scFv-FcH chains at similar amount and LC in excess was optimal for enhancing the yield of correctly assembled FabscFv-Fc. Compared to the use of IRES and MP, using 2A peptide for co-expression of the three polypeptides gave the highest BsAb expression level and proportion of correctly assembled product. However, incomplete cleavage at furin recognition site and 2A resulted in production of some products attached with 2A residues. Further engineering of furin recognition site and 2A for enhancing cleavage efficiency is needed to overcome this issue. The results in this study shed insights into the correlation between the optimum ratios of chain expression levels and the efficiency of BsAb assembly, which could be extrapolated to the production of other KIH-based BsAbs. In addition, the vector design strategies presented here can also be applied in optimizing the production of other formats of BsAbs in which the optimal chain ratios could be different and varied depending on the specific formats and molecules.Consistent with a previous study, we found out that equal expression of knob and hole chains was optimal for enhancing the yield of correctly assembled products for both KIH based one-armed model antibodies and 2-armed FabscFv-Fc BsAbs . DeviatiFor monoclonal antibodies (mAbs), it is recognized that expression of excess LC over HC is desirable for enhancing the expression levels and reducing aggregation . The int2022-08-22_R1_BsAb_vector_design_Sup_Materials_Clean_tbac025Click here for additional data file.


Optimizing the laser wakefield accelerator (LWFA) requires control of the intense driving laser pulse and its stable propagation. This is usually challenging because of mode mismatching arising from relativistic self-focusing, which invariably alters the velocity and shape of the laser pulse. Here we show how an intense pre-pulse can prepare the momentum/density phase-space distribution of plasma electrons encountered by a trailing laser pulse to control its propagation. This can also be used to minimize the evolution of the wakefield thus enhancing the stability of the LWFA, which is important for applications. Achieving high quality electron beams in laser wakefield accelerators requires stable propagation of the driving laser pulse18. This is usually challenging because of mode mismatching arising from relativistic self-focusing. Here we present a numerical study to show the wakefield properties can be altered by the presence of an intense pre-pulse. The role of the pre-pulse is to prepare the phase-space distribution of plasma electrons prior to the arrival of an intense trailing driver pulse. This enables the driver pulse to produce a well-matched plasma channel that can control or minimise wakefield evolution. This type of channelling differs from that of preformed plasma channels, which require excellent alignment between channel and laser (driver) pulse to be guided20. Controlling the intensity of laser pulses is an essential step in developing useful wakefield accelerators and compact radiation sources23. Stable channels are necessary for developing next generation compact plasma undulators for compact synchrotron sources25 and free-electron lasers27. Wide availability of ultra-compact accelerators and radiation sources could transform the way science is done28.The laser-plasma accelerator1 is due to the ponderomotive force of an intense, ultra-short duration driver pulse, which separates electrons from ions. The resulting ultra-high electric field, which can exceed 100 GV/m, is several orders of magnitude larger than possible in conventional accelerators. Displaced electrons follow trajectories that define a thin sheath surrounding the ion-filled plasma bubble and converge at the rear of the bubble. When their velocities exceed the velocity of the wake they are injected into the bubble and rapidly accelerated to high energies11 with excellent properties10. They can also be used as compact all-optical sources of brilliant X-ray24 and gamma-ray25 photons that have potential applications including spectroscopy and imaging for material sciences, warm dense matter studies for laboratory astrophysics and fusion, and for the biology and health sciences31. Ultra-short pulse LWFAs are being investigated as drivers of synchrotron sources22, free-electron lasers32 and ion channel lasers (ICLs)27, and for very high energy electron (VHEE) radiotherapy35, pulsed radiolysis36 and medical imaging31.Particle acceleration in the laser wakefield accelerator (LWFA)28. The bubble evolution directly influences self-injection38. Laser39 and plasma37 variations which cause bubble evolution, result in time-varying electrostatic fields that have a direct impact on electron bunch quality40. Stable propagation of the driver pulse is required to minimise bubble variations to ensure persistent acceleration and high quality beams.Many applications require high-quality electron bunches with low emittance, ultra-short duration and high peak current. Electron bunch properties depend on maintaining field uniformity41. It can also cause transverse oscillations of the bubble and collective transverse oscillations of the electron beam43 that increases the beam divergence and transverse emittance, and can reduce the dephasing length, which ultimately reduces the maximum energy gain.The propagation of intense laser pulses is a highly nonlinear process; the laser pulse and the plasma medium are mutually modified during propagation. Stable propagation of the intense driving laser pulse ensures stable wakefield evolution. Variations of the wakefield and evolution of bubble shape and velocity strongly affect electron bunch parameters, such as charge, emittance and energy spreadIn this article, we present a double-pulse method of preparing the electron momentum distribution of plasma in advance of the arrival of an intense trailing driver pulse. The short-duration pre-pulse enables the trailing driver pulse to form its own self-aligned channel. Optimum formation is achieved when the time delay between the two pulses is slightly less than the plasma period, Particle-in-cell (PIC) simulations have been performed see "' sectionx- and y-direction due to the laser polarization of pre-pulse and main pulse in the y-direction and x-direction, respectively. The delay is fine-tuned to ensure that electrons displaced by the pre-pulse interact with the driver pulse as they converge at the back of the pre-pulse wake, where they form a co-moving parabolic density channel  bubble velocity, 44 and therefore dephasing point) and the wakefield it generates. The increased stability of the wake using a double pulse will help with developing high-quality laser wakefield accelerators (LWFAs), and also for new applications of ion channels such as for use in the ion channel lasers (ICLs)27, which require the electron bunch to remain at the dephasing point to minimise energy variations. Matching the electron bunch velocity to that of the dephasing point is only feasible if the latter varies slowly, which can be achieved using the pre-pulse to ensure that electron bunches injected with the correct velocity and phase will remain near the dephasing point for an extended period.Figure 11. The scheme presented here does not require alignment or synchronization, which are usually challenging when using preformed plasma waveguides. The driver pulse energy coupling efficiency into the channel at 2.7 mm is 98.7%, with an overall transmission efficiency of 89.3% observed when the laser propagates from 2.7 mm to 12 mm (including the loss of energy to create the channel and wake). The scheme could be optimised by varying the laser pulse parameters, tuning the inter-pulse delay, or guiding the pre-pulse.We have demonstrated how the introduction of a lower intensity chaperone pre-pulse in the LWFA can prepare the electron phase-space so that a trailing driver pulse produce its own co-moving plasma channel. For the parameters investigated here, we find that a pre-pulse with a delay of 0.85It is important that the pre-pulse is well matched to the plasma or guided so that it persists over the required interaction length. Guiding the pre-pulse using a preformed channel does not negate the stability benefits offered by the double pulse configuration presented here. For the results obtained here, we found that the best channel and most stable propagation are observed when the pre-pulse amplitude The stabilizing effect of the chaperone pre-pulse significantly improves the overall stability of the plasma wake. The double-pulse configuration should contribute to the development of controllable LWFAs for high-energy, high-quality electron bunches for a wide range of applications, such as drivers of secondary radiation sources. We also note that the stability of the ion cavity, driver pulse amplitude and plasma density can be used to control the velocity of the dephasing point, making this an attractive ion channel for an ICL or plasma wiggler. To aid the design of experiments to verify the mechanism described here, we note that similar results are obtained using parallel polarizations for the pre-pulse and driver pulse.45, which is a quasi-3D PIC code that uses cylindrical geometry and azimuthal modal decomposition to represent the 3D domain. This significantly reduces the computational resources required, especially for large domains, while retaining important 3D effects. The spectral solver used by the code reduces numerical artifacts, such as noise, dispersion and numerical Cherenkov radiation. Simulations have been performed for various laser intensities for single and double laser pulses, using a moving window of size Computer simulations are performed using the open source code FBPIC46 when the delay length is equal to the plasma wavelength, We first consider the wakefield generated by a single Gaussian-profile driver pulse with an initial normalized amplitude 48 with the plasma and pre-pulse parameters. We consider a laser pulse propagating along the z-axis with Gaussian profile defined by As a guide to finding a starting point for the delay optimization, we use a nonlinear 1D theorySupplementary Information 1.


The study hypothesis was that non-hospitalised COVID-19 patients have greater contact with children compared with hospitalised patients. The study did not find an association between contact with children and rates of hospitalisation after adjusting for multiple covariates. We also note that the hospitalisation was lower for patients with multiple concurrent child contact types (home and school) when compared with patients with no contact (20 vs. 72). Results from this study were surprising as we expected opposite results because of the higher likelihood of infection from multiple sources. We hope authors will provide reasoning to help readers understand the conflicting results.In ‘the association between contact with children and the clinical course of COVID-19 [et al. illustrated that hospitalisation rates were higher in patients without household contact, which is different from Jannuzzi et al. findings. One of the reasons for the heterogeneity is that asymptomatic patients with a low-risk contact might not have been tested; some COVID-19 patients ‘without household contact’ were also untested, and the actual number of the patients ‘without household contact’ remained unknown [To understand the role of children in the spread of SARS-CoV-2, it is important to understand the transmission chain (dynamics) of SARS-CoV-2. Most infections in children are asymptomatic, and the number of real-time polymerase chain reaction assay confirmed cases of SARS-CoV-2 infection in children is underestimated because of the high ratio of mild and asymptomatic cases in which testing was not completed [ unknown .et al. illustrated the greater odds of household transmission by children aged 4–8 years after controlling for testing delays and household size [et al. demonstrated that secondary attack rates were lower in households where children rather than adults had transmitted SARS-CoV-2, and rates were lower during the school period when interactions with other children were expected to increase disease transmission [Transmission dynamics of SARS-CoV-2 infection in children are affected by age groups of household members, exposure intensity, duration of contact, family size and viral load. Paul smission . The agesmission , 7. Mult


Suicide is one of the most relevant cause of death especially in young populations. The Interpersonal Theory of Suicide  is an important contribution tends to explain variability in suicidal behavior, particularly the difference between suicidal ideation and suicide attempt.This study aimed at the Italian validation of the Acquired Capability for Suicide Scale – Fearlessness About Death (ACSS-FAD), assessing fearlessness about death, one of the facets of the acquired capability (AC) to commit suicide as postulated by the Interpersonal Theory of Suicide by Thomas Joiner.This cross-sectional research was conducted on a sample of university students (n = 458) assessed with a battery including ACSS-FAD. The sample was evaluated for the presence of suicidal ideation and suicide risk. Factor structure, internal consistency and convergent/divergent validity of the scale were assessed.One-factor structure with good internal consistency (Cronbach’s α: 0.84) was derived. ACSS-FAD correlated with suicidal ideation and suicidal risk, and there was a tendency towards significance considering its capacity to discriminate between those who had a history of suicide attempts and those who did not. 4.1% of the sample attempted suicide at least one time. The tool showed good convergent/discriminant validity results, but the relationship between ACSS-FAD and pain needs further investigations.ACSS-FAD seems to be a useful and valid measure of fearlessness about death especially in young adults, which could be really important to enhance suicide risk assessment.No significant relationships.