Additionally, the constructs of emotional transformation , practice for change , and changes in the social environment  were statistically significant and accounted for 45.1% of the variance in the likelihood of sustaining handwashing behavior. This study highlights the applicability and usability of the MTM in designing and testing behavior change interventions and media messaging in campaigns targeting college students.Amidst the COVID-19 pandemic, handwashing offers a simple and effective hygienic measure for disease prevention. Reportedly, a significant proportion of college students did not follow handwashing recommendations provided by the Centers for Disease Control and Prevention (CDC) in the pre-COVID era. The purpose of this cross-sectional study was to explore and explain the handwashing behavior among college students during the COVID-19 pandemic using a contemporary fourth-generation multi-theory model (MTM) of health behavior change. Data were collected from 713 college students at a large public university in the Southern U.S. in October 2020 using a validated 36-item survey. Statistical analyses included independent samples t-tests, Pearson correlation, and hierarchical regression modeling. Among students not following handwashing recommendations, the constructs of participatory dialogue (β = 0.152;  The novel coronavirus COVID-19) disease caused by the SARS-CoV-2 virus has rapidly spiraled across the world and took the course of pandemic 9 disease. To reduOne of the simple yet effective measures recommended by the government and public health agencies is frequent and thorough handwashing ,5. HandwCollege campuses and their surrounding communities became a central concern for COVID-19 prevention due to surging trends of COVID-19 among young adults aged 20–29 in the U.S. since June 2020 . The CDCRecent studies reported that female students and those with the positive attitudes and knowledge towards handwashing for COVID-19 prevention were more likely to adopt handwashing behavior ,19. FurtThe current study uses a contemporary, fourth-generation theoretical framework, the multi-theory model (MTM) for health behavior change, to explore handwashing behavior among college students. The MTM is a health behavior change theory with the unique ability to explain factors related to one-time  and long-term  engagement in health behavior . The conPrevious health behavior theoretical models focused on behavior acquisition rather than change have yielded mixed results when exploring behavior change, are documented to lack substantive predictive power, and are not suited to determine long-term behavior change . The MTMThis descriptive, quantitative, cross-sectional survey study was conducted from 8 October 2020, to 29 October 2020, among college students at a large public Southern US university. The survey was deemed the most appropriate design due to its inherent capacity to collect information with relative ease from large population groups within a given period . StudentThis study followed the Checklist for Reporting Results of Internet E-Surveys (CHERRIES) guidelines . This stThe convenience sampling method was used for sample recruitment. Participants were recruited through advertisements posted on the university’s daily e-news bulletin, intended to disseminate information to faculty, students, and staff with a valid university email address. The recruitment advertisement containing an anonymous survey link with complete details of the study was sent out every Thursday for three consecutive weeks to all currently enrolled students . Upon clicking the link, interested participants were connected to the Qualtrics  interface to access and fill out the web-based survey. As an incentive, participants were informed that by participating, they had the opportunity to enter a drawing to win one of five $20.00 Walmart gift cards. The final survey item asked students about their willingness to enter the drawing to receive one of the gift cards. Interested students clicked ‘yes’, which functioned as a link to a separate survey where an email address could be collected for contact purposes. This process preserved participant anonymity by making it impossible to link the provided email address to data previously disclosed during the data collection process. The Qualtrics survey options were set to prevent “ballot box stuffing.” This option restricts the same IP address from contributing in multiple surveys. Participants who accessed the survey link more than once would receive a message that their responses had already been recorded.Handwashing encompasses five steps from wetting the hands with running water, applying lather by rubbing soap, scrubbing hands thoroughly for at least 20 s, rinsing hands under running water, followed by drying with a clean towel or air drying them . UtiliziThe second element of MTM: sustenance has three constructs, namely emotional transformation , practice for change , and changes in the social environment . These constructs were measured using three items, each with a score range of 0–12 units . The insXM were exported to Microsoft Excel  and then imported to IBM SPSS version 26.0 . Confirmatory factor analysis using the maximum likelihood method and internal consistency diagnostic tests using Cronbach’s alpha were performed to check the construct validity and internal consistency of the tool. For establishing a one-factor solution following the Kaiser criterion of Eigenvalue greater than or equal to 1.0, factor loadings on each item greater than 0.326  were established a priori as per the generally accepted recommendations from previous literature [p-values less than 0.05 (two-sided) were considered statistically significant, and data were reported with 95% confidence intervals. Hierarchical multiple regression was utilized to predict the likelihood of initiation and sustenance of handwashing behavior based on multiple regressors or independent variables, such as age, gender, race/ethnicity, and individual constructs of the MTM. Gender and race/ethnicity variables were dummy coded. Assumptions of independence of observations , linearity , homoscedasticity, multicollinearity, and normality  were evaluated on the full model.Participants’ responses from Qualtricsterature ,37,38. Fterature . DescripG*power software (version 3.1) was used to perform a priori power analysis . The a pn = 503) (n = 483), with nearly one-fourth (n = 197) being enrolled in the graduate program  respondents identified as females and 176 (24.7%) were males . The aven = 503) . Most of program . Further program .Construct validation revealed a one-factor solution for each subscale and Eigenvalues greater than 1. All factor loadings were over the critical value of 0.326. The Cronbach’s alpha for all subscales was at least 0.70  = 62.157, p < 0.0001; adjusted R2 = 0.471  = 130.32, p < 0.0001. The addition of behavioral confidence to the prediction of initiation (Model 3) led to a statistically significant increase in R2 of 0.222, F (1407) = 170.97, p < 0.0001.Hierarchical multiple regression was run to determine if the sequential addition of participatory dialogue, changes in the physical environment, and behavioral confidence improved the likelihood of initiation over and above demographic variables . Among p = 0.471 . The addR2 = 0.434, F  = 52.953, p < 0.0001; adjusted R2 = 0.425  = 255.710, p < 0.0001. The addition of practice for change to the prediction of sustenance (Model 3) led to a statistically significant increase in R2 of 0.038, F  = 27.413, p < 0.0001.In the hierarchical regression with sustenance as the dependent variable, the full model (Model 4) was statistically significant,  = 0.425 . The addR2 = 0.295, F  = 12.615, p < 0.0001; adjusted R2 = 0.272  = 41.784, p < 0.0001  = 24.682, p < 0.0001. In the hierarchical regression with sustenance as a dependent variable, the full model (Model 4) was statistically significant, R2 = 0.468, F (6184) = 26.972, p < 0.0001; adjusted R2 = 0.451  = 100.790, p < 0.0001. The addition of practice for change to the prediction of sustenance (Model 3) led to a statistically significant increase in R2 of 0.080, F  = 26.772, p < 0.0001. The addition of changes in the social environment to the prediction of sustenance (Model 4) led to a statistically significant increase in R2 of 0.023, F  = 7.957, p < 0.05.Among participants not following handwashing recommendations, the full model (Model 4) to predict initiation was statistically significant,  = 0.272 . The add< 0.0001 . The add = 0.451 .The addiThe study utilized the MTM, a novel fourth-generation behavioral theory, to explain handwashing behavior among college students during the COVID-19 pandemic. Despite the aggressive media messages and recommendations from governmental authorities, the study revealed that 31.4% of the college students did not practice frequent handwashing in the recommended manner compared to 33.1% to 61.5% reported in pre-COVID-19 era ,13,14. Tp < 0.0001). Participatory dialogue will be higher in the magnitude if advantages outweigh disadvantages and will be lower in the reverse scenario. This can be validated by the participatory dialogue score among students not following the handwashing recommendations, who perceive handwashing having more disadvantages than advantages. Every unit increase in behavioral confidence resulted in a 0.474 unit increase in the likelihood of the initiation of handwashing behavior. Similarly, for every unit increase in participatory dialogue, a 0.152 increase in the likelihood of the initiation of handwashing behavior ensued. Among students following the handwashing recommendations  and participatory dialogue , among those not following the handwashing recommendations  and greater among those who were practicing the recommendations for handwashing compared to those who were not. This finding lends support to the predictability of MTM for the initiation of handwashing behavior among college students. Future research and individual-level behavior change interventions and media campaigns should utilize the constructs of participatory dialogue and behavioral confidence in promoting handwashing behavior among college students.Further, it is worth noting that significant differences between all three initiation constructs in the MTM for initiation were statistically significant (p < 0.0001), practice for change  (p < 0.0001), and changes in the social environment  (p < 0.05). For every unit increase in emotional transformation, there is a 0.33 unit increase in the likelihood of maintaining handwashing behavior; for every unit increase in practice for change construct, there is a 0.296 increase in the likelihood of maintaining handwashing behavior; and for every unit increase in the changes in the social environment score, there is a 0.180 increase in the likelihood of maintaining handwashing behavior. The findings are also supported by data of students practicing handwashing according to recommendations  and higher among those who were practicing the recommendations for handwashing as compared to those who were not, which further lends support to the predictive potential of MTM for the sustenance of handwashing behavior among college students.Regarding the MTM’s ability to predict maintaining handwashing behavior, the examination of the sustenance model among those not following the recommendations revealed 45.1% of the variance in the likelihood to continue the practice of handwashing. Additionally, sustenance significantly predicted by all three constructs: emotional transformation   plays an important role in the sustenance of a behavior ,47,48,49The study has important implications for designing handwashing promotion interventions, especially during the COVID-19 pandemic. The findings point to the fact that, despite the pandemic mounting at alarming rates, a substantial proportion of college students do not wash their hands as per the CDC recommendations. This issue can be addressed by designing individual-level behavioral change educational interventions and media campaigns  directed toward college students. Individual-level behavior change interventions can be delivered through classrooms or other university channels using the learning management systems (LMS) such as Blackboard, Canvas, Moodle, Brightspace, which almost all universities utilize to access course content. Further, as a policy measure supportive of behavior change, such interventions can be mandated by the university administration.MTM has been used in designing similar technology-based brief and specific interventions ,52,53 anConcerning sustenance constructs, emotional transformation emerged as the most influential construct in this study, and the importance of channeling one’s feelings cannot be overemphasized. To build this construct, the first step is to be cognizant of one’s feelings, which is vital in changing handwashing behavior and changing any behavior and self-improvement . After iTo our knowledge, this study is among the very few theory-based or evidence-based studies that have been performed on handwashing behavior among college students during the COVID-19 pandemic. This study offers a unique perspective by utilizing newer multiple theory models for studying handwashing behavior among college students. Among the few preventive measures that we have available to combat this pandemic, frequent and adequate handwashing seems to be an effective approach that a substantial number of college students are not practicing. Hence, this study provides direction for designing efficacious, brief educational interventions to promote handwashing. The scale utilized was psychometrically robust and met the acceptable criteria for validity and reliability and can be used for future cross-sectional and interventional studies.https://oir.uark.edu/quickfacts/factbook-2019-2020.pdf.However, this study has some limitations, which merit discussion. First, this study is based on only one large, public Southern U.S. university. Therefore, the findings may not be extrapolated to students of other institutions, and caution should be applied while interpreting the results. However, our sample was nearly representative of the institution where study took place. University racial breakdown for previous year was White/Caucasian (73.7%), Black/African American (4.4%), Hispanic (8.6%), and Asian (2.5%) (available at Second, the study relied on self-reported information, which can subject to measurement error. However, when it comes to measuring attitudes towards health behavior, this is the only method for the measurement. Third, in testing the instrument’s reliability, the instrument’s stability over time was not assessed. This offers a potential avenue for future research and will be especially important before conducting experimental studies. Fourth, actual availability of resources for handwashing were not measured in this study. Finally, the study used a cross-sectional design in which the independent and dependent variables are measured simultaneously, thereby preventing any causal inferences.Amid the COVID-19 pandemic, this is a timely study that identifies a newer fourth-generation behavior change theory, MTM, to address handwashing behavior among college students. This study found that a substantial number of college students are not following handwashing recommendations. Further, the study provided evidence that MTM can help promote handwashing behavior among college students. There is a need to design and test individual-level behavior change interventions and media campaigns based on MTM for efficacy and effectiveness to change handwashing behavior among college students.


Medulloblastoma (MB) is the most common type of brain malignancy in children. Molecular profiling has become an important component to select patients for therapeutic approaches, allowing for personalized therapy. In this study, we successfully identified detectable levels of tumor-derived cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) samples of patients with MB. Furthermore, cfDNA from CSF can interrogate for tumor-associated molecular clues. MB-associated alterations from CSF, tumor, and post-chemotherapy plasma were compared by deep sequencing on next-generation sequencing platform. Shared alterations exist between CSF and matched tumor tissues. More alternations were detected in circulating tumor DNA from CSF than those in genomic DNA from primary tumor. It was feasible to detect MB-associated mutations in plasma of patients treated with chemotherapy. Collectively, CSF supernatant can be used to monitor genomic alterations, as a superior technique as long as tumor-derived cfDNA can be isolated from CSF successfully. Currently, MB is recognized as an umbrella term that encompasses various molecular pathological entities. Based on 2016 World Health Organization (WHO) classification of central nervous system (CNS) tumors, MB was divided into four subgroups, i.e., WNT, SHH, Group 3 and Group 42. About one-third of children with MB have cerebrospinal fluid, cranial or spinal metastases at diagnosis3. Patients who suffer from recurrence or tumor progression, even with advanced treatment protocols, have dismal prognosis. Nowadays, cancer genome landscape is essential for diagnosis, prognostic evaluation and treatment selection. Unfortunately, for patients with tumor progression or recurrence, it is difficult to detect molecular alterations, because of unobtainable tumor tissue and lack of biopsy sample. Only advanced imaging technologies, such as MRI, CT and PET, are applied to diagnose and monitor progressive disease for MB.Medulloblastoma (MB) is the most common primary malignant tumor in children, accounting for 20% of all pediatric intracranial tumorsEGFR mutations in non-small cell lung cancer. However, blood–brain barrier impedes detection of ctDNA in blood of a patient with brain tumor4. Recently, accumulating evidence has supported that circulating cell-free nucleic acids including DNA (cfDNA) and RNA (cfRNA), and even proteins in cerebrospinal fluid (CSF) are potential to revolutionize diagnosis and clinical care for CNS tumors, especially for glioblastoma (GBM) and MB5. We aim to explore the feasibility of liquid biopsy using CSF to facilitate diagnosis and to predict prognosis in MB.The next-generation sequencing (NGS) has been applied to sequence circulating tumor DNA (ctDNA) from blood, especially for identifying To decipher pathogenesis of MB and apply targeted therapy, it is necessary to explore genetic background in different types of samples, such as tumor tissue and CSF, based on NGS platform. Because in our previous clinical genetic test reports, few MB-associated alterations were detected in tumor tissue. Therefore, deep sequencing on ctDNA in CSF and formalin-fixed paraffin-embedded (FFPE) tissue was performed to obtain comprehensive genome profiling. We hypothesized that genetic alteration in CSF may serve as a complementary role to tissue for monitoring disease progression.6. Thus detection of ctDNA is of great value for early diagnosis of malignancy. For detecting ctDNA in the blood, the content of ctDNA is lower at earlier stage, compared to relatively late-stage7. Additionally, the difference of fragments size in ctDNA and cfDNA was utilized for early diagnosis of malignancies8. Furthermore, high-grade (WHO grades III and IV) brain tumors were more likely to harbor detectable ctDNA in CSF than low-grade ones9. Therefore, ctDNA detection in CSF plays a predictive role in monitoring disease progression.Circulating cell-free DNA (cfDNA) is present in body fluid of healthy individuals as well as patients with cancer, such as plasma, urine and CSF. Circulating tumor DNA (ctDNA) is thought to be shed into circulation by apoptotic and necrotic tumor cells in patients with cancerHere, we determined the presence or absence of cfDNA in 58 CSF samples. In order to decipher medulloblastoma-associated alterations, deep sequencing based on NGS platform was applied to examine CSF, tumor tissue and blood in patients with MB. Notably, positive cfDNA in CSF is associated with disease progression or metastasis. It was more likely to detect medulloblastoma-associated alterations in CSF rather than tumor tissue. Shared alterations between CSF and matched tumor tissues could be detected when sample collected in short time interval.The protocol was approved by the institutional review board of Research Ethics Committee at Beijing Shijitan Hospital. Written informed consent was obtained from their parents of MB patients. All experiments were carried out in accordance with relevant guidelines and regulations.Beijing Tiantan Hospital of Capital Medical University and Beijing Shijitan Hospital of Capital Medical University between April 2019 and Dec 2019. For each patient, CSF was collected by lumbar puncture or at the time of surgery. Additionally, tumor tissue samples were collected from 4 patients on primary-care and 7 patients with relapse. The matched plasma was isolated from 5 patients with relapse and treated with adjuvant radiotherapy or chemotherapy. Furthermore, whole blood was obtained from each patient, serving as germline DNA control.In this prospective study, 58 patients with MB were recruited from The median volume of collected CSF is 5 mL. Fresh CSF was stored at 4 °C, and centrifugated  within 2–3 h after collection. Cellular pellet was discarded. The sterile centrifuge tube was pre-cooled at − 20 °C. CSF supernatant was transferred to a cryotubes and stored at − 80 °C. Immediately before use, CSF was thawed in a thermostat water bath at 37 °C. Then, cfDNA was extracted from CSF supernatant or plasma according to the manufacturer’s protocol using QIAamp Circulating Nucleic Acid Kit .Isolation of genomic DNA from FFPE tumor tissue or matched whole blood leukocytes was performed using QIAamp DNA FFPE Tissue Kit  and blood/cell culture DNA kit , respectively. Concentration of isolated (cell-free and genomic) DNA was quantified with Qubit 2.0 Fluorometer using Qubit dsDNA HS Assay kit  according to the manufacture’s protocol. DNA was stored at − 20 °C until it was used.10.The presence and absence of cfDNA was inferred according to isolated DNA fragment length distribution, instead of quantity. To verify isolated cfDNA, the size of cfDNA fragment was assessed using Agilent 2100 Bioanalyzer. If peak appeared at 150–200 base pair (bp), it is considered as cfDNA, otherwise (> 1500 bp) as CSF cellular genomic DNA. And NGS library construction was not performed against CSF cellular genomic DNA. The detection method cannot determine whether cfDNA is derived from tumor tissue. Therefore, we adopted the term “cfDNA”. The term “ctDNA” was used only when tumor-associated mutations were detected in CSF or plasma11. The SNVs and In/Dels were detected using Genome Analysis Toolkit (GATK)12 and VarScan213 and filtered out by dbSNP and 1000 Genome datasets. We selected at least 3 mutated reads with quality scores of > 30. In this study, we focused on mutations located in exons, such as nonsynonymous, synonymous, frameshift, non-frameshift and splicing sites. All the genes tested were verified with Integrative Genomics Viewer (IGV).First, tumor genomic DNA was fragmented into 150–200 bp fragments with Bioruptor Pico. Libraries of fragmented DNA and cfDNA were constructed with KAPA Library Amplification Kit (KK2611/KK2612) according to standard protocols. Next, cfDNA libraries were captured with a designed 500-gene or 952-gene panel (Agilent), which contained the majority of brain tumor related genes. The genomic DNA libraries were applied with whole exome sequencing (WES). The captured samples were subjected to paired-end sequencing on the Illumina NovaSeq 6000 platform. Finally, the low quality reads were removed based on bioinformatics; while the high quality reads were mapped on human reference sequence (hg19) (Human Genome version 19) using tool Burrows–Wheeler Aligner software (BWA)p ≤ 0.05 defined as statistical significance.Associations between CSF-cfDNA and clinical characteristics including age and quantity of DNA in CSF were evaluated by Mann–Whitney U tests. Clinical features such as recurrence, metastasis, tumor progression and quantity of DNA in CSF were compared between positive and negative CSF-cfDNA groups with Fisher’s exact tests or chi-square tests. All statistical tests were two-sided with a The patient population  included infants, children or teenager, aged at 2–15 years old (median: 7). Demographic and clinical features were summarized in Table p = 0.50, Mann–Whitney U test) or gender . However, the presence or absence of cfDNA in CSF was associated with several clinical features, including recurrence , metastasis , tumor progression , as well as quantity of DNA in CSF   Table . The med10. Medulloblastoma-associated alterations detected in ctDNA from CSF were more abundantly present than genomic DNA from tumor tissue , KMT2C (28.0%), SMARCA4 (24.0%), BCOR (20.0%), TP53 (12.0%), PTCH1 (8%), EP300 (8%), NF1 (8%), SETD2 (8%), MED12 (8%) and SPEN (8%). Notably, BCOR alterations were the most frequent in ctDNA from plasma. The presence of medulloblastoma-associated mutations in plasma may be due to the fact that patients with recurrent disease had been treated with adjuvant radiotherapy or chemotherapy. It was indicated that blood-brain barrier system could be damaged or destroyed in response to radiotherapy or chemotherapy17. It is surprising that medulloblastoma-associated somatic mutations from tumor tissues were detected in only 36% (4/11) of patients. We identified nonsynonymous SNVs in tumor tissues, such as SMARCA4 (in 192D0360T and 192D0641T), BCOR and LRP1B in 192D0641T, as well as frameshift mutations of KMT2D in 192D0695T and truncating mutations of PTCH1 in 192D0583T. Although it is possible that the low concordance between samples from the same individual could be resulted from the number of different detected alterations, the common medulloblastoma-associated alterations in tumor tissue were few in samples with more genes detected, such as 192D0583T, 192D0629T and 192D0674T , excluding from dominated somatic copy number aberrations (SCNA) in MB Group 419. Nevertheless, no shared alterations were found in the remaining seven patients (78%).NGS-based detection of alterations was performed against nine paired tumor DNA and CSF-ctDNA to explore shared genetic mutations between CSF and tumor tissue. As shown in Table To clarify the reason about the detectable shared mutations, we then collected clinical information of the above nine patients Table . We foun20. Therefore, detection of tumor-derived cfDNA in CSF has become a minimally invasive approach to dynamically monitor tumor progression, which can facilitate targeted therapy for cancer patients.It is challenging to monitor disease progression for patients with CNS tumors since repeated surgical resection is unfeasible, while the pathology diagnosis that distinguish proliferative/aggressive tumor, pseudoprogression or necrosis directly influenced treatment decision-making process9. In our patient population, tumor lesions interacted with CSF reservoir directly. The difference may be also caused by limitation of current detection techniques using CSF as liquid biopsy in childhood MB. Notably, CSF-cfDNA was undetected in the vast majority of patients who exhibited CR after surgery (without metastasis). cfDNA in CSF was detected in 15 patients. Among these 15 patients, five developed postoperative recurrence and PD, and 7 with initial therapy had varying degrees of metastasis (M1 or M3) or PD and even died of rapid PD. Only 3 patients achieved CR (without metastasis) after surgery in several months (range from 2 to 6 months). Furthermore, clinical stage, such as recurrence, metastasis and PD between positive and negative CSF-cfDNA was different statistically. Similar to previous studies, the levels of tumor-derived cfDNA in CSF reflected response to therapy or PD21. Additionally, the quantity of plasma-ctDNA was correlated with tumor burden, as a predictor for recurrence and progression10.In our study, cfDNA from 43 out of 58 CSF samples could not be detected. The presence of tumor-derived cfDNA from CSF was significantly related to primary location of tumor adjacent to CSF reservoir. The CNS neoplasms located in brain or spinal cord, where lesions adjacent to CSF reservoir were much more likely to harbor tumor-derived cfDNA in CSF22. From another perspective, the mean sequencing depth for liquid biopsy (2000×) is higher than tumor tissue (110×), so that more alterations may be captured. Therefore, more reasonable sequencing scheme should be designed to verify our findings. Additionally, medulloblastoma-associated alterations were detected in blood, possibly attributed to adjuvant radiotherapy or chemotherapy for patients with postoperative recurrence. We may infer that brain tumor-related gene mutations are detectable in the blood of patients with MB who have been treated with radiotherapy or chemotherapy. However, since we did not collect blood samples from patients who were not treated with chemoradiotherapy, it remained unclear if mutation detection in ctDNA from plasma is applicable to all kinds of treated patients with brain tumors.In our study, more medulloblastoma-associated gene mutations were identified in CSF than in tumor tissue Fig. . This diSMARCA4 forms a protein complex with CTNNB1, which remodels chromatin and participates in WNT pathway23. SETD8  regulates TP53 activity and expression24. KMT2D  acts as a universal oncogenic driver to regulate epigenetic alterations. SNCAIP encodes synphilin-1, by binding to α-synuclein, which promotes the formation of Lewy bodies in the brains of patients with Parkinson’s disease26. Interestingly, in both patients, time interval for sample collection between CSF and tumor tissue was very short. CSF was collected one month after operation for 192D0360 whereas during operation for 192D0695. By contrast, CSF was collected during operation for 192D0640 whereas one month after surgery for 192D0593, but no shared alterations were detected from these 2 cases. CSF was obtained from the remaining 5 samples after disease recurrence. It may be easier to detect shared mutations between CSF and matched tumor tissue when time interval for sample collection is relatively short. CSF-ctDNA from patients with MB provides a comprehensive and genetically representative genome profiling only at the time of CSF collection. Finally, this result is consistent with previous findings that genomic evolution of brain tumors can change significantly over time32.No or few medulloblastoma-associated alterations were detected from tumor tissue. Shared alterations were identified between CSF and matched tumor tissue in only 2 out of 9 patients. Shared missense mutations may contribute to pathogenesis of MB. For example, In conclusion, detectable cfDNA in CSF may predict disease progression. Deep sequencing on CSF-ctDNA derived from patients with MB may obtain more tumor-specific mutations than matched tissue. This finding may help targeted therapy and drug development. Furthermore, CSF samples collected at different time points could represent snap-shot genome landscape only at the time of acquisition. Our finding may help diagnosis and monitoring progressive disease for patients with MB. This research is still ongoing. Thus, prospective studies are needed to confirm if CSF-ctDNA detection as liquid biopsy could be used in routine clinical therapy for MB, and if CSF-ctDNA gene mutations could guide targeted therapy in recurrent childhood MB.Supplementary Information


D)-acceptor (A) polymers were studied by designing two types of polymers, (D-σ-A)n and (D-π-A)n, with non-conjugated alkyl (sp3) and π-conjugated (sp2) linkers using π-extended donor and acceptor monomers that exhibit planar A-D-A structures. The non-conjugated alkyl linker provides structural flexibility to the (D-σ-A)n polymers, while the π-conjugated linker retains the rigid structure of the (D-π-A)n polymers. Photoinduced energy transfer occurs from the large donor to acceptor units in both polymers. However, the photoinduced energy transfer dynamics are found to be dependent on the conformation of the polymers, where the difference is dictated by the types of linkers between the donor and acceptor units. In solution, intramolecular energy transfer is relatively favorable for the (D-σ-A)n polymers with flexible linkers that allow the donor and acceptor units to be proximally located in the polymers. On the other hand, intermolecular (or interchain) energy transfer is dominant in the two polymer films because the π-extended donor and acceptor units in polymers are closely packed. The structural flexibility of the linkers between the donor and acceptor repeating units in the polymers affects the efficiency of energy transfer between the donor and acceptor units and the overall photophysical properties of the polymers.The photophysical properties of donor ( In the past, various donor (D)- acceptor (A)-type dyad systems have been designed and synthesized as electronic and optoelectronic materials, including organic photovoltaic cells, organic field effect transistors, and organic light-emitting diodes, and their photophysical properties have been studied  and A-D1-D2-D1-A type acceptor monomers , and utilized them to synthesize two different polymers with non-conjugated alkyl linkers, (D-σ-A)n, and π-conjugated linkers, (D-π-A)n. The structural flexibility of (D-σ-A)n and (D-π-A)n is dictated by the linkers. Overall, (D-σ-A)n is the partially conjugated polymer in which the π-extended donor and acceptor monomers are connected through flexible non-conjugated sp3 linkers. In contrast, (D-π-A)n is the fully conjugated polymer in which the large donor and acceptor monomers are connected via conjugated sp2 linkers. It is very noteworthy that such polymer structures contain highly π-extended donor and acceptor monomers with a uniform molecular weight, and the large donor and acceptor monomers are alternatively arranged in the polymers. The photophysical properties of (D-σ-A)n and (D-π-A)n in toluene and films were investigated using UV-visible absorption, steady-state emission, and time-resolved fluorescence (TRF) spectroscopy, in conjunction with quantum chemical calculations. The photophysical properties of (D-σ-A)n and (D-π-A)n depend on the type of linker between the large donor and acceptor units in the polymers. The energy profiles of (D-σ-A)n and (D-π-A)n were found to be similar, but the photoinduced energy transfer from the donor to acceptor units occurred via different pathways. Photoinduced energy transfer in (D-σ-A)n and (D-π-A)n in toluene is mainly due to the intramolecular energy transfer, whereas photoinduced energy transfer in the (D-σ-A)n and (D-π-A)n films proceeds via both intramolecular and intermolecular (or interchain) energy transfer. This is the first report on the photophysical properties of partially and fully conjugated polymers containing well-defined π-extended donor and acceptor monomers. And our current results are expected to be applied to optoelectronic devices using π-conjugated donor-acceptor type polymers.In this study, we designed and synthesized large A-D-A type donor monomers , M2 , Pd2(dba)3 , and P(o-tolyl)3  were dissolved in toluene (4 mL). The degassed binary mixture was stirred at 180°C for 2 h and subjected to cross-coupling Stille reaction under optimized microwave irradiation. The synthesized crude polymers were purified by precipitation into methanol, followed by Soxhlet extraction to remove unreacted monomers, undesired byproducts, and low-molecular-weight materials, using acetone, hexane, and dichloromethane in succession. The polymer solution was precipitated from methanol, and the solid was filtered to obtain polymer (D-σ-A)n as a black solid . Elemental Anal. Calcd. for (C324H352N6O2S22)n: C: 76.82; H: 6.96; N: 1.66; S: 13.92. Found: C: 76.18; H: 6.83; N: 1.71; S:13.62.In a Schlenk tube, M1 , M3 , and Pd(PPh3)4  were dissolved in toluene (4 mL). The synthesis method and reaction conditions were the same as described for the microwave synthesis of (D-σ-A)n. (D-π-A)n is a black solid . Elemental Anal. Calcd. for (C304H324N6O2S20)n: C: 76.14; H: 6.86; N: 1.78; S, 9.55. Found: C:75.87; H: 6.76; N: 1.83; S: 9.32.In a Schlenk tube, (D-σ-A)n and (D-π-A)n were verified by gel permeation chromatography, 1H nuclear magnetic resonance n block and the final (D-π-A)n were 20.0 and 10.9 kg mol−1, respectively, and the corresponding PDI values were 2.4 and 2.3, respectively. Because the molecular weight of the repeating units consisting of the large monomers is over 5,000 g/mol, the molar ratio of the (D-σ-A)n and (D-π-A)n repeating units was determined to be 1:1 from NMR spectra and theoretical EA calculations.The chemical structures of nce NMR; , and eleb:4,5-b]dithiophene (BDT) unit used as the main core of π-extended donor and acceptor monomers in this study has become one of the most widely used polymer backbones because of its rigid planar conjugated structure, which can enhance electron delocalization and promote cofacial π-π stacking interactions in the solid state  backbone architecture of donor and acceptor monomers has drawn particular interest because of the easily tuned energy levels of these species. The benzodithiophene moieties as an additional donor unit (D1) and the end-capping groups of 1,1-dicyanomethylene-3-indanone (IC) as an accepting unit (A).A design strategy for preparing effective A-D-A type donor monomers with narrow bandgaps involves increasing the electron donating ability of the donor monomers by extending the effective π conjugation length. The extended π-conjugated structure with three thiophenes increases the stiffness of the donor backbone. (D-σ-A)n and (D-π-A)n polymers by connecting the donor (B3TP) and acceptor (B2IC) units using an sp2 type conjugated linker (thiophene) and sp3 type non-conjugated linker. A detailed investigation of the photophysical properties is presented in the following sections.In dyad systems, photoinduced energy transfer or photoinduced electron transfer is interesting photophysical phenomena that significantly depend on the linker type and length. To study such phenomena, we synthesized B3TP, B2IC, D-σ-A, and D-π-A were obtained. The frontier orbitals (HOMO and LUMO) and natural transition orbitals (NTOs) were calculated. B3TP and B2IC, respectively. The donor monomer, B3TP (A-D-A type), and the acceptor monomer, B2IC (A-D1-D2-D1-A type), are both planar and fully conjugated. Both B3TP and B2IC exhibit intramolecular charge transfer upon electronic transition  calculations [B3LYP/6-31g(d) and B3LYP-d3/6-31g(d)] were carried out. First, the optimized structures of ansition .D-σ-A and D-π-A are shown in D-σ-A in D-σ-A and D-π-A have the same donor and acceptor units, but different linkers (sp3 vs. sp2) between the donor and acceptor units, the calculated electronic energies of the HOMO and LUMO levels are almost the same n and (D-π-A)n, as discussed hereinafter.The optimized structures and frontier orbitals of the same . For botB3TP, B2IC, (D-σ-A)n, and (D-π-A)n in toluene are shown in (D-σ-A)n and (D-π-A)n are similar, with two absorption peaks near 480 and 660 nm, representing the donor and acceptor units (B3TP and B2IC), respectively. The emission spectrum of B3TP (ex = 520 nm), whereas B2IC shows very weak emission at 710 nm. B3TP is nearly planar and structurally rigid, which seems to give a relatively large fluorescence quantum yield. In contrast, B2IC contains 8 phenyl groups in the donor (D1) units, which are flexible and freely rotatable in toluene and might be responsible for significant fluorescence quenching.The UV-visible absorption and steady-state emission spectra of  of B3TP  exhibits(D-σ-A)n and (D-π-A)n exhibit two emission peaks at 590 and 710 nm (λex = 520 nm), resulting from B3TP and B2IC, respectively. However, the overall fluorescence of (D-σ-A)n and (D-π-A)n is significantly quenched when compared with that of B3TP. This observation can be readily explained based on the fluorescence properties of B3TP and B2IC as follows. The donor units (B3TP) in (D-σ-A)n and (D-π-A)n are directly excited by 520 nm photons. The excited donor units in (D-σ-A)n and (D-π-A)n undergo Förster resonance energy transfer (FRET) to the acceptor units (B2IC) in (D-σ-A)n and (D-π-A)n, owing to which, the fluorescence is quenched. FRET is readily possible in (D-σ-A)n and (D-π-A)n because the emission spectrum of the donor (B3TP) and UV-visible absorption spectrum of the acceptor (B2IC) overlap n, and (D-π-A)n in toluene were measured using the time-correlated single-photon counting method, as shown in B3TP, (D-σ-A)n, and (D-π-A)n in toluene were excited by 520 nm laser pulses and their TRF signals were measured at 590 nm to monitor the emission of the donor monomers (B3TP). The TRF signal of (D-σ-A)n (B3TP and (D-π-A)n in toluene. The TRF signals were fitted to a multi-exponential function, and the average fluorescence lifetimes are summarized in avg = 0.67 ns) of B3TP and (D-π-A)n in toluene are the same within experimental error, even though the initial TRF decay of (D-π-A)n is slightly faster than that of B3TP. The average fluorescence lifetime (τavg = 0.54 ns) of (D-σ-A)n in toluene is slightly shorter than that of B3TP and (D-π-A)n. The difference in the TRF signals of (D-σ-A)n and (D-π-A)n in toluene results from their structural difference, caused by the linkers between the donor and acceptor units, as illustrated in (D-π-A)n, the π-conjugated linker (sp2) between the donor and acceptor units is rigid, which fixes the donor and acceptor far from each other, as shown in (D-π-A)n is less likely. In contrast, in (D-σ-A)n, the sp3 alkyl chain between the donor and acceptor units is flexible; thus, a folded structure can feasibly be formed as shown in (D-σ-A)n is likely, as schematically illustrated in B3TP, B2IC, (D-σ-A)n, and (D-π-A)n in the films were further studied, as shown in B3TP, B2IC, (D-σ-A)n, and (D-π-A)n in the films were slightly red-shifted relative to those in toluene ((D-σ-A)n and (D-π-A)n. The steady-state emission spectra of (D-σ-A)n and (D-π-A)n in B2IC). This indicates completely efficient FRET from the large donor to acceptor units in (D-σ-A)n and (D-π-A)n. In the films, the donor and acceptor units are closely located so that intermolecular FRET from the donor and acceptor units in (D-σ-A)n and (D-π-A)n becomes very efficient. B3TP, (D-σ-A)n, and (D-π-A)n in the films. The TRF signal of B3TP in the films is found to decay faster than that of B3TP in toluene because of the “aggregation causing quenching” effect. The TRF signal of (D-π-A)n in the films decays much faster than that of (D-π-A)n in toluene, resulting from additional intermolecular (or interchain) FRET between the π-extended donor and acceptor units. The TRF signals of the films were fitted by a multi-exponential function, and the average fluorescence lifetimes are summarized in To further understand the fluorescence quenching, the TRF signals of (D-σ-A)n  decayed  toluene . The spe(D-σ-A)n (τavg = 0.54 ns) in toluene is shorter than that of B3TP (τavg = 0.67 ns) in toluene because of the intramolecular FRET from the donor units  to the acceptor units . The intramolecular FRET rate constant of (D-σ-A)n in toluene was estimated to be kFRET = 0.36 ns−1. In the same way, the FRET rate constants of (D-σ-A)n and (D-π-A)n in the films were determined to be kFRET = 5.09 and 3.69 ns−1, respectively. The FRET rate constant was found to be much greater in the films than in toluene. This is because the donor and acceptor units in (D-σ-A)n and (D-π-A)n are much more closely located in the films, and intramolecular and intermolecular FRET are both very efficient in the films, as illustrated in By using a simple kinetics model , the intB3TP, D) with an A-D-A structure and the π-extended acceptor monomers  with an A-D1-D2-D1-A structure were synthesized. These donor and acceptor units were linked together by two different linkers  to synthesize two types of D-A repeating polymers, (D-σ-A)n and (D-π-A)n.To study the role of linkers in energy transfer, we designed and synthesized two dyad polymer systems. First, the π-extended donor monomers ((D-σ-A)n and (D-π-A)n were comprehensively investigated by UV-visible absorption, steady-state emission, and TRF spectroscopy, in conjunction with DFT calculations. DFT calculations show that the π-extended donor and acceptor monomers (B3TP and B2IC) are planar; D-σ-A and D-π-A have similar energy profiles, originating from the donor and acceptor units, and exhibit intramolecular charge transfer characteristics upon electronic excitation. A folded structure is possible for (D-σ-A)n because of the flexible non-conjugated alkyl linkers. (D-σ-A)n and (D-π-A)n exhibit similar UV-visible absorption properties. Their fluorescence was found to be quenched due to FRET from the large donor to acceptor units in (D-σ-A)n and (D-π-A)n. In toluene, FRET is more efficient for (D-σ-A)n than for (D-π-A)n. It is likely that the donor and acceptor units are more closely located in the folded structure of (D-σ-A)n, which increases the speed of FRET. In the films, the fluorescence of (D-σ-A)n is more significantly quenched than that of (D-π-A)n, and intramolecular and intermolecular (or interchain) FRET occur more efficiently. The FRET rate constants were determined using a simple kinetics model.The photophysical properties of For the two conjugated polymers bearing π-extended donor and acceptor monomers, the linkers between the donor and acceptor units are shown to play an important role in determining the overall structure of the polymers, thereby influencing the optical properties. The flexible alkyl linkers in the repeating group of the polymers lead to more structural freedom in solution, while the rigid π-conjugated linkers provide less flexibility to the fully conjugated polymers. This structural flexibility can significantly impact the optical properties, and can be tailored depending on the application of the donor- and acceptor-based conjugated polymers for a wide range of research fields.The original contributions presented in the study are included in the article/DC and SP conceived the research. Chemical synthesis was carried out by NK, SHP, and MC. Spectroscopic experiments and DFT calculations were done by YK. YK and SP analyzed the spectroscopic data. NK, YK, DC, and SP wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Radiation protection on male testis is an important task for ionizing radiation‐related workers or people who receive radiotherapy for tumours near the testicle. In recent years, Toll‐like receptors (TLRs), especially TLR4, have been widely studied as a radiation protection target. In this study, we detected that a low‐toxicity TLR4 agonist monophosphoryl lipid A (MPLA) produced obvious radiation protection effects on mice testis. We found that MPLA effectively alleviated testis structure damage and cell apoptosis induced by ionizing radiation (IR). However, as the expression abundance differs a lot in distinct cells and tissues, MPLA seemed not to directly activate TLR4 singling pathway in mice testis. Here, we demonstrated a brand new mechanism for MPLA producing radiation protection effects on testis. We observed a significant activation of TLR4 pathway in macrophages after MPLA stimulation and identified significant changes in macrophage‐derived exosomes protein expression. We proved that after MPLA treatment, macrophage‐derived exosomes played an important role in testis radiation protection, and specially, G‐CSF and MIP‐2 in exosomes are the core molecules in this protection effect. Surprisingly, MPLA triggered the secretion of macrophage‐derived exosomes and these exosomes were accounted for the radioprotective effects. Exosomes are 30‐100 nm endosome‐derived vehicles,22.1Six‐week‐old male wild‐type C57BL/6 mice were obtained from Shanghai Ling Chang biological technology co., Ltd, and TLR4‐deficient mice and TRIF mutant mice were obtained from Model Animal Research Center of Nanjing University. Mice were kept in specific pathogen‐free (SPF) facility with sufficient sterilized mice food and water for all experiments. All the experiments associated with mice were approved by Second Military Medical University, China in accordance with the Guide for Care and Use of Laboratory Animals published by the US NIH (publication no. 96‐01). MPLA was bought from InvivoGen (Lot: MPL‐38‐02). For MPLA administration, mice were injected with diluted MPLA at dose of 50 µg/kg in 0.1 mL phosphate buffer saline 12 hours before irradiation through intragastric administration, whereas mice in control group were administrated with 0.1 mL phosphate buffer saline in the same way. After exposure to irradiation, mice were killed at different time‐points and subjected to related experiment as designed. For exosome inhibitor administration, GW4869  was injected through intraperitoneal injection (2 µg/g) 2 hours prior to MPLA administration. Neutralizing antibodies were used to neutralize specific proteins. Specifically, mouse MIP‐2 antibody ; mouse MDC antibody ; mouse RANTES antibody ; and mouse G‐CSF  were injected through intraperitoneal injection 2 hours prior to MPLA administration, and the dosage of antibodies refers to previous reports.2.22 humidified incubator. Cells were pre‐treated with MPLA at concentration of 1 µg/mL at 12 hours before irradiation and then, respectively, exposed to 0, 2, 4, 6 and 8 Gy irradiation and subjected to related experiments as designed. CHX  was used to inhibit protein synthesis. Briefly, RAW264.7 was cultured for 24 hours to reach the confluence of 60%‐70%. Cell culture medium was replaced by CHX‐contained medium (1 µg/mL) at 12 hours before MPLA administration. At 12 hours after CHX pre‐treatment, the original cell culture medium was removed, and fresh cell culture, containing with MPLA (1 µg/mL) and CHX (1 µg/mL), was added in cell culture plate for the next 24 hours. This cell culture was used for protein‐free exosome extraction. For neutralizing antibody administration, GC‐1 spg cells was treated with respective neutralizing antibody together with exosome treatment. The neutralizing antibody was used at a concentration three times to the neutralizing dose (ND50).GC‐1 spg cells mice spermatogonias was purchased from BeNa Culture Collection; RAW264.7 was purchased from American Type Culture Collection. DMEM cell culture medium (for both RAW264.7 and GC‐1 spg cells) with 10% foetal bovine serum (Gibco) and 1% penicillin‐streptomycin solution (Hyclone) was obtained from PAA Laboratories. All cells were incubated in 37°C in a 5% CO2.360Co‐γ rays is 1.25 MeV, and dose rate is 1 Gy/min. Mice and cells were exposed to different doses of radiation, depending on the requirements of experiments.60Co γ‐rays in Radiation Center  were used as irradiation generator. The average energy of 2.4g and re‐suspended with PBS for three times. Next, cell suspension was stained with mixed dye solution  for 15 min in 37°C. CytoFLEX (Beckman Coulter Company) was used for flow cytometry sample analysis.Six‐ to eight‐week‐old C57BL/6 male mice were killed 12 hours after 4 Gy irradiation, and then, testis from one side was isolated and grinded into cell suspension. Cell suspension was then centrifuged at speed of 8002.5C57BL/6 male mice were killed 21 days after 2Gy irradiation. Blood serum was isolated from blood drawn from angular vein venous before the animal was killed, and testis from one side was also isolated just after the animal was killed. Serum and testis homogenate were subjected to enzyme‐linked immunosorbent assay (ELISA) assay to determine testosterone level following the manufacturer's instructions (Westang Tech.).2.6To calculate epididymis sperm numbers, epididymis from one side was isolated and cut into tissue fragment in 2 mL 37°C normal saline. The sperm suspension was incubated for 10 minutes and then heated to 70°C in order to kill mice sperms. Sperms were counted by microscopic counting method.2.7For haematoxylin and eosin (H&E) staining, mice were killed at day 1, day 7 after 4 Gy irradiation and at day 21 after 2 Gy irradiation. Testis from one side was isolated and fixed with 4% paraformaldehyde. Next, the samples were embedded in paraffin, cut into thin sections (4 μm thick) and stained with the H&E for the final histopathological studies. For TdT‐mediated dUTP nick‐end labelling (TUNEL) stain, mice were killed at 16 hours after 4 Gy irradiation. Testis from one side was made into tissue sections as mentioned above and subjected to TUNEL staining by using IF TUNEL kit  according to manufacturer's protocol.2.85 RAW264.7 was seeded in transwell chambers, GC‐1 spg cells was cultured in the bottom of 24‐well plate, and transwell chambers and 24‐well plate were then combined according to manufacturer's instructions. For Western blot assay, 1.3*105 GC‐1 spg cells were seeded in 24‐well plates, and for clonal formation assay, 100, 200, 400 and 800 GC‐1 spg cells were seeded, respectively, for 0, 2, 4 and 8 Gy irradiation. RAW264.7 in transwell chamber or GC‐1 spg cells in 24‐well plate was treated with MPLA 12 hours before irradiation. Transwell chambers were removed immediately after exposure to irradiation. GC‐1 spg cells in 24‐well plates were then subjected to clonal formation assay or Western blot assay.The pore polycarbonate membrane  transwell chamber  was used for the co‐culture system. In brief, 1*102.9g to remove cell debris. The supernatants were then mixed with exosome concentration solution in a 4:1 ratio and rested for at least 2 hours in 4°C. The mixture was then centrifuged at 10 000 g for 1 hour to separate exosome from cell culture. Next, exosome preliminary extraction was obtained by re‐suspending exosome precipitate with PBS. Finally, we obtained purified exosomes by centrifuge re‐suspended exosome at 3000 g for 10 minutes in exosome purification filter. ZetaView® Nanoparticle Tracking Analyzer was used in exosome identification  Co., Ltd; Cat No: UR52101) was used for exosome extraction and purification. Briefly, RAW264.7 cell supernatants were isolated and centrifuged at 3000 n Figure C.2.10We obtained testis and cell protein samples by using M‐PER mammalian protein extraction reagent  followed by manufacturer's instruction. DNA‐PKcs T2609 , p‐ATR , γH2AX , TLR4 , Bax , Bcl2 , caspase3 , C‐caspase3  and β‐tubulin  were detected by Western blot assay, and the secondary antibody (1:5000) was purchased from Cell Signaling Technology.2.11Data were expressed as means ± the standard error of mean (SEM) for each experiment. The number of samples is indicated in the description of each experiment. We used an analysis of variance (ANOVA) followed by a Student‐Newman‐Keuls post hoc test for statistical analysis. Experiments for quantification were conducted in a blinded fashion, and all the experiments were repeated for at least 3 independent times.33.1To determine the radioprotective effects of MPLA on testis, we administrated MPLA at the concentration of 50 µg/kg per mice by intragastric administration 12 hours before 2 Gy irradiation. On 16 hours, day 1, day 7 and day 21 after irradiation, testis was isolated and subjected to H&E staining or TUNEL staining. We found that testis of MPLA administration group maintained a relatively complete structure. Less necrotic cells and cavities in convoluted seminiferous tubules were found than that in IR group on 1, 7 and 21 days Figure ,B. TUNEL3.2It is widely recognized that IR causes DNA strand breaks and subsequently induces cell apoptosis.3.3Next, we explored whether radiation protection effects of MPLA on testis are TLR4‐dependent. We isolated testis form TLR4‐deficient mice 16 hours and 7 days after irradiation and subjected tissue samples, respectively, to H&E staining Figure  and TUNEDeriving from spermatogonial stem cells, spermatogonias differentiate into primary spermatocytes and eventually into sperms; on the other hand, spermatogonias also maintain stable numbers through self‐renew.3.4Consistent to our in vivo data, MPLA showed little protective effect on murine spermatogonia GC‐1 cell lines, which showed low TLR4 expression level Figure A‐C. Next3.5To identify the key factor inside exosomes involved in radiation protection, we firstly used CHX to block the protein translation and found that after CHX treatment, the radioprotective effects of MPLA were abrogated Figure . Then, wAmong all, we selected four proteins with the most obvious changes in expression and used neutralizing antibody to validate their respective role in radiation protection both in vivo and in vitro. In histological examination, we observed that the protective effects of MPLA were inhibited in mice treated with G‐CSF neutralizing antibody and MIP2 neutralizing antibody Figure . We furt4To our knowledge, this was the first study that demonstrated TLR4 agonist MPLA‐stimulated macrophage‐derived exosomes participate in testis radiation protection. We detected that MPLA alleviated damage of testis and spermatogonias induced by IR, and maintained testis function by stimulating mice macrophage secreting G‐CSF– and MIP‐2–enriched exosomes. In in vivo study, we observed that MPLA treatment obviously alleviated testis structure damage both in short‐term (7 days) and long‐term (21 days). Consistently, testis endocrine function and sperm generation function also were maintained well in MPLA‐treated group. However, spermatogonias in testis express trace TLR4 as detected by testis sanction immunofluorescence staining and Western blot assay. Moreover, we observed significant TLR4 activation in spleen macrophages after MPLA administration, this give us an idea that distant cell/organ‐derived secretion might play roles in testis radiation protection effects. By using exosome inhibitor GW4869 in vitro, we preliminary confirmed that macrophage‐derived exosomes were accounted for testis radiation protection. Specifically, G‐CSF and MIP‐2 in macrophage‐derived exosomes were determined to be the core component in testis radiation protection as their neutralizing antibodies abrogated the radioprotective effects of MPLA.It is widely accepted that IR‐induced DSBs are the most important reason for cytotoxic lesion. DSBs can be induced by either IR direct damage or IR‐induced reactive oxygen species (ROS). Non‐homologous end joining (NHEJ) or homologous recombination (HR) are the two main repair methods. As NHEJ repair does not require a template and occurs in all stages of the cell cycle, NHEJ is the primary repair method in mammals.Apoptosis is a common way of programmed cell death when DNA damage cannot be repaired. DNA damage most often activates the extrinsic death receptor apoptosis pathway and/or the intrinsic mitochondrial apoptosis pathway. The activation of mitochondrial apoptosis pathway depends on the release of cytochrome c from the mitochondria into the cytoplasm, which is governed by pro‐apoptotic proteins, such as Bax (Bcl‐2–associated X protein) and Bak (Bcl‐2 homologous antagonist/killer), and anti‐apoptotic proteins such as Bcl‐2 (B‐cell lymphoma 2) and Bcl‐XL (B‐cell lymphoma‐extra large).Macrophages are derived from monocytes and participate in innate immunity, and the ability to secrete cytokines and chemokines is an important basis for macrophages in immune response and antigen presentation.It is widely reported that MPLA stimulates immune cells secreting plenty of pro‐inflammatory cytokines and chemokines, including IL‐1β, IL‐6, and TNF‐α.In a study conducted by Antonio Hernandez,Exosomes are widely studied as cellular communication mediator, and the lipid surface structure grants exosomes to be potential drug carriers in delivering drugs into blood‐brain barrier (BBB) or blood‐testis barrier. It has been demonstrated that cell‐derived exosomes can cross the BBB model under stroke‐like conditions in vitro,We believe that MPLA has great advantageous over other TLR4 agonists and is promising in clinical application as radiation protection drug on male reproductive system. MPLA is a low‐toxicity TLR4 agonist with TLR4 activation effect. According to our experimental results, MPLA produced significant testis radiation protection effects at the dosage of 50 mg/g, and there were no obvious side effects at the dosage 250 mg/g at least. This result suggested a wide range of safety dosage in clinical administration. It is reported that MPLA stimulates less pro‐inflammatory factors than LPS or other TLR4 agonists in endotoxin shock model or in macrophages, including IL‐6, IL‐12p40, TNF‐α and IL‐1β,Taken together, we firstly demonstrated that MPLA helped alleviate testis radiation injury. We found that this effect is mediated by MPLA‐stimulated macrophage‐derived exosomes. Furthermore, we identified G‐CSF and MIP‐2 were the two specific exosomes proteins participating in testis injury alleviation. These new findings will help us better understand the role of TLR4 in radiation protection and give us new thought on exosomes‐mediated protection against ionizing radiation injury.The authors have no conflicts of interest to disclose.FG, XS and JC contributed to study concept and design, carried out experiments, prepared the manuscript and obtained funding. Z Liu, KC, Z Liao and YY: carried out experiments, data analysis and figure preparation. QW, CL, KC and YC: carried out experiments. XL: contributed to study design and obtained funding.FigS1Click here for additional data file.FigS2Click here for additional data file.FigS3Click here for additional data file.


Pseudotumour cerebri syndrome  is characterised by the symptoms and signs of raised cerebrospinal fluid pressure (CSFp) in the absence of ventricular dilatation or an intracranial mass lesion. Its aetiology is unknown in the majority of cases but there is much evidence for impaired CSF absorption. Traditionally, sagittal sinus pressure has been considered to be independent of CSF pressure in adults. However, the discovery of stenoses of intracranial venous sinuses and introduction of venous sinus stenting has highlighted the importance of the venous drainage in PTCS. In this study, we have explored the relationship between CSFp and SSp before and during a CSF infusion test and during CSF drainage.Ten patients  with PTCS underwent infusion studies in parallel with direct retrograde cerebral venography. Both SSp and CSFp were recorded at a baseline and during CSFp elevation in a course of a CSF infusion test. The drainage of CSF after the CSF infusion was performed in 7 patients. In 5 cases, jugular venous pressure was also measured.R = 0.96; p = 0.001) and during infusion . During drainage, this correlation was maintained until SSp reached a stable value, whereas CSFp continued to decrease.CSFp and SSp including their amplitudes correlated significantly and strongly both at baseline (In this series of ten patients with PTCS, CSFp and SSp were coupled, both at baseline and during infusion. The implications of such coupling for the calculation of CSF outflow resistance are discussed. Pseudotumour cerebri syndrome (PTCS) is characterised by the symptoms and signs of raised cerebrospinal fluid pressure (CSFp) in the absence of ventricular dilatation or an intracranial mass lesion and often without a known aetiology , 27, 28.Potential underlying mechanisms for PTCS include impaired CSF drainage and raised intracranial venous sinus pressure , 25, 26.Many of these stenoses were not fixed but resolved, at least in part, with drainage of CSF. Such reversible stenoses contradict the traditional view that the intracranial venous sinuses are largely incompressible in adults with sagittal sinus pressure being independent of CSF pressure . FurtherIf + SSp, where Rout is the resistance to CSF outflow and If is the CSF formation rate [In this study, we have explored the degree of coupling between CSFp and SSp waveforms in adults suffering from PTCS by using lumbar CSF infusion studies to measure CSFp with simultaneous direct measurements of their SSp at baseline, during infusion, and during/after CSF drainage. In addition, we have examined the implications of such coupling for the calculation of CSF outflow resistance when using Davson’s equation: CSFp = Rout × ion rate .A preliminary account of this work has been published .Between 2004 and 2006, we investigated 10 patients (9F:1M) with the clinical features of PTCS who fulfilled the modified Dandy criteria , As part of their clinical investigation, they underwent two procedures: constant-rate lumbar CSF infusion studies, to assess the CSFp and CSF dynamics, and direct retrograde cerebral venography (DRCV) whereby a catheter was placed within the sagittal sinus under fluoroscopic guidance, in order to assess the significance of the stenosis.Patients undergo either or both procedures in our hospital routinely as part of their PTCS investigations, in order to establish a diagnosis and plan treatment. Both DRCV and lumbar infusions have been used safely in our centre as well as other centres internationally , 4, 18.TM manometry lines, length 180 cm and inner diameter 1.2 mm), and pressure amplifier (Spiegelberg or Philips) to the LP needle allowed for pressure recording at a frequency of 30–100 Hz, with following processing by ICM+ (University of Cambridge Enterprise Ltd.) [Access was gained via lumbar puncture using two 21-gauge Quincke needles at the intervertebral space L4-L5 using lidocaine local anaesthesia, with the patient lying on their side. A strict aseptic technique was used to keep all the pre-filled tubing and the transducer sterile. The skin was carefully prepared with antiseptic solution. Connection of a standard, disposable fluid-filled pressure transducer  were extracted through computer data analysis and recorded alongside CSFp.SSp was monitored and recorded with ICM+ in the same way as CSFp. The mean pressure level, slow vasogenic waves (period from 20 s to 2 min), and amplitude of pulse waveform   Fig. .Fig. 2CoR = 0.92; p < 0.003; N = 7) and the changes of both pressures correlated strongly . The slopes of the amplitude—pressure lines, calculated from a simple, linear regression model between CSFp and AMP of CSFp, and SSp and AMP of SSp—also correlated strongly during infusion   Fig. .Table The jugular venous pressure was measured in 5 patients and on average was 10.43 ± 3.8 mmHg. The jugular venous pressure (JVP) during one of the infusion tests is shown in Fig. R = 0.78; p = 0.065, N = 6 . During drainage, SSp appeared to stabilise at a level close to jugular vein pressure while CSFp continued to fall.During drainage, the overall correlation between the 2 pressures was = 6 Fig. . During Table a × CSFp + b . In the one patient, CVP was measured and found to be 11 mmHg; the intercept of the correlation was 9.2 mmHg, which is within the limits of measurement error .CSFp and SSp are coupled both statically  and dynamically .When CSFp increases during CSF infusion, it produces an increase in SSp and its vasogenic components.During drainage, both pressures decrease until a certain point (most probably JVP) when CSFp may decrease further while SSp remains constant.Our results indicate the following, in many cases of PTCS:Thus, venous sinus narrowing in PTCS generates significantly raised CSFp in contrast to healthy normal subjects , 22, 24.After the start of infusion, even though there is a direct coupling between changes in CSFp and SSp, the two pressures appeared to diverge compared with baseline , approximately 67% lower.In the ten patients with PTCS in this study, the Rout calculated without correction for changes in SSp is 16.1 ± 2.1 mmHg/(ml/min). Using the measured values of SSp, Rout is calculated as 5.2 ± 1.4 mmHg/(ml/min)  that may be described by a single parameter. If CSF absorption is split between upstream and downstream channels, Davson’s equation cannot be used as the relative proportion of ‘with IIH . InteresThe situation may become even more complex if the stenosis is reversible with CSF removal and behaves as a Starling resistor . If partAn important question that merits systematic study is whether the phenomenon of direct coupling of CSFp to SSp is limited only to PTCS or may also play a role in some cases of acute intracranial hypertension seen during brain swelling . A study in this direction from early work suggested that 60% of ICP should be attributed to vascular mechanisms, rather than CSF circulatory component , 22, 32.Finally, our statistically strong and significant findings could have important implications for PTCS patients, both in the adult and paediatric populations, and it would be worth designing future randomised trials aiming at treating PTCS patients by stopping the reported pathophysiological coupling of the two pressures.We did not collect information about arterial blood pressure during the infusion studies and therefore its role in the interaction between the CSF and arterial/venous blood flows for these PTCS patients.Unfortunately, full analysis of the frequencies of the CSFp and SSp/JVP is not possible using retrospective data because we did not have information about the frequency properties of the two pressure measurement systems used: in CSFp, a short and wide manometer line and a LP needle was used; however, in SSP, a long thin catheter and external transducer. This makes accurate spectral analysis on CSFp-SSp questionable.CSFp and SSp are coupled in PTCS, both at baseline and during infusion, forming a positive feedback loop that may be interrupted by CSF drainage. The implications of the calculation of CSF outflow resistance are discussed.


LIS1 p. Lys64* variant at 9.4% frequency; in a second patient with focal cortical dysplasia the TSC1 p. Phe581His*6 variant at 7.8% frequency; and in a third patient with ganglioglioma the BRAF p. Val600Glu variant at 3.2% frequency. To determine if cerebrospinal fluid cell-free DNA was brain-derived, whole-genome bisulphite sequencing was performed and brain-specific DNA methylation patterns were found to be significantly enriched (P = 0.03). Our proof of principle study shows that cerebrospinal fluid liquid biopsy is valuable in investigating mosaic neurological disorders where brain tissue is unavailable.Brain somatic mutations are an increasingly recognized cause of epilepsy, brain malformations and autism spectrum disorders and may be a hidden cause of other neurodevelopmental and neurodegenerative disorders. At present, brain mosaicism can be detected only in the rare situations of autopsy or brain biopsy. Liquid biopsy using cell-free DNA derived from cerebrospinal fluid has detected somatic mutations in malignant brain tumours. Here, we asked if cerebrospinal fluid liquid biopsy can be used to detect somatic mosaicism in non-malignant brain diseases. First, we reliably quantified cerebrospinal fluid cell-free DNA in 28 patients with focal epilepsy and 28 controls using droplet digital PCR. Then, in three patients we identified somatic mutations in cerebrospinal fluid: in one patient with subcortical band heterotopia the  et al. report a new strategy to use CSF cell-free DNA to detect brain-only somatic mutations as a surrogate for brain tissues.Somatic mutations can cause brain diseases. Currently, the only established route to study the brain-only somatic mutations is by sequencing brain tissues obtained from neurosurgery or autopsy. Ye   Cell-free DNA is externalized, fragmented DNA found in bodily fluids and is the product of programmed cell death. Fragments are predominantly short with a predominant peak at ∼167 base pairs (bp)  and as aWhile a significant proportion of patients with neurological and neurodevelopmental disorders have disease-causing germline mutations . Mosaic The Human Research Ethics Committees of The Royal Children’s Hospital (Project No. 29077F) and Austin Health, Melbourne, Australia (Project No. H2007/02961) approved this study. Written informed consent was obtained from all participants or their parents or legal guardians according to the Declaration of Helsinki.®  tubes via dural puncture immediately after craniotomy (epilepsy patients) or lumbar puncture (controls). Mean CSF volume collected was 2 ml .For initial CSF cell-free DNA quantitation, we studied 28 patients with epilepsy  and 28 controls without epilepsy . They were recruited through Austin Health, Royal Children’s Hospital, and Queensland Children’s Hospital, Australia. All patients had drug-resistant, lesional, focal epilepsy requiring surgical treatment . ControlFor molecular diagnosis, CSF samples were obtained by lumbar puncture  and peri-operative dural puncture  in patients with known or suspected brain mosaic mutations.g at 4°C for 10 min then extracted according to the manufacturer’s protocol. For blood and brain, genomic DNA was extracted using standard methods .Cell-free DNA was extracted from CSF using the QIAamp Circulating Nucleic Acid Kit : CSF was centrifuged at 1000 × GNAQ c.548G wild-type allele (NM_002072.5:c.548G) as a reference for cell-free DNA copies  that we previously optimized in-house  ddPCR assay (TSC1 NM_000368.5:c.1741_1742delTT (p.Phe581His*6) ddPCR assay , or a PrimerPCR™ BRAF NM_004333.6:c.1799T>A  assay   . Dropletles, CA) . The limles, CA) .To determine origin of CSF cell-free DNA, we examined cell-free DNA for brain-specific DNA methylation patterns. We analysed four control CSF samples and patient 1’s CSF sample by whole-genome bisulphite sequencing (WGBS) followed by methylK analysis . Sequencn = 11) using Kallisto software (n = 5) and plasma (n = 5) cell-free DNA were assigned  to their cell-of-origin by hash table lookup using Kallisto software  and central nervous system (CNS) cell types . A P-value of <0.05 was considered statistically significant.Student’s The authors confirm that the data supporting the findings of this study are available within the article and its GNAQ c.548G wild-type allele as a reference for cell-free DNA copies using ddPCR. Cell-free DNA copies were successfully detected and reliably quantified in all patient CSF samples  in blood (13% VAF)  detected at 2.8% VAF by deep sequencing in the resected lesion. We obtained 1.5 ml of CSF via dural puncture prior to resective surgery and isolated cell-free DNA (789 copies/ml). The TSC1 p. Phe581His*6 mutation was detected at 7.8% VAF in CSF; using the same ddPCR assay, we re-confirmed this mutation in brain tissue (2.3% VAF), and it was undetectable in blood  . Since 50–60% of patients with ganglioglioma have a brain-only recurrent somatic l600Glu) , we screl600Glu) .P = 0.03) enriched for unique reads pseudo-aligned to brain cells, whereas plasma-derived cell-free DNA had a significantly (P = 0.04) higher number of unique reads pseudo-aligned to blood cells  age: the patients were children while the controls were adults and apoptosis is elevated in the brain during development; (ii) CSF collection: patient CSF was collected via dural puncture whereas control CSF was collected via lumbar puncture. It would have been ideal to obtain CSF via dural puncture from some controls, but we did not have institutional ethics approval for this.Our findings suggest that CSF liquid biopsy is a viable surrogate for brain tissue to establish molecular diagnosis of brain somatic mosaicism, with implications for clinical care. These findings may provide new insights into the molecular genetic factors behind epilepsy and brain malformations, as well as other neurodevelopmental and neurodegenerative diseases potentially linked to brain somatic mutations where neurosurgery is not standard of care.There are three main challenges to clinical implementation of CSF liquid biopsy for diagnostic purposes. First, the technical issue of obtaining high-quality DNA from CSF. We have shown that the level of CNS-derived cell-free DNA in the CSF of epilepsy patients is sufficient to enable targeted molecular interrogation of somatic mutations. Second, the biological issue of detection of low concentrations of mutant DNA in CSF, especially if the mutation is presumed localized to a small lesion; detection of mutations in patients 2 and 3 shows that this challenge may be overcome. Third, we performed only targeted mutational analysis in three patients to establish the proof of principle of this approach. The remaining patients were not tested due to lack of a candidate variant from tissue analysis for targeted assays. In the future, unbiased, high depth screening, with optimized low-input genomic capture for CSF cfDNA, will be required to identify a novel somatic mutation genome-wide in establishing broader clinical utility.As precision treatments for neurogenetic disorders are being investigated, such as mTOR inhibitors for cortical dysplasia, implementing CSF liquid biopsy for aetiological diagnosis will become increasingly important as a surrogate for brain-derived DNA to diagnose brain-specific somatic disorders.Brain Communications online.fcaa235_Supplementary_DataClick here for additional data file.


Differences between the thickness of the anterior talofibular ligament and the calcaneofibular ligament (p < 0.001), the diameter of the fibular insertion of the anterior talofibular ligament (p < 0.001), the diameter of calcaneal attachment of the calcaneofibular ligament (p < 0.05), and tibiocalcaneal angle (p < 0.01) were statistically significant. The presence of the double fascicular anterior talofibular ligament and the calcaneofibular ligament fascicles correlate with connections to adjacent structures.The anterior talofibular ligament and the calcaneofibular ligament are the most commonly injured ankle ligaments. This study aimed to investigate if the double fascicular anterior talofibular ligament and the calcaneofibular ligament are associated with the presence of interconnections between those two ligaments and connections with non-ligamentous structures. A retrospective re-evaluation of 198 magnetic resonance imaging examinations of the ankle joint was conducted. The correlation between the double fascicular anterior talofibular ligament and calcaneofibular ligament and connections with the superior peroneal retinaculum, the peroneal tendon sheath, the tibiofibular ligaments, and the inferior extensor retinaculum was studied. The relationships between the anterior talofibular ligament’s and the calcaneofibular ligament’s diameters with the presence of connections were investigated. Most of the connections were visible in a group of double fascicular ligaments. Most often, one was between the anterior talofibular ligament and calcaneofibular ligament (74.7%). Statistically significant differences between groups of single and double fascicular ligaments were visible in groups of connections between the anterior talofibular ligament and the peroneal tendon sheath ( Meanwhile, the calcaneofibular ligament (CFL) is composed of the medial (M-CFL) and lateral (L-CFL)9. The interconnections between the ATFL, CFL, and the posteriori talofibular ligament (PTFL) provide integration between components making a complex of ligaments to a functional unit10. The most prominent interconnection is I-ATFL and the CFL, which is usually arch-formed11. The S-ATFL is considered as an intraarticular structure, while the I-ATFL is an extraarticular part. It is unclear if the S-ATFL is intrasynovial or only intracapsular. If the only S-ATFL is injured, healing potential is lower because of its intraarticular localization, and if the I-ATFL is preserved, a secondary microinstability may appear12. However, during injury propagating via the I-ATFL, forces may also be transmitted to the CFL, causing its injury, and the full instability may appear13.The ATFL is the weakest part of the lateral ankle ligaments, which is usually the first and most common injured ligament in ankle inversions. The anterior talofibular ligament (ATFL) is composed of the superior (S-ATFL) and inferior fascicle (I-ATFL)14. MRI criteria of injury to a ligament also include an assessment of dimensions; however, the cut-off values are not fully established. There is a significant discrepancy in the length, thickness, and width of the normal ATFL , a history of previous fracture (19 cases excluded), and obvious abnormality in relation to the lateral malleolus (8 cases excluded). In total, 198 MRI examinations of the ankle fulfilled the inclusion and exclusion criteria. All examinations were reviewed twice, and the final results were made by consensus.The MRI protocol may vary between cases, but inclusion criteria were the presence at least proton density (PD) or T2-weighted sequences without fat saturation in sagittal, axial, and coronal plane to assess ligaments structure. The other sequences, like T1-weighted turbo spin-echo (T1-TSE), PD with fat suppression, or short-T1 inversion recovery (STIR), were used to detect pathology, which might be the exclusion criterion. All MRI examinations were performed using the MRI machine Ingenia 3.0 T MR system  with a dedicated ankle coil.Our study included 110 females and 80 males with an age range of 18–58 years, and a mean age of 31 years. The right ankle was examined in 107 cases, and the left one in 91.24 was performed and correlated with CFL morphology and morphometry.The statistical correlations between of presence of connection to adjacent structures, the morphometry, side, gender, and presence of the interconnections between the ATFL and CFL in the groups of single and double fascicular ATFL and CFL were analyzed. The other lateral structures included in our study were the PTFL, the superior peroneal retinaculum (SPR), the peroneal tendon sheath (PTS), the fibulotalocalcaneal ligament (FTCL), the anterior inferior tibio-fibular ligament (AITFL), and the inferior extensor retinaculum (IER). The measurement of the tibiocalcaneal angle (TCA), according to Buck et al.24.The protocol and some of the measurements are presented in Fig. The Local Swedish Ethics Committee and Institutional Ethics Committee at the Medical University of Warsaw approved the study and waived the need for informed consent (Numbers 06177 and AKBE/258/2019) due to the retrospective and non-invasive nature of the study. The study was conducted in accordance with the Declaration of Helsinki. The anonymization of patient data in the research process ensured data protection in accordance with the European General Data Protection Regulation. The authors declare that this work has not received any funding before or during research. There are no relationships with any companies whose products or services may be related to the subject matter of the article.p > 0.05).Our MRI study revealed the presence of interconnections between the ATFL and CFL and with other lateral ankle structures included in the study Figs.  and 3. WThe connection of the ATFL with the CFL was the most common one and was found in 148 cases (74.7%), Figs. The most connections with non-ligamentous structures were seen in groups of double fascicular ligaments Figs. , 5, 6. IIn the group of the CFL, the other most common connections were with the PTFL 68.7%), the SPR (67.2%), and the FTCL (64.1%), Figs. %, the SPThe presence of the double fascicular ATFL was identified in 148 cases (74.7%), of which in 101 cases (76.3%), two fascicles were completely separated while in 47 cases (23.7%) were incompletely separated. The single fascicular ATFL was identified in 50 cases (25.3%). The double fascicular CFL was recognized in 131 cases (66.2%), whereas a single fascicular CFL was seen in 67 cases (33.8%). In 84 cases (42.4%), the LTCL was present and branched from the CFL in 69 cases (34.8%), while in 15 cases (7.6%), it originated from the calcaneus as a separate structure.The possibility to distinguish the medial (M-CFL) and the lateral (L-CFL) CFL fascicle was from the midportion and proximal to that. The L-CFL was always thicker than the M-CFL and had insertion on the inferoanterior outline of the apex of the lateral malleolus. We identified two variations of attachment of the M-CFL. The most often, one was seen on the fibula medial of the L-CFL in 84 cases (64.1%). The other one was found on the talus in 47 cases (23.7%) of which united with LTCL before it reaches the talus in 28 cases (14.1%) or ran directly to the talus in 19 cases (9.6%)  = − 0.25, p < 0.001. The differences in TCA were statistically significant, Table 19.We revealed a statistically significant negative correlation between the CFL angle and TCA The differences in diameters between groups of single and double fascicular ATFL and CFL.The average length of the ATFL was 24.2 ± 3.4 mm, the thickness in the midportion was 2.1 ± 0.3 mm, while the corresponding values for the CFL were 32.6 ± 4 mm and 2 ± 0.2 mm Tables . StatistThe average breadth of the midportion of the ATLF was 5.1 ± 0.7 mm, and the corresponding value for the CFL was 6.5 ± 1.4 mm; however, without statistically significant differences  MRI sequences when a collagen structure is oriented at 55 grades to the main magnetic field36. Our study revealed the fascicular structure of the ATFL also on long-TE MRI sequences, which negates the presence of this artifact. No absence of the ATFL was detected, which was reported previously in 5%22.The presence of connections revealed in our study is also important for radiologists during the assessment of an ankle using an MRI after injury and for orthopedists in planning the reconstruction of ruptured ligaments11. The close relation to the CFL indicates that the LTCL, if present, is part of the lateral ankle ligament complex.The LTCL is a variable structure located in direct relation to the CFL. Most of the cases found in our study branched directly from the CFL, while in others, authors reported that it was never found as a separate structure1. We believe that the presence of the connections described by us is also crucial in training and preventing sports injuries. Strengthening one structure by the presence of connections may improve the function of others. However, further research is needed to confirm our hypothesis. We believe that our results provide sufficient anatomical background for further functional studies.A ligament's fascicular structure allows different fiber orientations, functional differentiation, and better alignment of a joint. Due to interconnections, single smaller ligaments connect to form the larger anatomical-functional units resembling, for example, a cuff rotator in the shoulder. Functionally, this possibly results in better biomechanical joint stabilization20, whereas when compared to other authors who used larger material, our results were similar22 (Tables 37. It was reported that the wider and longer ligament’s fascicles are more prone to the pathological process than thinner ones38. Therefore, the diameters may have a clinical significance.Differences between the diameters of the ligaments between the single and double fascicular were noticed. To our knowledge, there is no anatomic or radiologic paper about the correlation of the length, the width, and the thickness of the ATFL and the CFL with its fascicular nature. The length of the ATFL reported previously seemed to be circa 10 mm shorter when compared to studies on smaller materials17. The ATFL, in clubfeet, was reported to be much wider than in the two normal feet, which indicates a developmental background27. The width of the midportion assessed in our study is slightly smaller than reported previously33.The thickness of the CFL shown in our study was consistent with what was previously reported32. We reveled that ligaments’ dimensions are related to their fascicular structure. We used 3 T MRI because it allows for good resolution and excellent tissue differentiation39. To our knowledge, there is no cut-off value of the normal variants of the ATFL. The average thickness of the midportion of the ATFL revealed by us corresponds to the values reported before41 thus, we recommend using 2 mm as a cut-off value of the normal ligament. It is difficult to find studies on all dimensions of the ATFL and CFL on the same material, which makes comparison difficult (Tables The thickness of the ligaments is one of the most important measurements for the radiologists because the ligamentous thickening and thinning is an MRI criteria for tears42. Most of the previous studies provide the value of the area of insertion, which makes it harder to compare. However, some reference can be made. There is a discrepancy in the size ratio of the attachments reported in the literature. Some authors report that size of the talar insertion of the ATFL is bigger than the fibular one9, while the other reported exactly the opposite18. It is difficult to solve this problem based on our measurements. However, the craniocaudal diameter revealed in our study was larger in the fibula, where the attachment is more elongated, while the talar attachment is more oval18.To our knowledge, there have been no previous large-scale MRI studies regarding measurements of bony insertions. MRI examinations were performed for clinical purposes, so the assessment of the two dimensions of the insertion was somewhat difficult. Therefore, we included the largest diameter of the attachment-based on the known shape of the attachment19, may be used with great sensitivity and specificity. The increased TCA is associated with a reduced CFL angle and may indicate a hindfoot19. Taking into consideration the presence of the connections between ligaments and non-ligamentous structures, fluctuations in tension in CFL translates to other structures causing changes in their function.An unrecognized hindfoot valgus may cause a chronic deformity and secondary osteoarthritis. Initially, the patient experiences only the pain associated with overuse, and an MRI is often performed. Early identification of a valgus malalignment on an MRI may be crucial for the patient’s outcome. The CFL angle and the tension of the CFL change during movements and deformation. The cut-off value for the suspicion for a valgus hindfoot, ≤ 119 grades18. The measurement errors may be related to the cutting angle and the measurement point of the single slice image. The retrospective character may also be a limitation of the study.We acknowledge several limitations in our present study. Only one dimension of the bony attachments was measured. We believe that the full assessment of the bony attachments area and more accurate measurements may be possible when a special protocol with ultra-thin layers or with 3D sequences can be used. However, we believe that the MRI assessment of the fibular attachment may be difficult due to the multiple connections describedThe presence of the double fascicular structure of the ATFL and CFL is related, more often, with the occurrence of interconnections between these two ligaments as well as with the adjacent structures.