The COVID-19 pandemic, as well as efforts to prevent its spread, have had a strong impact on the delivery of rehabilitative services in Germany. While several studies have addressed the impact of these developments on health service providers and COVID-19 patients, little is known about its impact on patients in need of rehabilitative treatment because of other conditions. This study aims to identify expectations, concerns and experiences of rehabilitation patients related to service delivery in this situation.Using a qualitative study design, user posts from six German online forums between March and Mid-November 2020 were systematically searched with respect to experiences, concerns and expectations of health care users toward receiving rehabilitative treatment. We used qualitative content analysis with inductive coding as our methodological approach.Users fearing physical or psychological impairment were concerned about not receiving timely or effective treatment due to closed hospitals, reduced treatments and limited admissions. In contrast, patients more concerned about getting infected with COVID-19 worried about the effectiveness of protective measures and being denied postponement of treatment by the funding bodies. During their stay, some patients reported feeling isolated due to contact restrictions and did not feel their treatment was effective, while others reported being satisfied and praised hospitals for their efforts to ensure the safety of the patients. Many patients reported communication problems before and during their treatment, including concerns about the safety and effectiveness of their treatment, as well as financial concerns and worries about future treatments. Several users felt that their concerns were disregarded by the hospitals and the funding bodies, leaving them feeling distressed, insecure and dissatisfied.While some users report only minor concerns related to the pandemic and its impact on rehabilitation, others report strong concerns relating not only to their own health and safety, but also to financial aspects and their ability to work. Many users feel ignored and disregarded, showing a strong need for more coordinated strategies and improved communication specifically with funding bodies like health insurance companies and the German pension funds. In addition to the COVID-19 pandemic itself, efforts to reduce the number of infections and to protect vulnerable populations have strongly impacted processes and structures in health care services in several ways. Following a rapid increase in infection rates, rehabilitation hospitals had to develop new strategies to deliver safe and effective services and to protect patients and health care workers. In response to this situation, the Deutsche Rentenversicherung Bund (DRV), i.e., the German Pension Insurance Association, which is the responsible funding organization for the majority of rehabilitative treatments provided in Germany, announced a reduction in operation of almost all hospitals nationwide in early March 2020 , 4.In the wake of the restrictions implemented to reduce or prevent the spread of COVID-19, the disruption of health care services, forced social isolation at home and movement restrictions also increased the need for rehabilitation persistently , 6. NeveWhile several studies focus on improving the treatment and rehabilitation of patients recovering from COVID-19 through newly developed procedures and specific regulations , few stuA qualitative design using inductive qualitative content analysis was employed to analyze posts on German online forums. We used a purposive sampling approach to identify relevant posts. In this respect, six German online forums (names withheld to ensure anonymity of participants) with different thematic foci were chosen for data collection to explore different perspectives of rehabilitation patients. Four of these forums mainly addressed patients with chronic conditions , one was a general health and lifestyle forum, and one forum focused on rehabilitation, retirement, and pension planning. Apart from their thematic focus, forums were chosen according to the numbers of active users, numbers of threads and posts, and recent activity (posts in relevant forum sections in the last 48 h before data extraction). Potentially relevant information in these forums was identified through keyword searches using the terms “reha”, “SARS-CoV-2”, “Covid”, “Corona”, “pandemi*” or “lockdown”, covering all postings from the beginning of March to mid-November 2020. The applied time frame was roughly chosen to include posts from the beginning of the pandemic in Germany up to the day of data extraction.Relevant content was extracted to a text document for data analysis until information saturation was reached and the collection of further user contributions did not provide any new findings. All German threads and comments were translated into English in the coding process, while usernames and nicknames obtained during the data collection were anonymized with a standardized signifier. This approach to create anonymity for the forum users is in line with other studies , 15; theAll posts and comments related to rehabilitation and SARS-CoV-2/COVID-19 discussed in the threads were imported to the qualitative data analysis software MAXQDA to organize and code the data. Qualitative content analysis was chosen for the analysis to systematically classify information and identify themes or patterns from text data . To answThrough investigator triangulation and purposive sampling, credibility was ensured . The recOverall, 99 threads and approximately 450 posts were obtained. In our analysis, we identified 9 codes and subcodes from the data, representing five general topics or themes.It must have escaped the employees that we are currently in a Corona pandemic. I am really outraged by this behavior. (User #31)After my hip replacement surgery, I was only able to spend 11 out of 20 days (15 + 5 extension days) in outpatient rehabilitation, because the facility was closed due to Corona. (User #4)In addition, other patients reported difficulties in getting an appointment due to increased waiting times, travel restrictions, limited admissions to hospitals, and the reduced number of opened hospitals. While patients were living in ongoing pain and with limited mobility after surgery and other treatments, they were unable to or advised not to participate in rehabilitation programs.I am not angry but rather sad that my treatment in a rehabilitation facility is interrupted. I‘m having a really hard time with my swollen and sore joints. Of course, the newly operated people should get treatment. They can’t go to rehabilitation and there is no follow-up treatment. (User #25)To prevent infections and stop the spread of COVID-19, many rehabilitation hospitals were closed temporarily. The cancellation of admissions and treatments led to anger and concern among health care users. Many health care users indicated anxiety and frustration after incomplete or delayed treatments.Despite the restrictions, there is a variety of therapies [available], and the hospital organized itself quite well. [ … ] Some leisure activities are not available, but that doesn’t particularly bother me now. The only criticism is the lack of variety in the food. (User #49)I can only recommend the hospital. At the moment, all employees are working so hard and everything is being done to prevent Corona. The group sizes have been reduced. There are two meal schedules, so that there aren’t too many people in the dining room at the same time. [ … ] The therapists working here are great. (User #50)In contrast, other users reported dissatisfaction with their stay because of too many restrictions and stated their fears of insufficient or ineffective treatments due to these restrictions. These patients perceived the regulations as potentially hampering their recovery. In consequence, many of these health care users either tried to extend their rehabilitation treatment or to delay it to a later date, hoping for a cessation of the restrictions. The measures were perceived as stressful for the patients. One health care user expressed the excessiveness of the measures while stressing the necessity of relaxation during the rehabilitation program.My mother is currently in [the hospital] for a convalescence treatment. It has been 4 weeks now, but she does not feel completely fit again. Due to Corona, very few activities were carried out - which prompted her to apply for an extension for medical reasons after consultation with a doctor. How long does it take for such an application to be approved? (User #52)No meetings, no strolling, and no coffee … I feel very constricted, especially at the Baltic Sea. The rehabilitation should improve psychological well-being, too! (User #51)Additionally, visiting bans at rehabilitation hospitals were reported as particularly burdensome for some patients, as well as their families and friends. In addition to patients feeling isolated and anxious during their stay, relatives and friends experienced difficulties in receiving information about the health and well-being of the patients directly.I am just panicking about this hospital and the 5 weeks I'm supposed to spend there in June. Unfortunately, the objection period has expired and I don't know what to do. (User #22)I am also concerned, because I will miss my husband very much. If you are not allowed to receive visitors because of Corona, this will certainly be very stressful. (User #10)Due to the Corona crisis I cannot visit him, which makes me so desperate. The only option was to ask the nurse to hold his phone in front of his face and make a video call. (User #11)In reaction to the persistent threat of the COVID-19 pandemic, several measures were taken to protect patients and staff and to enable hospitals to continue treatment, such as measurements of body temperature, entrance questionnaires about COVID-19 symptoms, visiting bans, a reduction of group therapies, and admission of fewer rehabilitation patients to the hospitals. While some patients starting their rehabilitation found these new regulations acceptable and efficient, others were more skeptical about the necessity and impact of these restrictions. Some current inpatients shared their experience through online forums, detailing their satisfaction with implemented changes and providing advice to other health care users regarding their experiences in the hospitals. These reports mostly focus on which measures were taken and their impact or the lack thereof on the quality of the treatment.Can I be sure that there are good hygienic measures in place to prevent the spread of the Corona virus and that sick people are tested? I have been in rehabilitation several times. Sick people come and go all the time. (User #12)In light of current events , the question arises, why are these facilities still open at all and why do they continue to accept new patients? (User #13)While patients had different opinions on whether the measures taken by the hospitals could have a negative impact on the quality and effectiveness of the treatment, their effectiveness in preventing infections with COVID-19 was doubted by several patients as well. Health care users who had already had prior experience with rehabilitation hospitals shared their observation of the spread of various diseases during their stay. These users were now more concerned about the threat of the COVID-19 pandemic. The fear of COVID-19 led these patients to demand better hygienic measures. In addition, health care users reported a lack of knowledge about the effectiveness of new hygiene regulations in the hospitals, which suggests that they felt unsafe and perceived a lack of relevant information. Several patients also signaled their hesitancy about continuing their treatments in the hospitals.I am supposed to start an inpatient rehabilitation [ … ]. Can the clinic even provide effective therapies, such as kinetotherapeutic baths, relaxation exercises, physical therapies and group therapies? There is a contact ban for public areas. How can that be maintained in this hospital? I am also very afraid of getting infected and of course of loneliness, because visitors are not allowed. So, what can I do to get a postponement for an indefinite period? (User #77)Due to the new situation caused by the Coronavirus, I am not sure whether I should do it at this time. Gatherings and sports facilities should be avoided. Since all of us in rehabilitation are older and we are already suffering from other illnesses, I think it would be better to postpone my stay. (User #29)Since I'm really scared about the pandemic, do I have to start a rehabilitation now which takes place in a high-risk area? I have read several times that people were sent home because of Corona. To drop out is not the point and purpose of rehabilitation. At the DRV nobody answers the phone because of Corona. I have already sent an email to move the rehab to a later date. Is it possible that they cancel my rehabilitation?? (User #23)Many health care users whose admissions were approved reported trying to postpone or cancel their upcoming stay due to fears of infection and perceived ineffectiveness of the treatment because of the restrictions. Many believed that as a result of the strict regulations, treatment would be inefficient and preferred to delay their appointment to a later date in hopes of a decreased COVID-19 incidence and a subsequent rollback of restrictions. In particular, older adults and patients with chronic illnesses appeared to feel more vulnerable to infections with COVID-19. Although many reported a distinct need for rehabilitation, they perceived delaying their treatment as a more reasonable option instead of risking infection or treatment interruption.I don't know what to do next. The DRV says there is no problem! My health insurance refuses helping me! I'm just gathering some information here and I know that nobody can help me here. I have already asked for help elsewhere. (User #61)I withdrew my application for medical rehabilitation due to the Corona pandemic, because I think that the safety precautions won’t be that effective, and rehabilitation would not be as relaxing as it would be under normal circumstances. In my letter, I explicitly pointed out that the pandemic is the reason. Now I have received a letter from the DRV stating that the approval of my treatment will be regarded as irrelevant, since I forfeit the approved treatment. My reasoning was not taken into account at all. (User #65)After the closure of hospitals and delays of treatments, many patients had to take action to re-arrange the agreement on coverage between insurance companies and rehabilitation hospitals. Since the approval patients received from the insurance organization was valid only for a specific time period, appointment delays led the patients to be concerned about insurance coverage. Similarly, unilateral changes by the insurance organization with respect to the services provided, e.g., offering only outpatient instead of inpatient care, were regarded by patients as health risks, disregarding their fears and safety concerns. These in turn led to additional financial concerns for some patients.I have received a disability pension due to being fully incapacitated to work for the last 7.5 years. When I applied for the continuation of my disability pension, I was asked to go to rehabilitation and received approval for an all-day outpatient treatment (this was my explicit request). Now the outpatient hospital is refusing due to Corona. Can I be forced to get an inpatient treatment now? My pension has been approved until October and will be discontinued if the treatment is not completed. If I cancel this pension and instead get my old-age pension, will it be as much as what I currently receive? [ … ] Am I obligated to do inpatient rehab even though I explicitly asked for outpatient rehab? What if the disability pension is canceled just because I would not agree to any inpatient rehab? (User #58)Here, after the new measures in the hospitals were implemented, the requests of health care users for outpatient rehabilitation were denied. The new regulations implemented to address the pandemic also affected the types of care services, the coverage for rehabilitation, and the types of insurance benefits such as disability pension or old-age pension.Several users reported that their concerns about potential infections, impaired quality of treatment due to restrictive measures, as well as financial concerns, were disregarded by the insurance organization when trying to cancel or postpone treatment. Furthermore, many users reported difficulties in getting relevant information on safety measures, infection risks and conditions of their treatment from rehabilitation hospitals and the insurance organization.Our analysis of posts in six online forums indicates that the COVID-19 pandemic and protective measures implemented to prevent infection create various areas of uncertainty for health care users during the entire process of rehabilitation. Many health care users tried to reduce this uncertainty by sharing their questions, knowledge, and their experiences gained with hospitals and/or the insurance organization online. By evaluating changes in the structure of therapies, rules approved by hospitals, and new regulations concerning hygiene or mealtimes, they provided recommendations and information for other health care users.Our analysis showed that health care users’ experiences varied greatly concerning several aspects of rehabilitation, but often led to very specific concerns. The cancellation of scheduled treatments and the closure of rehabilitation hospitals in the beginning of the pandemic led to anxiety, sadness, and frustration among several users. Many rehabilitation patients suffered from physical pain, impaired psychological well-being, and limited mobility, which were prolonged as a consequence of the aforementioned measures. Such delays have been shown to adversely affect quality of life, physical functioning and activities of daily living . After tWhen hospitals reopened, newly introduced protective measures caused further concerns. The perceived risk of a COVID-19 infection affected patients’ perceptions of protective measures introduced by the hospitals . While sHealth care users in some cases became skeptical of the necessity of many of these measures in the following months, when both the incidence and mortality of COVID-19 decreased. Other patients who had prior experience with rehabilitation treatments still perceived high subjective risk, citing previous infections with other respiratory diseases during rehabilitation as the reason. The perceived threat level of infection determined the perception of protective measures and led to lower acceptance among those feeling less at risk. This is in line with other studies that identified perceived severity of the pandemic as predictors of the perceived importance of hygienic precautions .This led some health care users to try to postpone or cancel their scheduled treatment. Some users reported feeling ignored or disregarded by the insurance organization, which is covering the cost of rehabilitation. In some cases, attempts to switch from inpatient to outpatient treatment or to postpone or cancel treatment were denied or disregarded by the insurance organization, adding additional distress. Users reported being afraid of financial consequences and of being denied treatment at a later point in time, if they chose to cancel their approved rehabilitation. As studies from the USA have shown, the inability to directly access providers for rehabilitation creates delays in treatment and, in our study, in conjunction with uncertain financial consequences of unilaterally canceling scheduled treatment, created additional barriers to utilization of these services . ConsideThese findings suggest distinctive difficulties for health care users in rehabilitation hospitals to obtain reliable, adequate and comprehensive information on central aspects of their rehabilitation in the current situation. The identified areas of uncertainty also indicate the importance of coordinated efforts of health communication to provide clear, unambiguous and consistent information that is easily accessible .To our knowledge, this is the first study focusing on the experience of patients undergoing or in need of rehabilitative treatment in Germany during the COVID-19 pandemic using online forums as its data source. Online forums and social media can contribute to the empowerment of health care users in obtaining and providing social support, consultation, self-preparation, self-screening, and giving feedback by creating new information –33. In tHowever, using social media as an instrument of analysis has some limitations that need to be mentioned, such as the difficulty of the generalization of the results and unclear characteristics of the population . Six socOur study shows that health care users undergoing or waiting for rehabilitation treatment have a multitude of concerns related to their treatment during a pandemic. The risk of infection with COVID-19, prolonged pain, physical and psychological impairment due to delayed or canceled treatments, as well as perceived limitations in the effectiveness of treatment are among the main concerns. Concerns about being infected with COVID-19 during rehabilitation were strongly related to the subjectively perceived threat of the virus and prior experience with rehabilitation treatments, while perceived limitations in the effectiveness of treatments were mostly related to the perception of implemented protective measures. In addition, communication problems and difficulties in the interaction with hospitals and the insurance organization added further concerns about treatment schedules and financial aspects. These findings suggest a strong need for more coordinated efforts and improved communication to mitigate concerns and provide clear information to health care users and patients.These findings suggest that the use of online forums and other types of social media as an additional tool to gather user feedback, improve communication strategies and provide support and guidance can be beneficial for rehabilitation hospitals, funding providers and other stakeholders. That way, direct access to formal, accurate, and clear information could be accelerated for a diverse range of health care users. Considering not only the communication between health care users and service providers or funding bodies, but also the relations of patients with other health care users, health personnel, and their families or friends, continuity of communication should be supported by digital communication tools. While online forums and social media are well suited for qualitative studies, limitations in generating statistically valid and representative data suggest that their use for quantitative studies is currently questionable. Studies from the USA have shown that urban and more densely populated areas were overrepresented on Twitter –39. Simi


The results indicate that PTSS and attachment are connected. Positive changes in attachment insecurities brought about by trauma-focused psychotherapy seem possible.Severe posttraumatic stress symptoms (PTSS) are connected to a variety of health-related and interpersonal problems, among them are the insecure attachment orientations. However, psychotherapy seems to improve not only PTSS but also attachment insecurities. In a large multicenter, randomized clinical trial, the attachment characteristics and PTSS of 85 adolescents and young adults (aged 14–21 years) with clinically relevant abuse-related PTSS were assessed at study entry, at the end of treatment, and 3 months after the end of treatment. Participants were randomized either to a developmentally adapted cognitive processing therapy (D-CPT) or to a wait-list with treatment advice (WL/TA). The purpose of the study was to analyze the association between PTSS and attachment at study entry as well as changes in attachment during the trial. We found that attachment-related avoidance (AR avoidance) was positively associated with PTSS from both self-reports and clinician ratings, whereas attachment-related anxiety (AR anxiety) was only related to self-reported PTSS (Pearson correlation coefficients between 0.37 and 0.46). Changes in AR anxiety occurred in both conditions at some point during the study ; Germanctr.de; identifier: DRKS00004787; date of registration: 18 March 2013.The online version of this article (10.1007/s00787-020-01637-x) contains supplementary material, which is available to authorized users. Sexual and physical abuse in childhood and adolescence is associated with severe negative consequences for mental health , 2. BesiTraditionally, measuring attachment was conceptualized using a categorical approach with a focus on caregiver relationships, and assessed in interviews, primarily the Adult Attachment Interview . More rOne of the key aspects of attachment theory of relevance for psychopathology is the hypothesis that attachment style influences an individual’s available emotion regulation strategies. During periods of stress, an anxiously attached person sees himself as relatively helpless and insistently attempts to obtain care and support from others through exaggerating threats, applying so called hyperactivating strategies. An avoidantly attached person who has no trust in others uses deactivating strategies, denying attachment needs and trying to cope with stressors on their own. Recent research showed that both of these strategies are related to psychopathology . This isNo study applied an interview-based approach for the assessment of PTSS. That is worth noting as there seem to be discrepancies in the relationship between attachment and PTSS depending on the assessment modality , 20: WooThe association between attachment, emotion regulation and PTSS further raises the question whether attachment, like the latter two, improves with effective psychotherapy. Although it is often assumed that attachment styles are stable throughout an individual’s lifespan, Bowlby  believedWhile results are mixed in terms of attachment, the efficacy of psychotherapy on PTSS is well established for different approaches, among them cognitive processing therapy CPT; , 33). CP. CP33]).In a recent multicenter randomized clinical trial, we compared a developmentally adapted CPT (D-CPT) to a wait-list condition with treatment advice (WL/TA) in a sample of adolescents and young adults (aged 14–21 years) with PTSD after abuse . CompareData were collected during a multicenter randomized clinical trial. A detailed description of the trial design and the results on treatment effects regarding primary (PTSS) and secondary outcomes can be found elsewhere , 38.The study was approved by the ethics committees of the universities of Eichstätt-Ingolstadt, Berlin and Frankfurt, and was therefore performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. We enrolled adolescents and young adults aged 14–21 years from July 2013 through June 2015 and obtained informed consents from all participants and, in the case of minors, from their respective parent or guardian. For inclusion, child sexual and/or physical abuse-related PTSD as a primary diagnosis was required, with a lowered threshold for avoidance symptoms . FurtheAfter baseline assessments, participants were randomized either to the D-CPT or WL/TA condition. Participants in D-CPT underwent an adapted form of CPT in thirty 50-min sessions and six optional joint sessions with the caregiver or for crisis intervention. The treatment duration was 16–20 weeks (for more information on D-CPT see ). In theAttachment was assessed using the German version of the Experiences in Close Relationships—Revised Questionnaire ECR-R; , 42). Th. Th42]).N = 225) and an age- and gender-matched non-clinical (N = 250) sample with the clinical group showing higher scores for AR anxiety (3.71 [SD = 1.41] vs. 2.61 [SD = 1.15]) and AR avoidance (3.08 [SD = 1.27] vs. 2.46 [SD = 1.10]).For the German version of the ECR-R, good psychometrical properties regarding internal consistency  and construct validity were reported . In the α = 0.71; avoidance, α = 0.80; intrusion α = 0.86), with a Cronbach’s alpha of 0.90 for the overall total score.Clinician-rated PTSS were measured using a structured clinical interview, the Clinician-Administered PTSD Scale for Children and Adolescents for DSM-IV , the avoidance (α = 0.81) and the intrusion subscale (α = 0.79). However, the hyperarousal scale had a low reliability (α = 0.66).To measure self-reported PTSS, we used the German translation  of the UWe employed a missing completely at random (MCAR) test to examine patterns of missing values . To analt-tests and significance levels were adjusted for multiple testing as described above. Respective effect sizes were calculated using Cohen’s d (earlier vs. later measurements). Due to dropouts during the course of the trial and significant variation in attachment scores at baseline, we decided to analyze the effect of the different treatment conditions using multilevel modelling, which is increasingly recommended in psychotherapeutic research [For a first descriptive overview, changes in attachment after treatment were analyzed separately for both conditions by dependent research . One majresearch , 52. MorTo compare improvements in both groups, we followed the suggestion by Morris  and calcAll analyses were performed using IBM SPSS Statistics for Windows, Version 25.0.N = 85). Table M = 4.09, SD = 1.24; D-CPT: M = 3.54, SD = 1.20; t83 = 2.06, p = 0.043) and prevalence of nicotine dependence .For the present analyses, we only used data from trial participants for whom ECR-R scores were available at least at baseline .The missing data were mostly due to study dropout or non-attendance at the respective measurement point. We lost 15 patients (35%) in D-CPT and 7 patients (17%) in WL/TA to the posttreatment assessment. Complete datasets were available for 49 participants (58%), 16 (19%) provided data at two of three assessment points and 20 (23%) contributed baseline data only. The MCAR test supported the assumption that data were missing completely at random , we calculated partial correlations for the significant associations. After controlling for AR avoidance and adjusting significance thresholds, AR anxiety was still significantly correlated with the UCLA-PTSD-RI avoidance scale  and the total score of the UCLA-PTSD-RI , but no longer with the CAPS-CA avoidance scale . The results were slightly different for AR avoidance after controlling for AR anxiety, resulting in significant partial correlations with the CAPS-CA avoidance scale  and the UCLA-PTSD-RI avoidance scale , but not with the total score of the UCLA-PTSD-RI .First, we looked at the correlations of age and gender with attachment to check for a confounding effect of these variables. There were no significant associations (data not shown). The results of the subsequent correlation analysis are given in Table t23 = 3.66, p = 0.001, d = 0.75). However, we obtained considerable effect sizes for the WL/TA group for AR anxiety  and for the D-CPT group for AR anxiety  and AR avoidance . The attachment scores for the total sample and both groups at all measurements as well as results of all paired-samples t-tests can be found in Online Resource 1.Posttreatment was reached on average after 27.59 SD = 8.10) weeks in D-CPT and 22.41 (SD = 3.98) weeks in TAU. The 3-month follow-up was reached on average after 40.93 (SD = 7.63) weeks in D-CPT and 34.67 (SD = 5.72) weeks in TAU. The course of AR anxiety is presented in Fig. 0 weeks iTo obtain a better understanding of the changes in attachment, the more advanced method of multilevel modeling was used to consider all measurement points simultaneously. To test whether the growth rate changes over time we entered a quadratic term to the model and inspected the AICs and BICs. For both AR anxiety and AR avoidance the quadratic term was not significant and model fit was not improved, so it was removed from the final model.The fixed effect parameters of the final models are presented in Table d = 0.07 from baseline to posttreatment and d = 0.36 from baseline to follow-up assessments, thus indicating small effects in favor of D-CPT in the long run. For AR avoidance, the effect from baseline to posttreatment was medium (d = 0.65), but a large effect size was obtained from baseline to follow-up (d = 0.80), also in favor of D-CPT.The effect sizes for improvement in AR anxiety between the two groups were The present study evaluated attachment styles and their changes during psychotherapy in a sample of adolescents and young adults with PTSD after childhood abuse. Baseline AR anxiety and AR avoidance scores were more comparable to an adult clinical validation sample with mixed diagnoses than to a non-clinical group . AR anxiFirst, we analyzed the association between PTSS and attachment, accounting for multiple testing and intercorrelation. We found positive associations between avoidance symptoms in the UCLA-PTSD-RI and both AR anxiety and AR avoidance. Only AR avoidance was significantly related to avoidance in the CAPS-CA, and only AR anxiety was significantly related to the total score of the UCLA-PTSD-RI. Regardless of clinician rating or self-report, there was no significant relation to the intrusion or hyperarousal symptom scales. The second aim was to evaluate changes in attachment during the course of the trial and by treatment group. In both treatment conditions, participants showed a decline in AR anxiety at some point in the study. However, there was no significant effect of treatment group. For AR avoidance, only participants in D-CPT showed a significant reduction, and we found a significant group-time interaction, indicating more change in attachment avoidance in the D-CPT group.Some of our results are in line with recent research like the finding of a stronger association between attachment and PTSS when attachment is assessed using a self-report instrument. This is also mentioned in the meta-analysis by Woodhouse et al. . There iMost studies find a modest association between AR avoidance and PTSS and instead a stronger association between AR anxiety and PTSS , 20. We . In our study, AR anxiety improved in both groups. However, with nearly half of our WL/TA participants receiving some kind of support or treatment and all of them having several appointments for study assessments, the participants in this group experienced interpersonal contacts that may have attenuated AR anxiety. For AR avoidance, there was a clear advantage of D-CPT. Only 8 patients in the WL/TA group reported that the trauma was directly targeted during their treatment. The focus on exposure with trauma-related memories and cognitions in D-CPT could perhaps explain the larger improvement in AR avoidance in this group.To evaluate the effect of D-CPT compared to WL/TA on attachment scores, we compared AR anxiety and AR avoidance of participants in both groups at baseline, posttreatment, and 3-month follow-up. In terms of AR anxiety, we did not find an effect for group on reduction of AR anxiety. For AR avoidance, we found a significant group-time interaction; only participants in D-CPT improved significantly from baseline to follow-up. Taylor et al.  reportedTo our knowledge, this is the first study to evaluate the effect of a trauma-focused behavioral therapy on the two attachment dimensions, AR anxiety and AR avoidance, in a sample of adolescents with PTSD. We were able to monitor changes in attachment for individuals being treated with an effective therapy approach in comparison to a control group where most participants did not receive any evidence-based treatment or no treatment at all. We only included participants with clinically significant PTSS that was assessed using clinician ratings and self-report.Despite these strengths, our study has several limitations. First, although our RCT has one of the biggest samples within PTSD trials on young patients, our sample size was quite small. For growth models, a sample size of at least 100 is preferred, but also models with much smaller sample sizes have been fitted successfully . As our The consideration of attachment in psychotherapy could produce beneficial effects for therapists and patients. On one hand, therapists should be aware that adolescents with PTSD after sexual and physical abuse display elevated AR anxiety and AR avoidance, and that these behavioral patterns may influence the outcome of psychotherapy. Sensitivity for attachment issues may help therapists to establish an optimum therapeutic alliance. On the other hand, attachment insecurities seem to improve with psychotherapy. The perspective of experiencing an improvement in interpersonal relationships might increase treatment motivation. However, especially in this sample of adolescents and young adults with abuse experiences, it should be noted that the aim of treatment is to change the representation of attachment and not to enter directly into a new intimate relationship. This population has an elevated risk of starting relationships with abusive partners, so a discussion about partner choice is crucial in this treatment. Nevertheless, attachment issues are highly relevant for case conceptualization, treatment planning and understanding the process of change .This is one of the few studies on attachment in adolescents diagnosed with PTSD. The findings shed light on the association between attachment insecurities and PTSS and its changeability during psychotherapy. Future studies should also use attachment measures based on blind expert ratings and should include significant persons in the patient’s environment in the assessments, e.g. via proxy-reported measures. Moreover, it would be interesting to also address comorbid disorders and to consider the relationship between early attachment, complex PTSD and reversibility through treatment. It could be of particular interest to identify which specific interventions are responsible for inducing changes in attachment. D-CPT combines classical CPT with techniques for enhancing treatment commitment and improving emotion management strategies while taking developmental tasks into account. Thus, it is still unclear whether CPT alone, one of these additional elements or a combination thereof encourages changes in self-reported attachment.Supplementary file1 (PDF 44 kb)Below is the link to the electronic supplementary material.


Da Costa et al., Druege et al., Mhimdi and Pérez-Pérez proposed AR formation as the default state of root development in plants, highlighting that root initiation from non-root tissues might be considered as an ancestral trait, as ARs are formed by default in lycophytes and ferns. According to the same authors, in many angiosperms, AR formation seems to be repressed through mechanisms that maintain competent cells in a quiescent state. Activation of competent cells is triggered by environmental conditions to which the cuttings or intact plants are exposed, like exogenous auxin treatment, wounding, waterlogging, or light exposure and mineral nutrition determining the activation of a complex network of signaling, metabolic, and transport pathways  formation. The first is known as the induction phase, in which high auxin levels are required for reprogramming of competent cells toward root development, which is further linked to other biochemical changes  genes, on the control of auxin or jasmonic acid conjugation, and the PIN, AUX/LAX, and ABCB genes, on the control of auxin transport.The articles of Aux/IAA and ARF genes that encode auxin signaling repressors and their target auxin response factors respectively. Downstream, genes of transcription factors (TFs) from the GRAS, SHORTROOT (SHR), SCARECROW (SCR), WUSCHEL RELATED HOMEOBOX (WOX), LATERAL ORGAN BOUNDARIES-DOMAIN (LOB) and APETALA2/ETHYLENE RESPONSIVE ELEMENT BINDING (AP2/ERF) gene families, control the auxin-dependent establishment of the AR founder cell and the new root apical meristem, the maintenance of the quiescent center (QC) and subsequent establishment and differentiation of the AR primordium . A coordinated change of expression of genes controlling the modulation of the cell wall, such as EXTENSIN and CYS ENDOPEPTIDASE genes, may probably also be involved (Li). Even though these reviews present different perspectives, they stay in line to each other in pinpointing to the core function of plant hormone crosstalk on regulating AR formation, within a process involving a hierarchy of transcription factors, that is linked to epigenetic modulation events, with reactive oxygen species (ROS) also being involved as messenger molecules.Important molecular factors that determine auxin signaling are specific Vilasboa et al. highlight the capacity to efficiently modulate redox conditions though high activity of SOD and soluble GPRX, as the main factor defining AR ability in Eucalyptus. The role of phenolic compounds acting as synergistic or antagonistic in AR formation has long been known, and its role as a species-dependent parameter was also highlighted by Vilasboa et al.The involvement of enzymes from ROS-scavenging mechanisms, and phenolic compounds working as a non-enzymatic antioxidant system, have been investigated across a diversity of plant species during the AR formation process. The activity of ascorbate peroxidase (APX), superoxide dismutase (SOD), and catalase (CAT) have been associated with improved rooting ability . The importance of ROS on AR formation was also explored by studying the involvement of the alternative oxidase (AOX), a key enzyme in the alternative respiratory pathway, using semi-hardwood cuttings .Within the different ROS molecules, the focus has been given to HAbarca suggested the epigenetic control of miR160/167-modulated auxin signaling, the TARGET OF RAPAMICINE (TOR) and possibly RETINOBLASTOMA-RELATED (RBR) dependent metabolic and auxin control of the cell cycle, and the non-cell autonomous regulation of WOX11/12 expression by CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) peptides, as three molecular checkpoints for the age-related decline in AR induction. In their original article, Pizarro and Díaz-Sala compared hypocotyl cuttings excised from Pinus radiata seedlings with different ages to identify genes that control the cell wall-plasma membrane-cytoskeleton continuum in the basal stem, in relation to rooting competence. The RECEPTOR-LIKE KINASE (RLK) and MECHANOSENSITIVE CHANNEL (MSC) genes, as well as genes encoding downstream regulators of the small GTP-binding protein family, were associated with rooting competence, probably via acting on the control of cell wall sensing and membrane disturbances during the essential cellular reorganization .The loss of rooting capacity with plant aging , a major problem in breeding and propagation of many tree species, has also been investigated. Regarding the relevant literature, Bannoud and Bellini, Abarca).Despite all the research carried out and the information achieved in the last years, the mechanisms underlying maturation-induced rooting recalcitrance and environmentally mediated rooting, as well as several other aspects of the AR formation process, are still far from being fully understood . The work of UD was funded by the German Federal Ministry of Food and Agriculture (BMEL) based on a decision of the Parliament of the Federal Republic of Germany, granted by the Federal Office for Agriculture and Food .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Early adiposity rebound (AR) has been associated with increased risk of overweight or obesity in adulthood. However, little is known about early predictors of age at AR. We aimed to study the role of perinatal factors and genetic susceptibility to obesity in the kinetics of AR.Body mass index (BMI) curves were modelled by using mixed-effects cubic models, and age at AR was estimated for 1415 children of the EDEN mother–child cohort study. A combined obesity risk-allele score was calculated from genotypes for 27 variants identified by genome-wide association studies of adult BMI. Perinatal factors of interest were maternal age at delivery, parental education, parental BMI, gestational weight gain, maternal smoking during pregnancy, and newborn characteristics . We used a hierarchical level approach with multivariable linear regression model to investigate the association between these factors, obesity risk-allele score, and age at AR.A higher genetic susceptibility to obesity score was associated with an earlier age at AR. At the most distal level of the hierarchical model, maternal and paternal educational levels were positively associated with age at AR. Children born to parents with higher BMI were more likely to exhibit earlier age at AR. In addition, higher gestational weight gain was related to earlier age at AR. For children born small for gestational age, the average age at AR was 88 [±39] days lower than for children born appropriate for gestational age and 91 [±56] days lower than for children born large for gestational age.The timing of AR seems to be an early childhood manifestation of the genetic susceptibility to adult obesity. We further identified low birth weight and gestational weight gain as novel predictors of early AR, highlighting the role of the intrauterine environment in the kinetics of adiposity. After birth, body mass index (BMI) increases to a peak during the first year of life and declines between age 4 and 6 years, then increases again. This BMI increase following the minimum is called adiposity rebound (AR) –3. This To provide a track to prevent the development of obesity, one must follow children’s BMI growth curve and better understand the determinants of age at AR. Furthermore, the DoHAD theory  suggestsGenome-wide associations studies have highlighted single-nucleotide polymorphisms (SNPs) implicated in increased risk of obesity , 22 and In this context, the purpose of this study was to investigate the role of parental education, perinatal factors, and genetic susceptibility to obesity in the kinetics of AR within the French EDEN mother–child cohort. We hypothesize that the association between age at AR and perinatal factors is independent of genetic susceptibility to obesity and that age at AR could be programmed by a set of factors involved as early as in pregnancy.The EDEN mother–child study is a prospective French cohort that aims to assess prenatal and postnatal determinants of child growth, health, and development. This cohort included 2002 pregnant women (before 24 weeks of amenorrhea) recruited from 2003 to 2006 in two university hospitals in Nancy and Poitiers. Exclusion criteria were <18 years of age, multiple pregnancies, known diabetes before pregnancy, illiteracy, and intention to give birth elsewhere than these two French hospitals or to move outside the region within the next 3 years. Details of the study protocol have been published elsewhere .The study was approved by the relevant ethical research Committee of Kremlin‐Bicêtre Hospital and by the Data Protection Authority. Both parents gave their written consent.z-scores were calculated according to Gardosi references taking into account physiological fetal  and maternal factors  )   was not associated with age at AR, we noted a statistically significant association for rs10913469 (at the locus harboring SEC16B) and an association at the limit of significance for rs713586 (at the locus harboring RBJ/POMC) but with reduced statistical power. In accordance with our results, the authors reported a negative association between an obesity risk-allele score, based on 97 SNPs, and child age at AR, but did not model the effects of other life factors [The genetic susceptibility to obesity was associated with an increased probability of early AR. The role of genetic factors in AR timing was investigated recently in a genome-wide association study including up to 7215 children in five European cohorts. In this study, Couto Alves et al. identified an association between two SNPs and age at AR. Although the rs1421085 (at the locus harboring  factors . AltogetConsistent with previous research, we found that high parental BMI predicted early AR in children. A longitudinal study of 248 families enrolled in North Carolina concluded high maternal BMI as the robust predictor of early AR . Two othOur results regarding gestational weight gain support this latter hypothesis. To our knowledge, only one study has considered gestational weight gain as a potential predictor of age at AR, but no significant association was shown . HoweverWe found a negative association between maternal smoking status during pregnancy and the timing of AR but not risk of early AR. A longitudinal analysis, based on mothers from the longitudinal Growth and Obesity Chilean Cohort Study, also showed no link between smoking status during pregnancy and risk of early AR . In prevFor the first time, we identified birth weight for gestational age as a predictor of age at AR. Some previous studies did include birth weight in their multiple models but did not find any significant association with age at AR. For the “Children in Focus” group from the ALSPAC cohort, no difference in birth weight was observed between the groups of very early, early, and later AR . In contEven if some of our results need confirmation and replication, they highlight a number of prenatal factors, including smoking during pregnancy, maternal prepregnancy BMI, gestational weight gain and birth weight for gestational age, possibly involved in the timing of AR. These findings strongly suggest that some intrauterine programming may also be at work in the etiology of this specific adiposity phenotype. Among the possible mechanisms, altered fetal nutrition could lead to persistent changes in the structure and function of adipose tissue, regulation of appetite and energy metabolism, which could affect the kinetics of adiposity and its rebound . EpigeneThe main strength of this study is that anthropometric data were assessed repetitively and prospectively from birth to age 13, which allowed us to estimate age at AR for a large number of children. As is often the case in cohort studies, the presence of selection bias at inclusion and missing data of covariates but also attrition bias limits the generalization of the results. However, multiple imputations and growth modeling of individual growth curves helped reduce the risk of these biases. Another limitation is that we could not consider ethnicity in our analyses, because the information was not collected in the EDEN cohort. Nonetheless, for 85% of children in the EDEN cohort, both parents were born in France and came from French regions where most of the population has a Caucasian origin. Moreover, the construction of the genetic score relies on the assumption of a co-dominant effect of each allele of a given SNP, which is not always true and could induce measurement errors. Our results, relying on observational data, need to be replicated in other studies with larger samples and with distinct designs to provide additional arguments for a causal interpretation of the observed associations. Further studies are needed to examine postnatal factors involved in the kinetics of BMI growth curve, to better characterize children at risk of early rebound. Indeed, the detection of early age at AR might be a useful tool for tailored and personalized child’s growth monitoring aiming at preventing overweight/obesity and chronic disease in later life.We confirmed that genetic susceptibility to adult obesity plays a predictive role in the timing of age at AR. Timing of AR in early childhood may contribute to the path from genetic susceptibility to obesity onset. Independent of this genetic background, we identified several prenatal predictors, including two novel factors, gestational weight gain and birth weight. These results strongly support that intrauterine programming may also be at work in the etiology of this specific adiposity phenotype.Table S1, Table S2, Table S3, Table S4, Table S5, Figure S1, Figure S2, Figure S3, Figure S4, Figure S5


Neovascularization is a key factor that contributes to tumor metastasis, and vasculogenic mimicry (VM) is an important form of neovascularization found in highly invasive tumors, including lung cancer. Despite the increasing number of studies focusing on VM, the mechanisms underlying VM formation remain unclear. Herein, our study explored the role of the HIF-1α/NRP1 axis in mediating lung adenocarcinoma metastasis and VM formation. HIF-1α, NRP1 expression, and VM in lung adenocarcinoma (LUAD) patient samples were examined by immunohistochemical staining. Quantitative real-time (qRT-PCR), western blot, transwell assay, wound healing assay, and tube formation assay were performed to verify the role of HIF-1α/NRP1 axis in LUAD metastasis and VM formation. ChIP and luciferase reporter assay were used to confirm whether NRP1 is a direct target of HIF-1α. In LUAD tissues, we confirmed a positive relationship between HIF-1α and NRP1 expression. Importantly, high HIF-1α and NRP1 expression and the presence of VM were correlated with poor prognosis. We also found that HIF-1α could induce LUAD cell migration, invasion, and VM formation by regulating NRP1. Moreover, we demonstrated that HIF-1α can directly bind to the NRP1 promoter located between −2009 and −2017 of the promoter. Mechanistically, MMP2, VE-cadherin, and Vimentin expression were affected. HIF-1α plays an important role in inducing lung adenocarcinoma cell metastasis and VM formation via upregulation of NRP1. This study highlights the potential therapeutic value of targeting NRP1 for suppressing lung adenocarcinoma metastasis and progression. NSCLC features uncontrollable mortality and invasiveness, high recurrence rates, and strong angiogenesis. Tumor angiogenesis is an independent prognostic factor in cancer and is associated with widespread hypoxia in tumors and a relatively inadequate blood supply2. Moreover, accumulating studies have also demonstrated a new method of angiogenesis under hypoxic conditions named vasculogenic mimicry (VM). VM is characterized by vascular-like structures and results in the formation of a new blood supply system in aggressive tumor cells, which is positive for periodic acid-Schiff (PAS) staining4. VM has been found in various malignant tumors, including NSCLC, indicating more aggressive tumor behavior5.Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide. And it also accounts for 30% of all lung cancer cases7. It has been well established that the transcription factor HIF-1α enhances tumor cell motility and invasiveness and contributes to the epithelial–mesenchymal transition (EMT) process, which is crucial for VM formation9. Overexpression of HIF-1α in primary gallbladder carcinoma is related to vasculogenic mimicry and unfavorable prognosis10. In gastric adenocarcinoma, STAT3, p-STAT3, and HIF-1α are associated with vasculogenic mimicry and impact patient survival11. Moreover, it has been reported that HIF-1α promotes vasculogenic mimicry formation in hepatocellular carcinoma through upregulated LOXL2 in the hypoxic tumor microenvironment12.Within the tumor microenvironment, hypoxia is the most common phenomenon because of the vast energy and oxygen consumption of tumor cells. Hypoxia, which is orchestrated by hypoxia-inducible factor-1α (HIF-1α), contributes to tumor metabolism, angiogenesis, and cell survival14. NRP1 is considered to be an important driver of malignant tumor progression. NRP1 is overexpressed in a variety of tumor tissues and is involved in the development of axon guidance and remyelination, immune response, angiogenesis, cell survival, migration, and invasion16. It has been reported that the NRP1+ cell subpopulation showed an increased expression of the pluripotency markers OCT-4, Bmi-1, and NANOG as well as higher cell migratory, clonogenic, and self-renewal capacities17. Our previous published study pointed out that NRP1 contributed to TGF-β1-induced EMT and metastasis in NSCLC by binding with TGFβRII18. In addition, we demonstrated that NRP1 promotes NSCLC metastasis via EGFR signaling19. The role of NRP1 in the hypoxic tumor microenvironment in mediating VM has remained largely unknown.NRP1 is a transmembrane glycoprotein that acts as a coreceptor for numerous extracellular ligands, including transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF)In our present study, we demonstrate that NRP1 is the direct target of HIF-1α that promotes VE-cadherin, MMP2, and Vimentin expression under hypoxic conditions. HIF-1α plays an important role in the development of VM in lung cancer by upregulating NRP1, indicating the potential therapeutic value of targeting NRP1 for the suppression of NSCLC metastasis and progression.A total of 169 primary lung adenocarcinoma specimens were obtained from the Affiliated Huai’an Hospital of Xuzhou Medical University. All participants were provided with written informed consent at the time of recruitment. The diagnosis of lung adenocarcinoma was verified for all of these samples based on their histological and pathological characteristics. Before tissue sampling, none of the patients had received any therapy, such as chemotherapy or radiotherapy. The present study was approved by the Ethics Committee of the Affiliated Huai’an Hospital of Xuzhou Medical University. All samples were stored frozen at −80 °C.20. The antibodies used in the current study were as follows: anti-HIF-1α (D1S7W) and anti-NRP1 . In addition, we also performed CD34/PAS double staining to detect VM expression in tumor tissues. CD34 staining  was performed on the sections prior to PAS staining . Then, all slides were treated with periodic acid solution for 10 min and submerged in Schiff solution for 15 min at 37 °C. After washing, all sections were counterstained with hematoxylin, dehydrated, and mounted. And we applied the quantitative scoring methods to evaluate the staining results. The total score was calculated by multiplying the percentage of positive cells (P) by the intensity (I). Formula: Q = P × I; P-score was assigned that represented the estimated proportion of antibody-stained tumor cells. For positive cells, 0 (<5%), 1 (5–25%), 2 (26–50%), or 3 (51–75%), 4 (76–100%) means the percentage of malignant cells staining within carcinomatous areas. I-score was assigned that estimated the average staining intensity of positive tumor cells. For intensity, 0 means no staining, 1 means weak staining, 2 means moderate staining, and 3 means strong staining. Finally the R language was applied to draw the correlation analysis.The detailed procedures were performed as previously described2)  was used to mimic hypoxic conditions. Under some conditions, cells were treated with 150 μM CoCl2 for 48 h and then subjected to RNA or protein extraction.The A549 and SPC-A1 lung adenocarcinoma cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences . All these cells were cultured in RPMI 1640 medium with additional 10% fetal bovine serum  and 1% penicillin–streptomycin solution . Moreover, cobalt chloride  were subcloned into the lentiviral vector pGMLV-SC5  by digestion with the endonucleases BamHI and EcoRI. To generate HIF-1α-overexpressing cells, a 2481-bp fragment of the HIF-1α coding sequence was synthesized , and subcloned into the PLVX-IRES-Neo vector using the endonucleases SwaI and NotI. HIF-1α-silenced or HIF-1α-overexpressing constructs or controls were cotransfected with packaging plasmids into human embryonic kidney 293 cells using Lipofectamine 2000 . Forty-eight hours later, we collected the supernatant to infect A549 and SPC-A1 cells at least three times and then selected positive cells with 0.4 µg/ml puromycin .The methods used to construct stable NRP1 knockdown and overexpression cell lines were reported in our previous study21. The following primers were used for qRT-PCR: for HIF-1α, 5′-ATCCATCTGACCATGAGGAAATG-3′ (forward) and 5′-TCGGCTAGTTAGGGTACACTTC-3′ (reverse); and for NRP1, 5′-GAAAAATGCGAATAll procedures were performed as previously describedGG CTGAT-3′ (forward) and 5′-AATGGCCCTGAAGACACAAC-3′ (reverse). β-actin was used as internal control. For β-actin, the primer sequences were 5′-CACAGAGCCTCGCCTT−ΔΔCt method.TGCC-3′ (forward) and 5′-ACCCATGCCCACCATCACG-3′ (reverse). Relative fold changes were calculated using the 222. The antibodies used in our current study were anti-NRP1 (A-12) , anti-HIF-1α (D1S7W), anti-VE-cadherin (D87F2), anti-MMP2 (D8N9Y) , and anti-Vimentin (RV202) . Anti-β-actin and anti-mouse or anti-rabbit secondary antibodies from Cell Signaling Technology were used.All procedures were performed as we previously reported4 cells/ml) were suspended in RPMI 1640 medium with 1% FBS. For the migration assay, 200 µl of the cell suspension was added to the upper transwell inserts. For the invasion assay, all inserts were precoated with Matrigel matrix  diluted in 0% medium and then incubated at 37 °C for 2 h. Then, 200 µl medium was added to the upper precoated inserts. All lower chambers were filled with 800 µl medium containing 10% FBS. Twenty-four hours later, the inserts were fixed with methanol and stained with 0.5% crystal violet. Images were acquired under a microscope .A549 and SPC-A1 lung adenocarcinoma cells .5 cells/ml. Two hours later, 100 μl of cell suspension was seeded onto the gel in each well and cultured for 24 h. The formation of VM structures was observed and captured under a microscope  at ×100 magnification.To monitor VM formation, 50 μl thawed Matrigel matrix  was added to 96-well plates and then kept at 37 °C for 2 h. A549 and SPC-A1 cells after the indicated treatments were suspended in complete medium at a density of 5–10 × 10Cells were seeded in 12-well plates that were preinserted with glass slides. Twenty-four hours later, when the cells reached 40–50% confluence, they were washed with PBS. The cells were then fixed with 4% paraformaldehyde for 30 min followed by permeabilization with 0.5% Triton X-100 solution for an additional 20 min. Next, 5% bovine serum albumin was added to function as the blocking buffer. The primary antibodies used in our experiment were anti-HIF-1α (D1S7W) and anti-NRP1 (A-12). The corresponding secondary antibodies tagged with Cy3 and FITC were used .A 2000-bp fragment of the NRP1 promoter containing potential HIF-1α binding sites was fused to the 3′-end of a luciferase reporter for the luciferase assay. Plasmids containing wild-type (WT) NRP1 promoter (pGL3-NRP1) or mutated fragments of the NRP1 promoter (pGL3-NRP1-mutant1/mutant2/mutant1 and 2) were directly synthesized . A549 and SPC-A1 cells were plated in a 24-well plate and cotransfected with the constructed plasmids (500 ng) together with a Renilla pRL-TK plasmid (40 ng) using Lipofectamine 2000 . Twenty-four to forty-eight hours later, the cell lysates were collected, and luciferase activity was examined with a Dual-Luciferase Reporter Assay Kit (Promega).A ChIP-IT Kit  was used to perform the ChIP assay. Briefly, cells were fixed with formaldehyde and then lysed. To precipitate the DNA fragment, 2 μg anti-HIF-1α antibody or normal IgG was used. The DNA-protein complexes were pulled down with magnetic beads and then de-crosslinked. The extracted DNA samples were then amplified with NRP1 promoter-specific primers.t-test. Significant differences between three or more groups were analyzed using one-way or two-way ANOVA followed by the Bonferroni post hoc test. All statistical tests were two-tailed. p < 0.05 was set as statistically significant.All experiments were independently performed in triplicate as a minimum. All statistical analyses were performed using GraphPad Prism 7.0  and SPSS 17.0 software . All data were presented as the mean ± SD. Significant differences between two groups were assessed by a nonpaired Student’s +CD34− tumor cells within the cavities were considered VM channels (red arrows). Both PAS and CD34 positive channels were defined as EDVs . NRP1 was highly expressed in 86 of the 169 LUAD sample tissues (50.88%)  for 48 h. The data showed that CoCl2 induced HIF-1α expression, and HIF-1α knockdown inhibited the CoCl2-induced upregulation of NRP1  to enhance angiogenesis, vascular permeability, and arteriogenesis via interaction with VEGF receptor 2 (VEGFR2)33. In addition, NRP1 regulates angiogenesis by activating the intracellular kinase ABL1 independent of VEGF signaling34. NRP1 also acts as a receptor for the class 3 semaphorin (SEMA3A) to regulate vascular permeability and vessel maturation during tumor angiogenesis13. However, until now, limited studies have reported the role of NRP1 in promoting VM formation. In the current study, we found that NRP1 overexpression is associated with VM and poor patient survival in LUAD patient specimens. We proved that NRP1 affects the migration and invasion of LUAD cells, which was in line with our previously published results. In addition, we also found that blocking NRP1 inhibits VM formation, while NRP1 overexpression promoted VM formation.NRP1 is a transmembrane cell surface coreceptor that serves as a receptor for the VEGF35. HIF-1α was also reported to promote glycolysis in pancreatic cancer by regulating NRP136. However, whether the HIF-1α/NRP1 axis functions in VM formation has not been reported. ChIP assay showed that NRP1 is a direct target of HIF-1α. Migration and invasion assays, wound healing assays, and VM formation assays showed that NRP1 knockdown counteracted the aggressive phenotype induced by HIF-1α, while NRP1 overexpression enhanced the aggressive phenotype under HIF-1α knockdown. All data suggested that the HIF-1α/NRP1 axis regulated LUAD cell migration and VM formation.HIF-1α plays a central role in bone marrow-derived EPC (bmEPC) homing and sprouting in the postacute stage of ischemic Sprague–Dawley (SD) rat brains by regulating NRP1 expression37. A hypoxic tumor microenvironment promotes HIF1-α-induced EMT and thus enhances the invasive and migratory abilities and VM in tumors. Vascular endothelial (VE)-cadherin is a major endothelial adhesion molecule that controls cell–cell junctions and blood vessel formation in tumor development38. It has been reported that HIF-1α binds to the promoter of VE-cadherin and promotes its expression at the transcriptional level39. In addition, VE-cadherin promotes vasculogenic mimicry by modulating the expression of kaiso-dependent genes, such as WNT11 and CCDN123. Therefore, we next verified whether the HIF-1α/NRP1 axis activated MMPs and VE-cadherin in LUAD. Interestingly, we found that the elevated VE-cadherin, MMP2, and Vimentin expression induced by HIF-1α was downregulated following NRP1 knockdown.Furthermore, we deeply explored the underlying mechanisms of HIF-1α/NRP1 axis-mediated cell migration and VM formation. It has been reported that high levels of matrix metalloproteinases, such as MMP-1, MMP-2, MMP-9, and MMP-14, produced by tumor cells participate in VMHere, we demonstrated a new regulatory axis in the hypoxic tumor microenvironment involving elevated NRP1 expression induced by HIF-1α. HIF-1α/NRP1 signaling can subsequently result in activation of MMP2, Vimentin, and VE-cadherin and thus promote EMT and VM, which ultimately contribute to tumor progression in LUAD Fig. .Fig. 6ThFigure S1Figure S2Figure S3Supplementary figure legends


Long non‐coding RNA growth arrest‐specific 5 (lnc‐GAS5) and its targets (microRNA [miR]‐21 and miR‐140) are involved in the development and progression of allergic rhinitis (AR). However, the correlation of lnc‐GAS5 with miR‐21 and miR‐140 and their associations with disease risk, symptom severity, and Th1/Th2 cytokines in AR remain unclear. Thus, this study aimed to investigate this topic.In total, 120 patients with AR and 60 controls were recruited. Nasal‐mucosa tissues were collected from all participants. Lnc‐GAS5, its targets (miR‐21 and miR‐140), interferon (IFN)‐γ, interleukin (IL)‐2, IL‐4, and IL‐10 were detected by reverse‐transcription quantitative polymerase chain reaction.p < 0.001). In AR patients, lnc‐GAS5 was negatively correlated with miR‐21 (p < 0.001), miR‐140 (p < 0.001), IFN‐γ (p = 0.019), and IL‐2 (p = 0.039) and positively correlated with IL‐4 (p = 0.004) and IL‐10 (p < 0.001), individual nasal symptom scores (INSSs) for itching, sneezing, and congestion (p < 0.05), and total nasal symptom score (TNSS) (p < 0.001). Moreover, miR‐21 and miR‐140 were negatively correlated with some INSSs, total TNSS score, and IL‐10 and positively correlated with IFN‐γ and IL‐2 (p < 0.05).Lnc‐GAS5 was elevated, while miR‐21 and miR‐140 was downregulated in AR patients than in controls (Lnc‐GAS5 is negatively correlated with that of its targets (miR‐21 and miR‐140) in AR; meanwhile, lnc‐GAS5, miR‐21, and miR‐140 are correlated with disease risk, symptom severity, and Th1/Th2 imbalance in AR, suggesting the potential of these biomarkers in the development and progression of AR. p < 0.001). In AR patients, lnc‐GAS5 was negatively correlated with miR‐21 (p < 0.001), miR‐140 (p < 0.001), IFN‐γ (p = 0.019), and IL‐2 (p = 0.039), while positively correlated with IL‐4 (p = 0.004) and IL‐10 (p < 0.001), as well as some of individual nasal symptom score (INSS) scores   and total nasal symptom score (TNSS) score (p < 0.001). Moreover, both miR‐21 and miR‐140 were negatively correlated with some of INSS scores, TNSS score, and IL‐10, while positively correlated with IFN‐γ and IL‐2 .Conclusion: Lnc‐GAS5 is negatively correlated with its targets (miR‐21 and miR‐140) in AR; meanwhile, they are all correlated with disease risk, symptom severity, and Th1/Th2 imbalance of AR, suggesting the potential of lnc‐GAS5, miR‐21, and miR‐140 as biomarkers for development and progression of AR.Objective: Long non‐coding RNA growth arrest‐specific 5 (lnc‐GAS5) and its targets (microRNA (miR)‐21 and miR‐140) take part in the pathology of allergic rhinitis (AR) development and progression. However, the correlation of lnc‐GAS5 with miR‐21 and miR‐140, as well as their associations with disease risk, severity, and Th1/Th2 cytokines of AR is unclear. Thus, the aim of this study was to investigate this topic.Methods: Totally, 120 AR patients and 60 controls were recruited. Besides, nasal mucosa tissues were collected from all participants. Then, lnc‐GAS5, its targets (miR‐21 and miR‐140), interferon (IFN)‐γ, interleukin (IL)‐2, IL‐4, and IL‐10 were detected by reverse‐transcription quantitative polymerase chain reaction.Results: Lnc‐GAS5 was elevated, while miR‐21 and miR‐140 were downregulated in AR patients compared to controls (all  In total, 120 patients with AR were consecutively enrolled between March 2019 and June 2020. The main inclusion criteria were as follows: (1) diagnosis of AR based on the criteria of the Chinese Society of Allergy Guidelines for Diagnosis and Treatment of Allergic Rhinitis2.2®  was added to the specimens to protect the RNA samples from degradation by RNase. Finally, the specimens were stored at −70°C for subsequent detection.After recruitment, the characteristics of all participants were recorded. After the participants provided informed consent, nasal‐mucosa tissue samples were collected under local anesthesia, washed using normal saline, and sliced into pieces. RNAlater2.3The individual nasal symptom score (INSS) was used to assess disease severity in AR patients. The INSS comprises scores for four symptoms, namely rhinorrhea, sneezing, itching, and congestion. The detailed scoring method was as follows: 0, no symptoms; 1, mild symptoms; 2, moderate symptoms; and 3, severe symptoms. The total nasal symptom score (TNSS) was calculated as the sum of the scores of the four symptoms and ranged from 0 to 12. A higher TNSS indicated a more severe disease.2.4® SYBR® qPCR Mix . The primers used in RT‐qPCR are listed in Table −ΔΔCt method.The expression of lnc‐GAS5, miR‐21, and miR‐140 and the expression of Th1 cell‐secreted cytokines (interferon [IFN]‐γ and interleukin [IL]‐2) and Th2 cell‐secreted cytokines (IL‐4 and IL‐10) were determined by RT‐qPCR. Briefly, total RNA was extracted using TRIzol™ Reagent  and reverse transcribed using the iScript™ cDNA Synthesis Kit . Afterward, qPCR was performed using THUNDERBIRD2.5t test, Wilcoxon rank‐sum test, and chi‐square test were used to compare data between AR patients and controls. Receiver operating characteristic (ROC) curve analysis and the derived area under the curve (AUC) were used to estimate the accuracy of a marker for identifying different subjects. The Spearman rank correlation test was used to perform correlation analysis. SPSS software  and GraphPad Prism 8.01 software  were used for statistical analysis and graph making, respectively. Statistical significance was set at p < 0.05.Continuous variables and categorical variables were presented as mean with standard deviation (SD), median with interquartile range (IQR), or count (percentage), as appropriate. Student's 33.1p > 0.05). Median values of serum IgE, IFN‐γ, IL‐2, IL‐4, and IL‐10 expression were all higher in AR patients than in controls (p < 0.001). Among AR patients, the median values of the rhinorrhea score, itching score, sneezing score, congestion score, and TNSS were 2.0 (1.0–3.0), 2.0 (1.3–2.0), 2.0 (2.0–3.0), 2.0 (2.0–3.0), and 8.0 (7.0–9.0), respectively  of these 120 AR patients were men, and 66 (55.0%) were women. Among the 60 controls, the mean age was 28.9 ± 7.6 years; 25 (41.7%) were men, and 35 (58.3%) were women. There was no significant difference in age or sex distribution between patients with AR and controls (3.2p < 0.001)   Figure . Meanwhi) Figure . Moreove) Figure .3.3r = –0.441, p < 0.001)   Figure .3.4r = 0.201, p = 0.028), sneezing score , congestion score , and TNSS score    Figure .3.5r = –0.214, p = 0.019) and IL‐2  expression  and IL‐10  expression , sneezing score , TNSS , IL‐4 expression , and IL‐10 expression   and IL‐2 expression    Figure . No corr) Figure .3.7r = –0.189, p = 0.038), sneezing score , congestion score , TNSS , and IL‐10 expression   and IL‐2 expression    Figure . No corr) Figure .4With regard to lnc‐GAS5, miR‐21, and miR‐140 expression in immune diseases, previous studies have shown that lnc‐GAS5 expression is increased in T cells in patients with rheumatoid arthritis (RA) compared to that in individuals without RA.via Enhancer of Zeste Homolog 2 (EZH2) and T‐bet, which would decrease immune function, promote inflammation, and consequently aggravate AR severity.Regarding the correlation between lnc‐GAS5 expression and immune‐disease severity, a positive correlation has been found between lnc‐GAS5 expression and the Expanded Disability Status Scale score for multiple sclerosis.Previous studies have reported that lnc‐GAS5 is associated with an imbalance in Th1 and Th2 in immune disorder‐related diseases, such as recurrent pregnancy loss.Regarding the association of miR‐21 and miR‐140 with the severity of immune diseases, a previous study has demonstrated that miR‐21 expression is negatively associated with chronic health evaluation II and sequential organ failure assessment scores in sepsisThere were several limitations in our study. First, the sample size was relatively small, which could result in insufficient statistical power. Second, the study did not explore the association of lnc‐GAS5 and its targets (miR‐21 and miR‐140) with the prognosis of AR, such as treatment response and recurrence; therefore, longer‐term observation is needed in future studies. Third, we only detected the expression of lnc‐GAS5 and its targets (miR‐21 and miR‐140) as well as the expression of Th1/Th2 cytokines in nasal‐mucosa tissue samples; analysis of their expression in other sources of samples, such as the blood, to verify their correlation with AR is required. Fourth, the clinical value of lnc‐GAS5, miR‐21, and miR‐140 expression in blood samples of AR patients should be explored in future studies. Finally, the mechanisms underlying the roles of lnc‐GAS5, miR‐21, and miR‐140 in the pathogenesis and progression of AR should be investigated in the future.In conclusion, the expression of lnc‐GAS5, miR‐21, and miR‐140 was correlated with disease risk, symptom severity, and Th1/Th2 cytokine expression in AR, indicating that lnc‐GAS5, miR‐21, and miR‐140 may be potential biomarkers for AR.Authors declared that they have no competing interests.Table S1Click here for additional data file.


Saccharina japonica is the most abundantly cultured brown seaweed in the world, and has been consumed in the food industry due to its nutrition and the unique properties of its polysaccharides. In this study, fucoidan (LJNF3), purified from S. japonica, was found to be a novel sulfated galactofucan, with the monosaccharide of only fucose and galactose in a ratio of 79.22:20.78, and with an 11.36% content of sulfate groups. NMR spectroscopy showed that LJNF3 consists of (1→3)-α-l-fucopyranosyl-4-SO3 residues and (1→6)-β-d-galactopyranose units. The molecular mechanism of the anti-inflammatory effect in RAW264.7 demonstrated that LJNF3 reduced the production of nitric oxide (NO), and down-regulated the expression of MAPK  and NF-κB (including p65 and IKKα/IKKβ) signaling pathways. In a zebrafish experiment assay, LJNF3 showed a significantly protective effect, by reducing the cell death rate, inhibiting NO to 59.43%, and decreasing about 40% of reactive oxygen species. This study indicated that LJNF3, which only consisted of fucose and galactose, had the potential to be developed in the biomedical, food and cosmetic industries.Seaweed of  Saccharina japonica is the most abundantly cultured brown seaweed in the world, and has been consumed in the food industry due to its nutrition and the unique properties of its polysaccharides. Polysaccharides that are found in S. japonica mainly refer to alginate, fucoidan, and laminarian, which have attracted widespread attention in biochemical and medical areas, offering different molecular structure characteristics and biological activities.Seaweed of Saccharina [Sargassum [Alaria [Costaria [Hizikia [Hormophysa [Kjellmaniella [Lobophora [Undaria [Scytosiphon [Spatoglossum [Fucoidan is a water-soluble polysaccharide with a sulfate group, and its main structural unit is α-1,3-linked or α-1,4-linked L-fucose residue. The monosaccharide composition of fucoidan is complex, mainly including fucose, galactose, mannose, fructose, xylose, and so on. Galactofucan is a novel fucoidan, containing only fucose and galactose in the monosaccharide composition, which have been found in brown seaweeds. Though there were numerous researches related to fucoidan, only a few galactofucans were reported to be purified from brown seaweeds, such as ccharina ,6,7,8, Sargassum ,16,17,18 [Alaria ,20, CostCostaria , Hizikia[Hizikia , Hormophrmophysa , Kjellmamaniella , Lobophoobophora , Undaria[Undaria ,27, Scyttosiphon , and Spaoglossum ,30, withoglossum , anti-inoglossum , antiviroglossum ,28,31, aoglossum ,10,13,19oglossum , and antoglossum .Saccharina spp. and Sargassum spp. The researches of galactofucan from Saccharina spp. investigated the antiviral and antitumor activities, and explored the relationship between the structure and biological activities [The present researches mainly studied the biological activities of galactofucan from tivities ,4. For etivities . Besidestivities .S. japonica. Therefore, it is necessary to explore the anti-inflammatory mechanism of galactofucan obtained from S. japonica, and evaluate the structural characteristics and biological activities of galactofucan.An excessive inflammatory response would lead to the release of a large number of pro-inflammatory cytokines, which have a serious impact on host cells and tissues, and then cause a variety of chronic inflammation . FucoidaS. japonica, which was composed of fucose and galactose. The anti-inflammatory activity and mechanism of LJNF3 as galactofucan were studied by RAW 264.7 cells and zebrafish models in vitro and in vivo, and the relationship between the structure and anti-inflammatory activity was explored, so as to provide a theoretical basis for the role of LJNF3 as an anti-inflammatory component in pharmaceutical, functional food, and cosmetics.In this study, we investigated the physicochemical properties of a purified fucoidan named LJNF3 (with a molecular weight of 261.7 KDa) from S. japonica was extracted by hot water, with a yield of 5.96 ± 1.35%, based on dried weight. After purification on a DEAE-Sepharose Fast Flow column  and galactose (20.78%). The monosaccharide composition and biological activities of the reported galactofucans were summarized in −1 and signal at 1077 cm−1 represented the stretching vibration of O–H linkage, which indicated strong inter- and intra-molecular interactions of the polysaccharide chains. A narrow weak peak around 2942 cm−1 resulted from C–H stretching vibrations. The absorption band at 1260 cm−1 was related to S=O stretch. The signal at 964 cm−1, derived from the asymmetrical stretching vibration of C–O–S bonds, appeared in the spectra [−1 represented the C–O–S stretching vibration, which showed the position of the sulfate groups. The absorption at 852 cm−1 was suggested to be due to sulfate groups at the axial C4 position [The IR spectra of LJNF3 was shown in  spectra . The ranposition .l-fucopyranose units. The position of H1/H2 cross-peaks were 15.05/3.93 ppm related to the 4-sulfated fucosyl residue [d-galactopyranose residues B [ residue . Besidessidues B .13C NMR spectrum  and high-field (16.8‒17.5 ppm) regions. The signal 100.77 ppm was assigned to anomeric C1 carbon, and signals in the region 61‒82 ppm were assigned to the C2–C5 carbons of the pyranoid ring [1H/13C NMR correlation spectra HMBC -β-d-galactopyranosyl- by LPS induction, while the production of NO treated with LJNF3 decreased (p < 0.05) in a dose-dependent manner, and NO was decreased by 40% more than that of the LPS-only treated group, with 25 μg/mL of LJNF3. With the NO inhibition experiment (the data were not shown), the inhibitory effect of LJNF3 on NO was significantly higher than that of LJNFF1 and LJNF2. Therefore, the following experiments focused on the study of the inflammatory mechanism of LJNF3.It is well known that NO, as a biological mediator, played an important role in the inflammation response caused by microbes and tumors . In addiS. japonica showed the optimal anti-inflammatory activity in concentrations of 50–200 μg/mL [→3)-α-l-fucose residues with abundant branches consisting of α-l-fucose and β-d-galactose residues [S. japonica, had anti-inflammatory activity and was composed of fucose (79.49%), galactose (16.76%), xylose (1.08%), mannose (1.84%), and rhamnose (0.82%) [S. japonica had relatively superior biological activity, with a higher content of sulfate groups and fucose. Besides, Murmansk et al. (2020) have studied the anti-inflammatory activity mechanism of fucoidan from Fucus vesiculosus, mainly discussing the anti-inflammatory activity of fucoidan by regulating COX-1/2, hyaluronidase, and the MAPK signaling pathway. In addition, it found that a higher content of sulfate group and fucose of fucoidan generally had superior anti-inflammatory activity [According to previous reports, fucoidan in brown seaweed generally plays a good anti-inflammatory effect in 200 or 400 μg/mL ,50, and 00 μg/mL  and 50–400 μg/mL ,52,53. Y00 μg/mL . Wang et (0.82%) . From thactivity . CompareS. japonica inhibited the secretion of TNF-α and IL-1β in LPS-activated RAW 264.7 cells, and the inhibition rates were about 48% or 38% at 50 μg/mL, respectively [It has been suggested that pro-inflammatory cytokines secreted by macrophages in innate immunity as well as adaptive immune responses, exhibit the abilities to stimulate apoptosis of abnormal cells (infected and transformed), digest harmful microbes, and destroy the extracellular matrix . IL-6 isp < 0.05) decreased in a dose-dependent manner. Researches have shown that excessive COX-2 expression can lead to a variety of chronic diseases, and inhibiting COX-2 overexpression can be an effective way to prevent and treat inflammation and tumors. Furthermore, COX-2 catalyzes the production of prostaglandins (PGs), which represents an important step in the inflammatory process. A dose-dependent inhibition of the protein level of COX-2 was observed , obviously. It was found that the content of proteins notably increased, by approximately 3.98-fold (p-ERK), 5.81-fold (p-JNK), and 7.25-fold (p-P38), respectively, compared with the control group (without LPS-treated group). While LJNF3 showed an inhibition of ERK, JNK and P38 phosphorylation, and the inhibitory effect enhanced with the increase in the concentration of LJNF3. When the cells were treated with the highest testing concentration of LJNF3, compared to the LPS-only treated cells, the phosphorylation of ERK  are phosphorylated and activate factors that trigger the transcription of inflammatory mediators. In the present researches, MAPK signaling pathways can be activated by LPS, and the inhibition of MAPK phosphorylation is an effective way to cure inflammation . From Fin of ERK a, JNK  when incubated with a tested concentration (3.125‒25 μg/mL) of LJNF3. LJNF3 of 25 μg/mL induced a dramatic decrease in p-P65 expression, from 1.22-fold to 1.04-fold, by LPS activation. IKKα and IKKβ consist of IκB kinases (IKKs), which are responsible for the phosphorylate IκB. During this study, the phosphorylation level of IKKα/IKKβ was determined to be 2.63 ± 0.26-fold in LPS-induced macrophages as a positive control (p < 0.05) inhibited, by 37.38% at 12.5 μg/mL, LJNF3 compared to the positive control. All the results demonstrated that LJNF3 had a great potential to inhibit the inflammation reaction via suppressing the phosphorylation of MAPK and NF-κB. Compared to the above reports [The transcription factor NF-κB has been considered as the most important transcription factor involved in immunity, inflammatory responses, cell proliferation, and cell death . In a no control e. The pr reports ,50, our p < 0.05) of LHNF3. Therefore, LJNF3 had an obvious protective effect on zebrafish embryos and could reduce LPS-induced inflammation.In this research, the toxicity test showed that LJNF3 had no toxicity to zebrafish embryos at 2.5–50 μg/mL. Therefore, the study evaluated the protective effect of LJNF3, with 12.5–50 μg/mL, on LPS-induced toxicity in zebrafish embryos. In p = 0.696) at the concentration of 50 μg/mL. The study indicated that the cell death of zebrafish larvae was inhibited by LJNF3 pretreatment.In p < 0.05). Sanjeewa et al. (2019) reported that the purified fucoidan at 12.5–50 μg/mL showed a protective effect against cell damage in LPS-induced zebrafish larvae, while suppressing the NO production caused by LPS [The production of ROS is an important factor for confirming the mechanism of inflammatory regulation. As shown in d by LPS . AccordiS. japonica was harvested on 20 June 2019 in Rongcheng, Shandong province, China. The strain is named Yanza, which is the traditionally hatched and cultured strain in Rongcheng S. japonica cultural area. Diaminofluorophore 4-amino-5-methylamino-2′7′-difluorofluorescein diacetate (DAF-FM DA), 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA), and acridine orange were from Sigma company . The monosaccharide standards of D-fucose (Fuc), galactose , L-arabinose (Ara), D-mannose (Man), L-d-rhamnose (Rha), D-glucose (Glc), and D-xylose (Xyl), as well as Griess reagents, lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO), and 3--dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were also obtained from Sigma Chemical Co. . Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were from company of Gibco . ELISA kits were purchased from Dakewei .S. japonica was washed with tap water followed by distilled water to remove sand and salt on the surface. The cleaned sample was air-dried in an oven at 60 °C. The dried sample was grounded to powder, and the fractions smaller than a size of 60 mesh were collected and stored in −20 °C until use. In order to remove the polyphenols, pigments and lipids, the sample was extracted with 95% ethanol at 40 °C for 2 h. The residue was collected by filtration. The defatted sample was incubated with ultrapure water  at 120 °C for 2 h, and the supernatant was obtained by centrifugation (2000× g for 20 min). Then, 2% CaCl2 solution was added to the supernatant to remove alginate and the derived alginate precipitate was removed by centrifugation at 2000× g for 15 min. The supernatant was concentrated by a rotary evaporator, dialyzed against distilled water and lyophilized by a freeze-drier to obtain the crude LJN.By anion-exchange chromatography, the crude LJN was purified by AKTA purifier . Three f−1.Nicolet 67 spectrometer  was used to analyze LJNF3 in the range of 400–4000 cmThe chromatographic column was TSK Gel GMPWXL (300 mm × 7.8 mm). DAWN HELEOS-II 18 angle laser detector and differential detector made by Wyatt Company .The monosaccharide composition was analyzed according to the reference reported by previous report using 6890 N gas chromatographic system  .1H NMR, 13C NMR, COSY, HSQC and HMBC).Agilent DD2 500 MHz  recorded on NMR spectra  were cultured at 37 °C in a humidified incubator  with 5% COne serum . The effne serum .RAW 264.7 cells were pretreated with various concentrations of LJNF3 for 1 h and then stimulated with or without LPS (1 μg/mL) for 24 h. The supernatants were collected for the analysis of pro-inflammatory cytokines release. The TNF-α, IL-1β, and IL-6 were determined by ELISA kits  ,51.The Western blot was analyzed according to the reference .The sources and cultivation methods of adult zebrafish are referred to by Ni et al. . The embSurvival rate and heart-beating rate were measured by the method described by reference .Intracellular cell death, ROS, and NO in zebrafish embryos were estimated according to the method by Zou et al. .p < 0.05 were significantly different. Different letters indicate a significant difference between the compared pairs of individual means, while the same letter means no significant difference between the compared pairs of individual means [All experiments were carried out in independent triplicates, and each experiment had at least three parallel groups. All results were expressed as the mean ± standard error. Significant differences were determined via IBM’s SPSS  statistics by one-way analysis of variance (ANOVA). Values with al means ,51.S. japonica, and evaluating the anti-inflammatory effect in vitro and vivo. The FTIR and NMR analysis demonstrated that LJNF3 contained a main chain of (1→3)-α-l-fucopyranosyl residues, with sulfate groups mainly at the C4 positions, and 6-linked β-d-galactopyranose units. The anti-inflammatory effect of LJNF3 was measured in vitro by RAW 264.7 cells. LJNF3 had no toxicity at the concentration of 3.125‒25 μg/mL in macrophages, and further suppressed the production of NO, TNF-α, IL-1β, and IL-6 in a dose-dependent manner, which was related to the translational down-regulation of iNOS, COX-2, MAPK, and NF-κB signaling pathways. Moreover, it possessed the protective effect of LJNF3 by zebrafish, through decreasing the cell death and production of ROS and NO. This study provides scientific evidence and advances in the potential application of LJNF3 from S. japonicas, an attractive anti-inflammatory agent for biomedical, food and cosmetic industries.It is the first time discovering LJNF3 as galactofucan extracted from