These mild injuries are not visible on routine clinical imaging. The potential clinical role of a specific imaging biomarker be it diagnostic, prognostic or directing and monitoring progress of personalised treatment and rehabilitation has driven the exploration of several new neuroimaging modalities. This systematic review examined the evidence for magnetoencephalography (MEG) to provide an imaging biomarker in mild traumatic brain injury (mTBI).meta-analysis was not possible, based on the heterogeneity of reported outcomes, we provide a narrative synthesis of results.Our review was prospectively registered on PROSPERO: CRD42019151387. We searched EMBASE, MEDLINE, trial registers, PsycINFO, Cochrane Library and conference abstracts and identified 37 papers describing MEG changes in mTBI eligible for inclusion. Since The two most promising MEG biomarkers are excess resting state low frequency power, and widespread connectivity changes in all frequency bands. These may represent biomarkers with potential for diagnostic application, which reflect time sensitive changes, or may be capable of offering clinically relevant prognostic information. In addition, the rich data that MEG produces are well-suited to new methods of machine learning analysis, which is now being actively explored.MEG reveals several promising biomarkers, in the absence of structural abnormalities demonstrable with either computerised tomography or magnetic resonance imaging. This review has not identified sufficient evidence to support routine clinical use of MEG in mTBI currently. However, verifying MEG’s potential would help meet an urgent clinical need within civilian, sports and military medicine. At leasPost-concussion symptoms (PCS) include headache, dizziness or balance disorders, and cognitive impairments including attention, concentration, memory and speed of information processing problems. Symptoms can also include sleep disturbances, blurred vision, photosensitivity, tinnitus and neuropsychiatric symptoms including personality change, irritability, anxiety, and depression that can develop following mTBI . WhetherMagnetoencephalography (MEG) is a functional neuroimaging technique that measures the magnetic induction produced by electrochemical current flows within the brain . CurrentEarlier neurophysiological research in mTBI comes from EEG studies. They demonstrated focal abnormalities in the delta and theta frequency bands as well as posterior alpha peak slowing; however, there is little evidence for correlation of either routine or quantitative EEG with clinical features of mTBI . There i2A systematic review of the literature was conducted with planned narrative synthesis, and possible meta-analysis dependent on data availability. The protocol was prospectively registered on PROSPERO CRD42019151387. A literature search of the electronic databases EMBASE, MEDLINE and PsycINFO via Ovid was conducted on 4th December 2020. The complete search strategies are listed in the supplementary material. All relevant papers published prior to the search date were included. References were screened for additional papers and searches of grey literature were conducted on Web of Science, ProQuest, World Health Organisation clinical trials registry, ISCRTN clinical trials registry and the US National Library of Medicine clinical trials registry.After de-duplication two authors screened the 466 abstracts independently. The inclusion criteria were human research, in adults aged over 16 years, who were clinically diagnosed with mTBI according to recognised criteria with post-traumatic amnesia ≤ 24 h, Glasgow Coma Scale ≥ 13, and loss of consciousness ≤ 30 min. MEG was used as an imaging modality and comparison was made between the mTBI participants and either a normative database or a case control design was used. Outcome assessments included symptom scores, neuropsychological test scores, or clinical diagnosis. The exclusion criteria were papers not available in English, mTBI was not diagnosed by recognised criteria, paper examining pharmacological interventions, mixed diagnoses with mTBI results not published as a subgroup analysis, mixed ages with adult results not published as a subgroup analysis, and review articles, single case reports, and duplicate papers. All disagreements were resolved by discussion and 383 abstracts were rejected, leaving 83 remaining. Two authors then conducted a full text screen independently, 46 papers were rejected, leaving 37 for final inclusion in the narrative synthesis. The Scottish Intercollegiate Guidelines Network critical appraisal checklists for either case-control or cohort study designs were used to appraise risk of bias and quality of individual studies . After r33.1In total, 37 papers were identified through text searching, detailed in Twenty-five papers examined a civilian population with mixed mechanisms of injury, in five papers the population recruited from was unclear. Five papers examined a military population with two of these specifically focussed on blast injury. Two papers include both military personnel and civilians. Ten of the papers recruited only patients with mTBI and persisting PCS. The study sizes ranged from six to 84 participants with mTBI. Mean time between injury and MEG assessment ranged from six days to 13 years but was unreported in nine papers. There was a male bias in the mTBI population of all included papers, with 17 reporting exclusively male participants. The mean mTBI sample age ranged from 25 to 42 years. Year of publication spanned 1999 to 2020.Ten papers reported sensor space analyses while the remaining 27 reported findings after source reconstruction. Fourteen papers presented analysis of resting state spectral power. Seventeen papers presented connectivity analyses or report network metrics. Nine papers presented analyses of task-based MEG recordings. Symptom severity was correlated with MEG findings in twelve papers, and neuropsychological test scores in five papers. Thirteen papers attempted classification metrics, most of these being machine-learning algorithms. Several papers reported multi-modal imaging, but only two presented associations between MRI abnormalities and their MEG findings.3.2MEG demonstrated improved ability to detect spectral power differences over EEG when utilising multimodal imaging . The mosThe alpha frequency band was reported to show increased power in mTBI participants compared to controls in three papers and the oKaltiainen et al. noted that only MRI T2 hyperintense lesions within 3 cm of the cortex were associated with aberrant theta frequency band activity . Similar3.3Combining both intra and cross-frequency analyses the most frequently reported band specific connectivity analysis was in the delta frequency band, in nine of the 17 papers. Of these, three reported an increase in delta frequency band connectivity in participants with mTBI relative to controls , and twoAlpha frequency band connectivity analysis was reported in seven papers. Four papers from the same group reported an increase in mTBI participants relative to controls . One papBeta frequency band connectivity was reported in five papers. Three reported an increase , in the Alternative network metrics included calculating coefficients of: small-worldness , rich cl3.4Of the nine papers that included task-based analyses; three assessed working memory , two setIn the set-shifting tasks, mTBI participants had longer reaction times and poorer performance in the extradimensional shift condition compared to controls. However, both set-shifting conditions showed mTBI participants had an aberrant sequence of brain area activation. This was significant in the right frontal and bilateral parietal lobes . The sam3.5Five papers reported the correlation between their MEG results and clinical interview results or symptom questionnaire scores, as a surrogate for clinical outcome. Two papers reported the sum of all regions with excess LFA positively correlated with symptom score on the Head Injury Symptom Checklist and symptom severity in a structured clinical interview, respectively . On the There can be diagnostic uncertainty when attempting to differentiate PCS and PTSD. While not the focus of this review, four of the included papers reported correlations between their MEG results, predominantly in the alpha frequency band and co-morbid PTSD symptoms . PopescuThree of the included papers reported on the correlation between MEG findings and symptoms of depression or anxiety . Huang e3.6There was marked variability in approach when correlating MEG data with neuropsychological testing data. Some papers used resting state data, while others used task specific data, e.g., from an N-back working memory task and both spectral power and connectivity analyses were used. The most reported neuropsychological assessments were the Trail Making Test Part B within the Delis Kaplan Executive Function Score (DKEFS), and the Digit Symbol Coding task within the Weschler Adult Intelligence Scale. Four papers reported correlations between these test scores and either power or connectivity of specific frequency bands in the frontal MEG results . For theFor the Digit Symbol Coding task, the right prefrontal cortex power in all frequency bands and low frequency power in right temporal gyri were negatively correlated with test performance . Huang e3.7Fifteen papers described methods to determine participant classification between mTBI participants and controls. From the reports, it is unclear if any of these used methods that were set prospectively, prior to data collection. Four of these used resting state LFA . Lewine Ten papers applied a machine learning approach to distinguish the connectivity analysis of mTBI participants from controls . Most pa3.8While the mean time between MEG assessment and injury ranged from six days to 11 years in the papers incorporated in this systematic review, five papers included repeat MEG imaging sessions . Three p3.9Individual studies did not report the ability to detect differing MEG abnormalities dependant on the mechanism of injury. The mechanisms were divided into sports-related concussion, any other civilian injury, or those suffered by military personnel, which could be further split into blast (from explosive blast waves) and non-blast trauma. In total 16 papers reported mTBI participants from more than one of these groups, though small sample sizes may have led to underpowered comparison.3.10Three papers were sufficiently detailed to complete at least two thirds of the relevant Scottish Intercollegiate Guidelines Network critical appraisal checklist and judged to be at the lowest risk of bias . Seven p4This review has identified that while MEG has demonstrated clear promise as a functional neuroimaging modality, it is not yet a diagnostic or prognostic clinical tool in mTBI of sufficient sensitivity and specificity. However, MEG is one of the most sensitive imaging modalities for the evaluation of mTBI, considering the very low sensitivity of CT, structural MRI and EEG. There is growing consensus around key features such as an increase in LFA power and widespread connectivity changes following mTBI. The consistently high prevalence of MEG abnormalities across several studies, and the initial successes of AI algorithms to classify participants, implies that MEG is one of the most sensitive neuroimaging modalities to investigate this condition. Future work should concentrate on harmonising biomarkers and data analysis methods, so that different groups can generate a robust evidence base quickly. Harmonisation should also aim to build on the current published longitudinal studies to establish the natural history of these changes in the weeks, months and years following injury. Current evidence indicates that task-based MEG data, with cognitive loading, are also an important tool to improve our understanding of the impact of mTBI on neural activity and could possibly play a role in guiding therapeutic interventions.Increases in LFA power have been reported with a frontal predilection. This correlates with acute changes in mTBI in the corpus callosum seen using DTI. In addition, when abnormal LFA and MRI lesions appear to co-localise, it suggests that LFA may arise from partial cortical deafferentation . DespiteMany papers have examined the role of network metrics, connectivity, and machine learning. There is a lack of methodological homogeneity across papers, and studies have not addressed the direction of observed effects. However, an increase in delta and theta connectivity is reported, including in four of the papers at minimum or intermediate risk of bias. Authors have suggested that these effects are reflective of plasticity in recovery, and symptoms may be related to an inability to deactivate the default mode network. Network metric studies often used machine learning to report high levels of classification accuracy, but frequently used convenience samples of unmatched controls, making them vulnerable to spectrum bias. While not yet being clinically useful, this shows a potential role for machine learning, which should be explored further.The most common risks of bias identified in this review related to clarity of outcome measures, likely retrospective unblinded analysis and a lack of clinical description of participants, leading to the possibility of confounding. Most studies were small, the largest included 84 participants with mTBI. Additionally, the analysis performed was heterogeneous, with the most common type of analysis  being performed in only 17 of the 37 included papers. There was a wide intra-study and inter-study range of time interval between injury and MEG scanning, which may mask some of the temporal evolution of MEG changes following mTBI.For future studies, collaboration across sites should be encouraged. This will increase sample size and power, and prospective registration with clear quantifiable outcome measures would limit bias. These should align with recommended core outcome sets for mTBI research . An appr5meta-analysis of MEG data, based on the available literature. For example, different mechanisms of injury could not be differentiated by MEG within individual studies. If this data could be pooled, and assessed with a pre-specified analysis method, we could definitively answer this question. This issue arises because of the broad definition of mTBI, the complex nature of the MEG datasets and variety of analysis methods available and reported. It is likely that a pooling of original study datasets will be required to overcome this, but this was beyond the scope of this review. The review’s strengths include the prospectively registered systematic design and independent rating of papers, which should limit the risk of bias in its conclusions. Additionally, this review has identified and made recommendations to improve study methodology, frequently judged suboptimal by clinical critical appraisal tools.The major limitations of this review were being unable to resolve its broad questions into quantitative measures and the inability to perform a 6To the best of the authors’ knowledge, this is the first prospectively registered systematic review of MEG studies focused on adult mTBI. This review has not identified sufficient evidence to support routine clinical use of MEG in mTBI currently. This is due to study heterogeneity, a lack of diagnostic test accuracy studies, and underpowered longitudinal studies of low quality. Despite this, some key areas of progress have been identified. These include the two most promising biomarkers of excess resting state low frequency power, and connectivity changes in all frequency bands. These may represent biomarkers, with potential for diagnostic application, which reflect time-sensitive changes, or may be capable of offering clinically relevant prognostic information. Verifying these findings would help meet an urgent clinical need within civilian, sports and military medicine to identify and characterise mTBI, and to allocate neurorehabilitation resources of differing nature, complexity and cost. This is best done with prospective clinical studies, using pre-defined protocols and drawing on the research guidelines highlighted in this review. Collaboration across sites would help standardise analysis methods and reporting, allowing quantitative comparison of findings across studies.


Glaucoma is a group of eye conditions that damage the optic nerve head and affect visual function, potentially leading to blindness. These conditions include adult and child chronic forms as well as acute forms. The prevalence of glaucoma is increasing: currently, about 60 million people are affected worldwide, and this number is expected to increase up to 110 million by 2040 [https://pubmed.ncbi.nlm.nih.gov using “glaucoma” as the keyword). To address these changes, this Special Issue in the Journal of Clinical Medicine (JCM) is dedicated to collecting high-quality scientific contributions focusing on diagnostic and clinical modifications of glaucoma management.The scene of glaucoma diagnosis and management is rapidly changing. A great number of innovations and technological developments have advanced over the last two decades, and this is reflected in the publication trends in the field: 1299 papers were published in 2001, compared with 4665 in 2021  using the Compass fundus automated perimeter . The staThe following three papers focused on the diagnostic performance on specific forms of glaucoma, also analyzing the impact of risk factors. Dr. Stingl and coauthors published the first data from a German Registry of childhood glaucoma . They prThe remaining seven papers explored different therapeutic options for glaucoma. Interestingly, most of these studies focused on surgical management, a fact that clearly reflects the shift from medical to surgical indications occurring in many glaucoma patients in the attempt of guaranteeing them a better quality of life. Treatment-related side effects and quality of life were explored in three papers. Park et al. performed a randomized trial to explore the changes occurring in the macular thickness of patients treated with prostaglandin analogues (PGA) and receiving uncomplicated cataract surgery . The autIn their prospective randomized trial, Kopsinis and coauthors elegantly showed that the intraoperative use of bevacizumab obtains similar efficacy to mitomycin C (MMC) during trabeculectomy . OverallFinally, three papers evaluated the clinical impact of minimally invasive glaucoma surgery (MIGS) devices. The efficacy of XEN, one of the most commonly used MIGS devices, was explored by two papers. Lewczuk and coauthors  retrospeJCM team for their robust support. Finally, I sincerely thank all of the authors for their valuable contributions.Several interesting findings are derived from this collective body of work. It is clear that, today, glaucoma experts are focused on preserving not only the visual function but also the quality of life of patients. This is possible thanks to the better comprehension of detrimental factors affecting the quality of life of people with glaucoma as well as the introduction of less invasive laser and surgical options. Additionally, diagnostics are becoming progressively more effective, but there are still many questions that need more evidence in order to be answered, for example, the role of the integration between morphology and function, or the impact of big data analyses on glaucoma. Considering the limited resources and the dramatic increase in glaucoma patients that we will have to manage in the future, new models of care should be explored, including screening of high-risk subjects and a more widespread use of telemedicine. As the Guest Editor, I give special thanks to the reviewers for their professional comments; in many cases, their volunteer work was valuable to improving the quality of the papers. I am also grateful to the


The pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a public health emergency, and research on the development of various types of vaccines is rapidly progressing at an unprecedented development speed internationally. Some vaccines have already been approved for emergency use and are being supplied to people around the world, but there are still many ongoing efforts to create new vaccines. Virus-like particles (VLPs) enable the construction of promising platforms in the field of vaccine development. Here, we demonstrate that non-infectious SARS-CoV-2 VLPs can be successfully assembled by co-expressing three important viral proteins membrane (M), envelop (E) and nucleocapsid (N) in plants. Plant-derived VLPs were purified by sedimentation through a sucrose cushion. The shape and size of plant-derived VLPs are similar to native SARS-CoV-2 VLPs without spike. Although the assembled VLPs do not have S protein spikes, they could be developed as formulations that can improve the immunogenicity of vaccines including S antigens, and further could be used as platforms that can carry S antigens of concern for various mutations. Coronaviridae family, emerged in 2019 and caused a public health emergency of international concern. Many companies and researchers have developed various types of vaccines that are now being supplied around the world. Over one hundred vaccines using varied approaches including protein subunit, viral vector, RNA, DNA, inactivated virus, attenuated virus, and virus like particles (VLPs) platforms are in clinical trials. Among these, viral vector and RNA vaccines are currently in use. SARS-CoV-2 is composed of four essential structural proteins: the spike (S) glycoprotein, membrane (M) protein, envelope (E) protein, and nucleocapsid (N) protein, as well as multiple accessory and non-structural proteins1. These protein subunits can assemble into virus particles without viral RNA genome in mammalian cells to study virus-cell entry2 and to develop vaccines3. Xu et al.4 developed a mammalian expression system to produce VLPs of SARS-CoV-2 for medical applications, and they confirmed that VLP vaccines provide a safe protection. VLPs self-assembled into empty shells can be used safely as vaccines without the risk of replication or infection due to the lack of viral nucleic acid. VLPs constitute attractive vaccine platforms in view of their safety and ease of production. VLPs are highly immunogenic and can induce elevated titers of neutralizing antibodies, even without adjuvants, thanks to the maintenance of the native conformation of viral proteins presenting repetitive epitopes to the host immune system cells. Moreover, VLPs offer interesting biotechnological advantages as they can serve as scaffolds for presenting heterologous antigens capable of inducing immune responses against other infectious diseases. Further, VLPs have the advantage that, contrary to inactivated or attenuated viruses that must be prepared in mammalian cell lines, they can be produced in heterologous systems, such as bacteria, yeasts, insect cells, or plants.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a member of 5, more studies and cases have been reported. Various VLPs such as bluetongue virus (BTV)6, HPV7, hepatitis B core antigens (HBcAg)10, Norovirus (NV)11, Dengue virus12 have been successfully produced in plants. These plant-produced VLPs are capable of inducing protective immunity. In the above examples, VLPs of HBcAg are also used as effective carriers of foreign antigen10. Most recently, Ghorbani et al.13 predicted the VLP of HBcAg-exposing epitopes of SARS-CoV-2 by an immunoinformatics approach towards the development of a vaccine against SARS-CoV-2. During this SARS-CoV-2 pandemic, many studies on the development of plant-derived vaccines and antibodies are underway. The most advanced is the clinical phase 3 trial using plant-derived VLPs vaccine by Medicago Co. .Plants are being considered as an alternative vaccine/mAb production system, especially in unexpected or emergency situations that cannot be solved by existing systems. Since a plant-derived antibody cocktail to Ebola virus in 2014 had been shown to be effectiveAgrobacterium-mediated delivery. This self-assembled VLPs can be purified and will be useful as a promising carrier platform for immunogenic SARS-CoV-2 spike-derived epitopes.Here, VLP of SARS-CoV-2 composed with M, E, and N were constructed by co-expression of two different vectors, one for M and E and the other for N, in a species of tobacco using IRESE and pBYR2fp-NFLAG, were then transfected into separate Agrobacterium (GV3101) lines, which were used for transient VLPs expression by co-delivery.For formation of VLPs in plant cell, three major proteins M, E, and N were co-expressed by agroinfiltration with separate vectors. M and E were expressed using a single expression construct. The coding sequences of M and E were linked by an IRES sequence from the tobamovirus coat protein, using the native M and E signal peptides and transmembrane domains Fig. a. The coIRESE and pBYR2fp-NFLAG in N. benthamiana leaves using a syringe , extraction buffer 2 , and extraction buffer 3 , browning of total soluble protein extracted from EB2 and EB3 buffers without 2-mercaptoethanol was observed  with plant-derived VLPs (CoVLPs). They demonstrated that the first trial of CoVLPs was immunogenic even at low doses20. Besides, RBD SARS-CoV-2 HBsAg VLP vaccine , an enveloped VLPs of SARS-CoV-2 S glycoprotein , etc. are under phase 1 to 2 . VLP is an attractive vaccine platform.Various approaches using recombinant hosts to produce SARS-CoV-2 vaccines are being attempted. These researchers have showed that S protein of SARS-CoV-2 is the suitable candidate for recombinant vaccine development as it can elicit potent immune responses and is the major target of neutralizing antibodiesN. benthamiana plants. M and E genes were linked by internal ribosome entry site (IRES) sequence. The IRES sequences are very useful system to introducing the coordinated expression of multiple genes22. GFP and LUS linked by encephalomyocarditis virus (EMCV) IRES were detectable in tobacco plant. On the other hand, EMCV IRES sequences are limited in their ability to induce multigene expression in rice22. Here, we applied tobamovirus (TMV)-derived IRES to coexpress of M and E proteins. The activity of TMV IRES was confirmed through multigene expression using GUS or LUC in Arabidopsis thaliana24. In addition, co-expression of phytoene synthase (Psy) and carotene desaturase (Crtl) linked to TMV IRES was also confirmed in rice endosperm25.The M, N, and E genes were codon optimized and cloned into the geminiviral vector pBYR2fp for plant expression, and these proteins were transiently expressed in 27, and these results were consistent with our purification conditions.Here, we designed SARS-CoV-2 VLPs composed with M, E, and N proteins without S protein, and showed their self-assembled VLP structure. To determine optimal conditions for VLPs purification, 10–60% sucrose gradient sedimentation showed VLPs in the fraction of > 40% concentration. The highest amount of VLPs was in the 60% fraction, otherwise no VLPs were detected in fractions 10–30%. Therefore, to harvest VLPs for TEM analysis, VLPs were purified by 40% sucrose cushion method. Previously, many types of VLPs including SARS-CoV-2 VLPs derived from mammalian or insect expression systems were detected in fraction of 30–40% sucrose concentration28. VLPs without S protein expressed in HEK-293 T human cells or Vero E6 animal cells were 90.33 ± 32.45 nm and 71.02 ± 21.98 nm27, respectively. Therefore, in our results, it was confirmed that the shape and size of plant-derived VLPs were similar to those of native SARS-CoV-2 VLPs without spike.Without the S glycoprotein in our constructs, the usual SARS-CoV-2 ‘crown’ was not detected in TEM images. Sizes of assembled particles were in the range of 50–130 nm with an average of 75.20 ± 19.72 nm , these genes were optimized to plant preferred-codons and inserted in pBYR2fp plant expression vectors using XbaI and SacI restriction sites31 23 under the single promoter, and N protein was constructed separately, resulting in pBYR2fp-MIRESE and pBYR2fp-NFLAG, respectively.Coding sequences of SARS-CoV-2 M, E and N protein were synthesized from the Bioneer co. . Based on full genome sequence of the virus , 10 mM MgSO4, 200 μM acetosyringone, at pH 5.7) to an OD600 of 1.0. Both A. tumefaciens strains were mixed together with same ratio, and bacterial suspensions were syringe-infiltrated into the abaxial side of 6-week-old N. benthamiana plant leaves which located at 4 and 6 numbered from the bottom and maintained at 24 °C growth chamber  sucrose were layered into ultracentrifuge tubes  and a volume of 700 µl of the crude extract was loaded on top of the gradient. After centrifugation in a Beckman TLA 100.3 rotor at 90,000×g for 2 h at 4 °C, 8 fractions were collected from the top to the bottom of the tube. The commercial recombinant M proteins form E. coli were used as the positive control  .A total of 25 µg TSP of each sample from 1 g fresh weight leaf discs was loaded onto SDS-PAGE gels to analyze protein separation. SDS-PAGE analysis was performed using pre-cast gel Mini-PROTEAN TGX 4–20% gradient Gels . The samples to be loaded were prepared by mixing 4 × Laemmli sample buffer  with 2-mercaptoethanol  in 25 μg TSP. Western blot analysis was performed according to the manufacturer’s instructions. Briefly, western blot in the denaturation condition was performed by heat-treatment of the prepared samples, and in non-reducing condition, the prepared sample was mixed with Native sample buffer  except for 2-mercaptoethanol and then performed without heat treatment. The loaded gels were transferred to a 0.45 µm PVDF membrane  for 90 min, and then the membranes were blocked with a blocking solution containing 0.1% Tween-20 and 5% skim milk in PBS for O/N at 4 °C. Membranes were incubated with Rabbit anti-M antibody , Rabbit anti-N antibody , or anti-Flag antibody . Then, the membrane was incubated with a goat anti-Rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody  diluted 1:10,000 for detection of primary antibody. After repeated washing steps, specific recombinant protein complexes were detected using a Pierce™ ECL substrate . Quantitative image analysis was carried out using the ImageJ software  at the UNIST . Concentrated VLPs were subjected to negative staining and examination by transmission electron microscopy. Two microliters of each removed salt component suspensions were placed on 200-mesh copper grids  and allowed to incubate at room temperature for 1 min in order for the particles to adhere. Two microliters of 1.5% uranyl acetate were added to the attached particles on the grid, grids were then analyzed by using a transmission electron microscope JEM-2100 , and images were acquired through a CCD camera SIS veleta  at the UNIST .To increase purify of VLPs for VLP imaging, an additional 35% sucrose sedimentation was added to the extraction procedure as mentioned in Moon et al.The use of plants or plant materials in the present study complies with international, national and/or institutional guidelines.Supplementary Figures.


Social-ecological network (SEN) concepts and tools are increasingly used in human-environment and sustainability sciences. We take stock of this budding research area to further show the strength of SEN analysis for complex human-environment settings, identify future synergies between SEN and wider human-environment research, and provide guidance about when to use different kinds of SEN approaches and models. We characterize SEN research along a spectrum specifying the degree of explicit network representation of system components and dynamics. We then systematically review one end of this spectrum, what we term “fully articulated SEN” studies, which specifically model unique social and ecological units and relationships. Results show more focus on methodological advancement and applied ends. While there has been some development and testing of theories, this remains an area for future work and would help develop SENs as a unique field of research, not just a method. Authors have studied diverse systems, while mainly focused on the problem of social-ecological fit alongside a scattering of other topics. There is strong potential, however, to engage other issues central to human-environment studies. Analyzing the simultaneous effects of multiple social, environmental, and coupled processes, change over time, and linking network structures to outcomes are also areas for future advancement. This review provides a comprehensive assessment of (fully articulated) SEN research, a necessary step that can help scholars develop comparable cases and fill research gaps. EnvironA network perspective focuses on relationships. For example, network studies might investigate how one kind of relationship, like trust, explains another relationship, such as interjurisdictional watershed collaborations .1 studies are looking to network concepts and methods to advance their work. Examples include, but are not limited to, teleconnections and ecosystem services research  connected to a network of harvested species  and their food-web  . This fuFully articulated SENs open up numerous conceptual and analytical possibilities to develop and test theories and measure social-ecological patterns, because all system relationships and dynamics can be considered. For this reason, we restrict our systematic component of the review to studies of fully articulated SENs ; however2.2.single-layer network. It is the kind with which most people are familiar, such as a stakeholder network or a food web. Two nodes in a single-layer model may only share one relationship; however, social and ecological nodes and edges can be differentiated graphicly using attribute values  using search terms that represent a variety of ways for talking about coupled human-environment or social-ecological systems. Joined by Boolean “OR”, we used the following search terms: social-ecological AND network, socio-environmental AND network, socio-ecological AND network, human-environment AND network, “coupled human and natural systems” AND network, CHANSThe lead author manually screened all papers to remove those that did not specifically address network science and only, for example, talked about the importance of networks or networking in social-ecological systems. Papers were screened using the abstract or main text as necessary; 338 were retained as candidate papers for the review. From this, the team reviewed and discussed an initial subset of purposely selected papers to pilot and refine the inclusion criteria and coding protocol.Papers were included in the final review if they met the following criteria: 1) They were empirical or substantive studies based on primary field or desk research, case-study synthesis, or computational modeling. Review and opinion papers were not included. 2) Papers needed to undertake the “fully articulated” approach, i.e., have a clear set of social and ecological nodes and contain edges within and between these social and ecological sub-components. These subcomponents did not need to be specifically identified by paper authors, but needed to be identifiable to the coders based on their interpretation. 3) Papers needed to include networks as a system driver or outcome. An agent-based model, for example, that simply included a network component in the model background in order to simulate a more realistic universe, but did not seek to understand how the network shapes environmental outcomes, or how human-environment problems shape the network, would not have been included. For this reason, papers using Bayesian belief networks, or concept mapping, tended to be omitted despite using networks. The 338 papers were reviewed and classified for inclusion in the review by two independent coders, who made a final consensus-based decision.We complemented our topic search with our own knowledge of the literature and included any articles that were not returned in the WOK search, such as new articles that had not been indexed. As a young and growing body of scholarship, including these most recent papers is essential to understand SEN research’s trajectory. In addition, we reviewed the reference list of all included papers to check whether other papers should be included. No additional papers were identified through this procedure. We acknowledge that there may be papers that were neither included in the WOK search, the reference lists of included papers, or through our own knowledge, but through this triangulation of search approaches we believe to have compiled most, if not all, fully articulated SEN studies in peer-reviewed journals. While omissions are possible, our review illustrates the diversity of uses of the fully articulated SEN approach and the rich sets of possibilities it offers.3.2.To understand our first line of inquiry, how fully articulated SEN studies have been framed and conducted, we coded the papers’ objectives, theoretical framing, how the study was bound, the kind of evidence, and methods used. These variables  directlyAll SEN papers included in the review were coded by two independent coders. The three first authors then reviewed and resolved any discrepancies among coders through a consensus decision and ensured the codes were applied consistently. Some codes refer to the overall paper , while others addressed the SEN . Though infrequent, a paper could describe more than one SEN, for example in a comparative case analysis. Therefore, the sample size varies among reported results depending on if they describe the paper or the SEN(s) described in the paper.3.3.We also constructed and visualized a citation network to determine if SEN papers were closely linked to each other to provide a broad picture of SEN research. We did not expect all papers to be linked through direct citations, but expected papers to draw from common theoretical works. We therefore visualized SEN papers and their common citations and identified if common citations were key works of theory or simply methodological. See the 4.0From the original pool of 1,232 papers, we identified 22 fully articulated SEN papers  that draWhile 22 papers represent a small scholarly corpus, they nevertheless comprise a large enough body of work to distill emerging patterns and learn how authors approach fully articulated SENs. We must be cautious, however, not to overreach when drawing conclusions. The following 4.1.Perhaps indicative of a budding research field, the majority of the 22 fully articulated SEN papers (86.4%) had clearly stated methodological aims. No paper, however, solely hung its hat on methods. Theory testing or applied ends were always motivators, though the latter was much more frequent .Fully articulated SEN papers were split between taking a diagnostic or inferential approach (n = 17 and 15), with ten papers combining the two . InferenThe majority of fully articulated SEN papers address the problem of spatial or functional fit among social governance systems and the environment with a scattering of other theoretical framings , while sTo analyze these data and address objectives, papers used a number of analytical approaches. The most common involved motif frequency counts e.g., , and Qua4.2.7 elements (n = 1). Ecological nodes were more evenly distributed among a range of concepts including groups of plants or animals (n = 4), habitat patches (n = 7), biophysical places (n = 6), and concepts of habitats and ecosystem types (n = 5). Several other categories were also considered , where ecological materials or functions flow to social entities regardless of active social agency, were also considered, as were several reciprocal relationships (n = 5). While many papers accounted for multiple social-ecological relationships , these were mostly aggregated into a single edge, or in the cases of the ecosystem service cascade , may be a way forward. Such a heuristic might help work through case by case idiosyncrasies to arrive at a more generalizable and causal understanding . Logistics and the lack of longitudinal data, however, may not be the only barrier. Differences in the time-scales at which activities such as collaboration and information sharing occur versus when ecological outcomes are observable, as well as myriad co-dependent factors, may also present barriers.Another area for future work will be to link SEN structures to environmental outcomes. We found that few studies concretely made this link, an observation that is consistent with broader social network studies on environmental issues  will be to focus on what unique conceptual and theoretical contributions SEN research offers. 5.2The fully articulated SEN papers in this review represent a diverse range of approaches to conceptualizing and analyzing social-ecological systems. Two fundamental aspects of any network study are how to model and bound a study system. Addressing these aspects undoubtedly affects how the studied phenomena is conceptualized and quite possibly the kinds of results that can be deduced.Model approach  affects Representing detailed and specific places or organization tends to require more complex models such as multiplex and multi-level. Still, in three of the multi-level landscape models that we documented  that can hold the attribute of both social and ecological entities. Operationalizing the world as a SEN is slightly limited in multiplex models. Still, the multiplex networks are probably underutilized  and if so, how best to work with SENs.In the past half-decade, there has been significant development of fully articulated SEN ideas and applications, with the qualification, of course, that the current pool of substantive studies is small (but growing). Potential future applications and advancements are promising and ample as outlined throughout this review. How these advancements take shape may potentially move SEN research beyond a way of thinking and set of methods into something more, though we refrain from speculating exactly what that may be. Amassing more case studies that firmly link SEN structure to outcomes would provide an evidence base to test theories, perhaps develop new ones rooted in network structure, and guide environmental management.The quest for inference, however, should not overshadow the demonstrated and potential strength of SEN research to diagnose conditions that (proven or theorized) enhance environmental governance and management. Under this applied research trajectory, scholars may wish to explore how best to communicate SEN concepts and ideas, or how to put network science tools in the hands of practitioners and stakeholders. Through research and professional appointments, many of the authors here have observed that the practitioner community is often enthusiastic about networks, but sometimes lacks exposure beyond activities such as stakeholder mapping. While anecdotal, this evidence suggests that SEN scholars might do more to transfer and translate the full richness of network ideas and methods to those working in and managing the systems that we all study. Transdisciplinary collaborations may be an important step to advance the applied and policy theme that, as we documented, runs strong within fully articulated SEN research.SI


As this radiopharmaceutical shows rapid metabolic inactivation, we aimed at developing new improved tracers.Adrenal tumors represent a diagnostic and therapeutic challenge. Promising results have been obtained through targeting the cytochrome P450 enzymes CYP11B1 and CYP11B2 for molecular imaging, and [123I]IMTO and the most promising compound (R)-1-[1-(4-[123I]iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinylamide ([123I]IMAZA) for scintigraphy. Subsequently, a first endoradiotherapy with [131I]IMAZA in one of these patients was performed.Several IMTO derivatives were newly designed by replacing the unstable methyl ester by different carboxylic esters or amides. The inhibition of aldosterone and cortisol synthesis was tested in different adrenocortical cell lines. The corresponding radiolabeled compounds were assessed regarding their stability, in vitro cell uptake, in vivo biodistribution in mice, and their binding specificity to cryosections of human adrenocortical and non-adrenocortical tissue. Furthermore, a first investigation was performed in patients with known metastatic adrenal cancer using both [131I]IMAZA induced a 21-month progression-free interval in a patient with rapidly progressing ACC prior this therapy.We identified three analogues to IMTO with high-affinity binding to the target enzymes and comparable or higher metabolic stability and very high and specific accumulation in adrenocortical cells in vitro and in vivo. Labeled IMAZA exhibited superior pharmacokinetic and imaging properties compared to IMTO in mice and 3 patients, too. An endoradiotherapy with [123/131I]IMAZA with superior properties compared to the reference compound IMTO and promising first experiences in humans.We developed the new radiopharmaceutical [The online version contains supplementary material available at 10.1007/s00259-021-05477-y. In a recent series of 11 patients with advanced ACC, we demonstrated a therapeutic potential of treatment with [131I]IMTO, resulting in a lasting stabilization of the disease in the majority of patients, with only few adverse effects [High uptake of [ effects .123I]IMTO represents a suitable tool for adrenal imaging and therapy, studies of the metabolic stability revealed rapid degradation in patients by esterases. IMTO derivatives resistant to rapid degradation but still maintaining high avidity to adrenal CYP11B enzymes could allow lower radiation exposure during imaging and improved imaging quality due to lower background activity and higher effective doses in targeted treatment of ACC lesions. Therefore, we aimed at establishing a new theranostic tool targeting adrenocortical tumors by development of IMTO derivatives with improved properties regarding target binding, tracer stability, and target to background ratios.Although IMTO  or the 1123I]IMAZA  and subsequently by [123I]IMTO . The time interval between the imaging studies was at least 5 days. Scintigraphic imaging was performed using a Siemens Symbia dual-head gamma camera equipped with medium-energy parallel-hole collimators [Three patients  were investigated by [limators . Activit131I]IMAZA 1 month and with 30.5 GBq [131I]IMAZA 7 months after the evaluation with [123I]IMAZA. Prior to the first treatment, the absorbed dose to the blood was measured with a tracer activity of 72 MBq [131I]IMAZA as a surrogate for the exposure of the dose-limiting organ, the red bone marrow. The absorbed dose to the blood was estimated from assessments of the activity kinetics in total body and blood up to 48 h when the mean activity concentrations had dropped to 5% of the values at 5 min after the administration in the total body and to 1% in the blood. During therapy, the total body activity retention was measured with a survey meter in the ceiling over the patient’s bed. Gamma camera whole body scans accompanied by blood sampling were acquired starting 4 days post-administration after the first and 2 days after the second therapy until dismissal. SPECT/CT imaging was performed after 4 and 5 days, respectively.Patient #1 had experienced disease progression with a more than fourfold increase in her dominant lesions within only 4 months after a phase of disease stabilization after standard treatment with mitotane and eight courses of combination chemotherapy with etoposide, doxorubicin, and cisplatin. She received therapeutic doses with 28.2 GBq [18F]FDG PET/CT at baseline and 2, 5, 8, 11, 14, and 21 months after administration of the first treatment dose. Subsequently, the patient declined further follow-up imaging or additional therapeutic interventions.Tumor evaluation was performed by IMTO and three new tracers was determined by radio-HPLC; results are listed in Table 3 and 4 exhibited nearly equal or even better metabolic stability than [125I]IMTO. The most stable compound was [125I]IMAZA showing a more than threefold longer half-life than [125I]IMTO.The metabolic stability of 15IIMTO an3 and 2,2-difluoroethylester 4 and IMAZA exhibited both strong enzyme inhibition and at least comparable metabolic stability to IMTO, so they were additionally investigated in NCI H295 cells. An accumulation of the upstream steroids 17-OH-progesterone and androstenedione was observed at lower concentrations of the compounds (0.1–10 nM) due to the block of both CYP11B enzymes. At higher concentrations, the production of the investigated hormones was completely stopped, most likely due to additional inhibition of side chain cleavage enzyme (CYP11A1), similar to the known effect of metomidate  and a lower accumulation in the nuclei, cell debris subfraction (33.6%), and in the cytosolic fraction (7%). Results were similar for [125I]IMAZA, while [125I]3 and [125I]4 showed a lower accumulation in the mitochondrial subfraction. Thus, particularly for [125I]IMTO and [125I]IMAZA, a high and specific binding to the CYP11B enzymes as targets localized in the mitochondrial cell fraction may be assumed  and distributes to the rapidly exchangeable fraction of the total body water. After injection of [123I]IMTO, activity concentration in plasma was 1.16 times higher than in whole blood . For both compounds, the effective half-life in tumor tissue was only slightly shorter than the physical half-life of I-123. The biological half-lives were too long to be derived reliably from the 123I data.Uptake in large tumor lesions was visible already at 10 min after tracer injection with both 12IIMTO and131I]IMAZA, and the kinetics recalculated for [131I]IMAZA corresponded to a mean blood absorbed dose of only 0.03 Gy per GBq compared to 0.11 Gy/GBq for [131I]IMTO. Areas under the curve of both tracers are presented in Supplementary Table The residence time of the activity in the whole blood was shorter for [131I]IMAZA in patient #1, the residence time in the total body was 10.9 h and in the whole blood 0.17 h per liter of whole blood, indicating an absorbed dose to the blood of 0.032 Gy/GBq or 2 Gy after 63 GBq [131I]IMAZA. The corresponding values for [131I]IMTO, derived from diagnostics with [123I]IMTO, were much higher with 0.55 h residence time per liter of whole blood and a blood absorbed dose of 0.17 Gy/GBq.In the pre-therapeutic kinetics assessment with IMAZA were 133 Gy for the recurrent primary and 117–265 Gy in 3 evaluable metastatic lesions  due to a loss of renal clearance. The metastases known from the [123I]IMAZA scans show comparable high uptake in the dosimetric and post-therapeutic [131I]IMAZA scans; see Supplementary Fig. The estimates for the mean absorbed tumor doses actually administered in the first therapy with 28.2 GBq IMTO and endoradiotherapy with [131I]IMTO, we started with the hypothesis that both higher avidity to the target enzymes and higher metabolic stability would result in an increased and longer lasting specific binding to target tissue and improved target to background ratios.Our article describes the successful development of a novel theranostic tool for adrenal tumors. In order to improve diagnostic imaging with [Starting from IMTO, a total of 50 new derivatives were synthesized and characterized. Of the most promising three new substances, we synthesized the corresponding trimethylstannyl precursors and used them to establish the radiosyntheses of the tracers. The chemistry and radiochemistry of the remaining new derivatives will be published elsewhere.3 and 4 and the small carboxylic acid amide IMAZA indicating high-affinity binding of these compounds to the respective enzymes. Considering the IC50 values for inhibition of cortisol and aldosterone secretion in the low nanomolar range up to only 0.3 nM, even higher affinity to the CYP11B enzymes compared to IMTO may be assumed for two agents.Cortisol and aldosterone synthesis were potently inhibited by the small aliphatic esters 3, 4) or higher (IMAZA) stability than IMTO. It is noteworthy that these three compounds have comparable  or lower (IMAZA) lipophilicity estimated from HPLC retention times. This fits well to the observation that the binding sites of metabolizing enzymes are generally lipophilic in nature [In vitro analysis of metabolic inactivation after preincubation with human liver microsomes revealed only three compounds with comparable Below is the link to the electronic supplementary material.


Our simulations show that under conditions that existed prior to the COVID-19 outbreak, extremely high rates of COVID-19 infection can result in a dialysis unit. In simulations under worst-case modeling assumptions, a combination of relatively inexpensive interventions such as requiring surgical masks for everyone, encouraging social distancing between healthcare professionals (HCPs), slightly increasing the physical distance between dialysis stations, and—once the first symptomatic patient is detected—isolating that patient, replacing the HCP having had the most exposure to that patient, and relatively short-term use of N95 respirators by other HCPs can lead to a substantial reduction in both the attack rate and the likelihood of any spread beyond patient zero. For example, in a scenario with R0 = 3.0, 60% presymptomatic viral shedding, and a dialysis patient being the infection source, the attack rate falls from 87.8% at baseline to 34.6% with this intervention bundle. Furthermore, the likelihood of having no additional infections increases from 6.2% at baseline to 32.4% with this intervention bundle.This paper describes a data-driven simulation study that explores the relative impact of several low-cost and practical non-pharmaceutical interventions on the spread of COVID-19 in an outpatient hospital dialysis unit. The interventions considered include: ( As we write this, the COVID-19 pandemic has essentially taken over the world, with more than 20 million cases spread over 216 countries. A big concern for policy makers all across the world has been the impact of COVID-19 on healthcare systems and whether these systems can cope with the enormous strain placed on them by COVID-19. In this paper, we consider the spread of COVID-19 in a specific healthcare setting: the outpatient dialysis unit. Hemodialysis patients are extremely vulnerable to infections in large part due to multiple immune-system deficiencies associated with renal failure and hemodialysis. Hemodialysis facilities also increase the risk of COVID-19 transmission because each patient is in frequent, close contact with other patients and healthcare personnel. Thus, a dialysis unit can be seen as a microcosm for the worst-case impacts of COVID-19 in a healthcare setting. In this manuscript, we show via high-fidelity modeling and simulations that under pessimistic modeling assumptions, there is a combination of relatively simple, inexpensive, and practical non-pharmaceutical interventions that can substantially lower the impact of COVID-19 in the dialysis unit. Our simulations are based on fine-grained healthcare personnel movement data that we make available for other modelers to use. As of August 13, 2020, 5.2M people in the United States were infected with the COVID-19 virus, with more than 166K of these cases resulting in death of the patient . These were based on estimates of R0 for COVID-19 in the general population. There are almost no prior studies that can be used to estimate an R0 value or an attack rate in a dialysis unit. One exception is a study that performed contact tracing of a COVID-19 outbreak at a single hemodialysis center in Toronto, Canada [R0 of 3.5 (2 seeds leading to 7 secondary cases). This trend—of an infected patient leading to fewer secondary cases compared to an infected HCP—is what we observe in our simulations as well. The overall attack rate reported in this study was low (0.07), due to an extensive intervention upon detection of a few COVID-19 cases. These interventions included universal droplet and contact precautions including gloves, face shields, surgical masks, and isolation gowns, that were imposed on top of the interventions that were already in place .In our modeling and simulations, we used , Canada . There wIn addition to COVID-19, dialysis patients are at risk for other respiratory infections spread by droplets. Accordingly, our results have implications for how to prevent the spread of other respiratory infections. For example, the acquisition of MERS, a virus similar to COVID-19, was also associated with treatment in dialysis centers , 55. In Some of our results also have implications for healthcare-associated infections spread by close contact . Specifically, patients on hemodialysis are at risk for a wide range of multidrug resistant organisms (MDROs) including MRSA, vancomycin-resistant Enterococcus (VRE), and carbapenem-resistant Enterobacteriaceae (CRE)  and MDROA comprehensive study on the comparison of dialysis facilities showed that the mean of the number of stations over 5,193 facilities was 18.9 (std dev 7.8) . So the Our results also have broad applications for other healthcare settings beyond dialysis centers where patients repeatedly receive care for prolonged periods. Specific examples include chemotherapy units where patients come regularly and sit in chairs for several hours at a time. In addition, patients receiving parenteral antibiotics through peripherally inserted central catheter (PICC) lines arrive daily and can stay up to several hours, receiving monitoring and care from healthcare professionals. Like dialysis, for both chemotherapy and parenteral antibiotic treatment, the longest durations of interactions between healthcare professionals and the patients under their care occur at the beginning and end of a therapy session.e.g., assigning each patient to a unique HCP in order to reduce redundant transmission paths. Second, we have limited our model to consider only person-to-person respiratory droplet transmission of the virus. However, aerosol and fomite transmission are also plausible, as SARS-CoV-2 can survive in aerosols for several hours and on surfaces for several days [Our work is subject to a few limitations. First, although we have explored interventions that reduce HCP-HCP contacts and patient-patient contacts, we did not explore interventions that reduce or modify HCP-patient contacts. Since most of these result from direct patient care activity, it would not be reasonable to expect a uniform reduction in these contacts as we did for the other types of contacts. We could, however, consider reorganizing dialysis schedules so as to reduce the diversity of HCP-patient contacts by, ral days . Detaileral days . Our papS1 AppendixR0 = 3.0, and the exp/exp (60%) shedding model). Here, in the Appendix, we show results on other settings of the parameters as follows:exp/exp (60%) shedding model, R0 ∈ {2.0, 2.5}exp/exp (60%) shedding model, R0 = 3.0, γ ∈ {1.5, 2.5}exp/exp (20%) shedding model, R0 = 3.0Additional analyses on voluntary self-isolationexp/exp (60%) shedding model, R0 = 3.0, on the other days of dataR0 = 2.0, and the exp/exp (60%)Fig A. Cumulative distributions of transmission events over 30 days in Baseline simulation on . (A) Scenario 1: dialysis patient is the infection source. (B) Scenario 2: HCP is the infection source. R0 = 2.0, and the exp/exp (60%)Fig B. Frequency of replicates as a function of attack rates for different NPIs on . (A) Scenario 1. (B) Scenario 2. R0 = 2.0, exp/exp (60%)Fig C. Attack rates for individual NPIs and selected NPIs in combination in Scenario 1 , . (A) Voluntary self-isolation vs. active syndromic surveillance and compulsory isolation. (B) Use of masks and respirators. (C) Improved social distancing among HCPs. (D) Increased physical separation of dialysis stations. (E) Patient isolation and preemptive isolation of exposed HCPs. (F) Combinations of inexpensive NPIs. R0 = 2.0, exp/exp (60%)Fig D. Attack rates for individual NPIs and selected NPIs in combination in Scenario 1 (infection source: HCP), . (A) Voluntary self-isolation vs. active syndromic surveillance and compulsory isolation. (B) Use of masks and respirators. (C) Improved social distancing among HCPs. (D) Increased physical separation of dialysis stations. (E) Patient isolation and preemptive isolation of exposed HCPs. (F) Combinations of inexpensive NPIs. R0 = 2.5, and the exp/exp (60%)Fig E. Cumulative distributions of transmission events over 30 days in Baseline simulation on . (A) Scenario 1: dialysis patient is the infection source. (B) Scenario 2: HCP is the infection source. R0 = 2.5, and the exp/exp (60%)Fig F. Frequency of replicates as a function of attack rates for different NPIs on . (A) Scenario 1. (B) Scenario 2. R0 = 2.5, exp/exp (60%)Fig G. Attack rates for individual NPIs and selected NPIs in combination in Scenario 1 , . (A) Voluntary self-isolation vs. active syndromic surveillance and compulsory isolation. (B) Use of masks and respirators. (C) Improved social distancing among HCPs. (D) Increased physical separation of dialysis stations. (E) Patient isolation and preemptive isolation of exposed HCPs. (F) Combinations of inexpensive NPIs. R0 = 2.5, exp/exp (60%)Fig H. Attack rates for individual NPIs and selected NPIs in combination in Scenario 1 (infection source: HCP), . (A) Voluntary self-isolation vs. active syndromic surveillance and compulsory isolation. (B) Use of masks and respirators. (C) Improved social distancing among HCPs. (D) Increased physical separation of dialysis stations. (E) Patient isolation and preemptive isolation of exposed HCPs. (F) Combinations of inexpensive NPIs. Fig I. Viral shedding models. The bar charts show the shedding level of an infected individual whose presymptomatic period and symptomatic period are 6 and 7 days, respectively. The percentage in the parenthesis of each model corresponds to the percentage of shedding during the presymptomatic state (P) relative to the total shedding volume. The peak shedding level differs in the two models, but the total shedding volume remains the same. The shedding models are calibrated by setting the shedding ramp down parameter of the exp/exp (20%) shedding model γ = 1.5. R0 = 3.0, and the exp/exp (60%) shedding model where γ = 1.5Fig J. Cumulative distributions of transmission events over 30 days in Baseline simulation on . (A) Scenario 1: dialysis patient is the infection source. (B) Scenario 2: HCP is the infection source. R0 = 3.0, and the exp/exp (60%) shedding model where γ = 1.5Fig K. Frequency of replicates as a function of attack rates for different NPIs on . (A) Scenario 1. (B) Scenario 2. R0 = 3.0, exp/exp (60%), where γ = 1.5Fig L. Attack rates for individual NPIs and selected NPIs in combination in Scenario 1 , . (A) Voluntary self-isolation vs. active syndromic surveillance and compulsory isolation. (B) Use of masks and respirators. (C) Improved social distancing among HCPs. (D) Increased physical separation of dialysis stations. (E) Patient isolation and preemptive isolation of exposed HCPs. (F) Combinations of inexpensive NPIs. R0 = 3.0, exp/exp (60%), where γ = 1.5Fig M. Attack rates for individual NPIs and selected NPIs in combination in Scenario 1 (infection source: HCP), . (A) Voluntary self-isolation vs. active syndromic surveillance and compulsory isolation. (B) Use of masks and respirators. (C) Improved social distancing among HCPs. (D) Increased physical separation of dialysis stations. (E) Patient isolation and preemptive isolation of exposed HCPs. (F) Combinations of inexpensive NPIs. Fig N. Viral shedding models. The bar charts show the shedding level of an infected individual whose presymptomatic period and symptomatic period are 6 and 7 days, respectively. The percentage in the parenthesis of each model corresponds to the percentage of shedding during the presymptomatic state (P) relative to the total shedding volume. The peak shedding level differs in the two models, but the total shedding volume remains the same. The shedding models are calibrated by setting the shedding ramp down parameter of the exp/exp (20%) shedding model γ = 2.5. R0 = 3.0, and the exp/exp (60%) shedding model where γ = 2.5Fig O. Cumulative distributions of transmission events over 30 days in Baseline simulation on . (A) Scenario 1: dialysis patient is the infection source. (B) Scenario 2: HCP is the infection source. R0 = 3.0, and the exp/exp (60%) shedding model where γ = 2.5Fig P. Frequency of replicates as a function of attack rates for different NPIs on . (A) Scenario 1. (B) Scenario 2. R0 = 3.0, exp/exp (60%), where γ = 2.5Fig Q. Attack rates for individual NPIs and selected NPIs in combination in Scenario 1 , . (A) Voluntary self-isolation vs. active syndromic surveillance and compulsory isolation. (B) Use of masks and respirators. (C) Improved social distancing among HCPs. (D) Increased physical separation of dialysis stations. (E) Patient isolation and preemptive isolation of exposed HCPs. (F) Combinations of inexpensive NPIs. R0 = 3.0, exp/exp (60%), where γ = 2.5Fig R. Attack rates for individual NPIs and selected NPIs in combination in Scenario 1 (infection source: HCP), . (A) Voluntary self-isolation vs. active syndromic surveillance and compulsory isolation. (B) Use of masks and respirators. (C) Improved social distancing among HCPs. (D) Increased physical separation of dialysis stations. (E) Patient isolation and preemptive isolation of exposed HCPs. (F) Combinations of inexpensive NPIs. R0 = 3.0, and the exp/exp (20%) shedding modelFig S. Cumulative distributions of transmission events over 30 days in Baseline simulation on . (A) Scenario 1: dialysis patient is the infection source. (B) Scenario 2: HCP is the infection source. R0 = 3.0, and the exp/exp (20%) shedding modelFig T. Frequency of replicates as a function of attack rates for different NPIs on . (A) Scenario 1. (B) Scenario 2. R0 = 3.0, exp/exp (20%)Fig U. Attack rates for individual NPIs and selected NPIs in combination in Scenario 1 , . (A) Voluntary self-isolation vs. active syndromic surveillance and compulsory isolation. (B) Use of masks and respirators. (C) Improved social distancing among HCPs. (D) Increased physical separation of dialysis stations. (E) Patient isolation and preemptive isolation of exposed HCPs. (F) Combinations of inexpensive NPIs. R0 = 3.0, exp/exp (20%)Fig V. Attack rates for individual NPIs and selected NPIs in combination in Scenario 1 (infection source: HCP), . (A) Voluntary self-isolation vs. active syndromic surveillance and compulsory isolation. (B) Use of masks and respirators. (C) Improved social distancing among HCPs. (D) Increased physical separation of dialysis stations. (E) Patient isolation and preemptive isolation of exposed HCPs. (F) Combinations of inexpensive NPIs. G on Day 2Fig W. Contact network . We gathered ten days of HCP movement and interaction data where six days had 14.5-15 hours of observation . HCP nodes, MWF patient nodes, and TThS patient nodes are depicted in blue, chocolate, and burlywood colors, respectively. We observe a total population of 42 agents (14 HCPs and 28 patients). Contacts within the same type of nodes are represented as edges colored according to node type. Grey colored edges represent HCP-patient contacts. The thickness of the edge corresponds to the contact duration. R0 = 3.0Fig X. Cumulative distributions of transmission events over 30 days in Baseline simulation on Day 2 and . (A) the exp/exp (20%) shedding model on Scenario 1. (B) the exp/exp (20%) shedding model on Scenario 2. (C) the exp/exp (60%) shedding model on Scenario 1. (D) the exp/exp (60%) shedding model on Scenario 2. R0 = 3.0Fig Y. Attack rates for combinations of inexpensive NPIs on Day 2, . (A) the exp/exp (20%) shedding model on Scenario 1. (B) the exp/exp (20%) shedding model on Scenario 2. (C) the exp/exp (60%) shedding model on Scenario 1. (D) the exp/exp (60%) shedding model on Scenario 2. R0 = 3.0Fig Z. Frequency of replicates as a function of attack rates for combinations of inexpensive NPIs on Day 2, . (A) the exp/exp (20%) shedding model on Scenario 1. (B) the exp/exp (20%) shedding model on Scenario 2. (C) the exp/exp (60%) shedding model on Scenario 1. (D) the exp/exp (60%) shedding model on Scenario 2. G on Day 6Fig AA. Contact network . We gathered ten days of HCP movement and interaction data where six days had 14.5-15 hours of observation . HCP nodes, MWF patient nodes, and TThS patient nodes are depicted in blue, chocolate, and burlywood colors, respectively. We observe a total population of 47 agents (13 HCPs and 34 patients). R0 = 3.0Fig AB. Cumulative distributions of transmission events over 30 days in Baseline simulation on Day 6 and . (A) the exp/exp (20%) shedding model on Scenario 1. (B) the exp/exp (20%) shedding model on Scenario 2. (C) the exp/exp (60%) shedding model on Scenario 1. (D) the exp/exp (60%) shedding model on Scenario 2. R0 = 3.0Fig AC. Attack rates for combinations of inexpensive NPIs on Day 6, . (A) the exp/exp (20%) shedding model on Scenario 1. (B) the exp/exp (20%) shedding model on Scenario 2. (C) the exp/exp (60%) shedding model on Scenario 1. (D) the exp/exp (60%) shedding model on Scenario 2. R0 = 3.0Fig AD. Frequency of replicates as a function of attack rates for combinations of inexpensive NPIs on Day 6, . (A) the exp/exp (20%) shedding model on Scenario 1. (B) the exp/exp (20%) shedding model on Scenario 2. (C) the exp/exp (60%) shedding model on Scenario 1. (D) the exp/exp (60%) shedding model on Scenario 2. G on Day 7Fig AE. Contact network . We gathered ten days of HCP movement and interaction data where six days had 14.5-15 hours of observation . HCP nodes, MWF patient nodes, and TThS patient nodes are depicted in blue, chocolate, and burlywood colors, respectively. We observe a total population of 46 agents (10 HCPs and 36 patients). R0 = 3.0Fig AF. Cumulative distributions of transmission events over 30 days in Baseline simulation on Day 7 and . (A) the exp/exp (20%) shedding model on Scenario 1. (B) the exp/exp (20%) shedding model on Scenario 2. (C) the exp/exp (60%) shedding model on Scenario 1. (D) the exp/exp (60%) shedding model on Scenario 2. R0 = 3.0Fig AG. Attack rates for combinations of inexpensive NPIs on Day 7, . (A) the exp/exp (20%) shedding model on Scenario 1. (B) the exp/exp (20%) shedding model on Scenario 2. (C) the exp/exp (60%) shedding model on Scenario 1. (D) the exp/exp (60%) shedding model on Scenario 2. R0 = 3.0Fig AH. Frequency of replicates as a function of attack rates for combinations of inexpensive NPIs on Day 7, . (A) the exp/exp (20%) shedding model on Scenario 1. (B) the exp/exp (20%) shedding model on Scenario 2. (C) the exp/exp (60%) shedding model on Scenario 1. (D) the exp/exp (60%) shedding model on Scenario 2. G on Day 8Fig AI. Contact network . We gathered ten days of HCP movement and interaction data where six days had 14.5-15 hours of observation . HCP nodes, MWF patient nodes, and TThS patient nodes are depicted in blue, chocolate, and burlywood colors, respectively. We observe a total population of 45 agents (11 HCPs and 34 patients). R0 = 3.0Fig AJ. Cumulative distributions of transmission events over 30 days in Baseline simulation on Day 8 and . (A) the exp/exp (20%) shedding model on Scenario 1. (B) the exp/exp (20%) shedding model on Scenario 2. (C) the exp/exp (60%) shedding model on Scenario 1. (D) the exp/exp (60%) shedding model on Scenario 2. R0 = 3.0Fig AK. Attack rates for combinations of inexpensive NPIs on Day 8, . (A) the exp/exp (20%) shedding model on Scenario 1. (B) the exp/exp (20%) shedding model on Scenario 2. (C) the exp/exp (60%) shedding model on Scenario 1. (D) the exp/exp (60%) shedding model on Scenario 2. R0 = 3.0Fig AL. Frequency of replicates as a function of attack rates for combinations of inexpensive NPIs on Day 8, . (A) the exp/exp (20%) shedding model on Scenario 1. (B) the exp/exp (20%) shedding model on Scenario 2. (C) the exp/exp (60%) shedding model on Scenario 1. (D) the exp/exp (60%) shedding model on Scenario 2. G on Day 9Fig AM. Contact network . We gathered ten days of HCP movement and interaction data where six days had 14.5-15 hours of observation . HCP nodes, MWF patient nodes, and TThS patient nodes are depicted in blue, chocolate, and burlywood colors, respectively. We observe a total population of 39 agents (13 HCPs and 26 patients). R0 = 3.0Fig AN. Cumulative distributions of transmission events over 30 days in Baseline simulation on Day 9 and . (A) the exp/exp (20%) shedding model on Scenario 1. (B) the exp/exp (20%) shedding model on Scenario 2. (C) the exp/exp (60%) shedding model on Scenario 1. (D) the exp/exp (60%) shedding model on Scenario 2. R0 = 3.0Fig AO. Attack rates for combinations of inexpensive NPIs on Day 9, . (A) the exp/exp (20%) shedding model on Scenario 1. (B) the exp/exp (20%) shedding model on Scenario 2. (C) the exp/exp (60%) shedding model on Scenario 1. (D) the exp/exp (60%) shedding model on Scenario 2. R0 = 3.0Fig AP. Frequency of replicates as a function of attack rates for combinations of inexpensive NPIs on Day 9, . (A) the exp/exp (20%) shedding model on Scenario 1. (B) the exp/exp (20%) shedding model on Scenario 2. (C) the exp/exp (60%) shedding model on Scenario 1. (D) the exp/exp (60%) shedding model on Scenario 2. rVI∈ {0.4, 0.5, 0.6, 0.7, 0.8} under different modeling assumptionsTable A. Attack rates for voluntary isolation compliance rate . “SM” is short for “shedding models”, “B” is short for “Baseline,” in Scenario 1 the infection source is a dialysis patient and in Scenario 2 the infection source is a HCP.In the paper, we showed simulation results focusing on the worst case modeling assumptions Click here for additional data file.


Moreover, we established a rat needle puncture model and injected mangiferin into the intervertebral discs to verify its protective effect on IVDD. Furthermore, the activity of the NF-κB signaling pathway was tested in vitro. Our results indicated that mangiferin alleviated the inflammatory response and reversed the loss of major intervertebral disc components. Besides, mangiferin reduced reactive oxygen species production, ameliorated mitochondrial damage, and decreased the expression of apoptosis-related parameters in stimulation of TNF-α. In addition, mangiferin antagonized the activation of the NF-κB signaling pathway induced by TNF-α. Collectively, mangiferin antagonized mitochondrial ROS in NP cells and protected against IVDD by suppressing the activation of the NF-κB signaling pathway, which might provide a potential therapeutic instrument for IVDD.Intervertebral disc degeneration (IVDD), one of the most common clinical diseases worldwide, causes disc herniation and sciatica. Recent studies have identified the involvement of mitochondrial dysfunction, inflammatory responses, and extracellular matrix degradation in IVDD. Mangiferin is known to protect against various diseases by inhibiting oxidative stress, suppressing inflammation reaction, and relieving mitochondrial dysfunction. Whether mangiferin can alleviate IVDD remains to be elucidated. In the present study, human nucleus pulposus cells (HNPCs) and mouse intervertebral discs were cultured and stimulated with TNF- AccordiVD cells –8. Mitocα is a critical proinflammatory molecule involved in IVDD. TNF-α leads to a reduction in aggrecan and type II collagen (Col-2) levels and promotes the degradation of the extracellular matrix (ECM) by inducing the production of matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) [α is known to promote ROS production and the release of various inflammatory cytokines, which potentially enhance cell apoptosis and senescence [α ultimately lead to the occurrence and development of IVDD.Inflammation is reported to be a major contributor to IVDD . TNF-α iADAMTSs) –13. Morenescence . These pβ-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone), which has a C-glucosyl linkage with multiple -OH groups, possesses strong free radical-scavenging activity [Mangiferin  with IVDD  who underwent lumbar spine surgery at Qilu Hospital of Shandong University, Jinan, China. The degree of IVDD was assessed using the modified Pfirrmann grading system . All pat2 and 95% air at pH 7.2. The culture medium was replaced once every 3 days. All HNPCs were passaged when they reached approximately 80–90% confluence, and the indicated experiments were subsequently conducted.We collected the human IVD samples during spine surgeries. All procedures involved in the isolation and culturing of primary HNPCs were performed as previously reported . We wash6 cells/ml). Then, the cells were treated with PBS, TNF-α (20 ng/ml) , TNF − α (20 ng/ml) + mangiferin (100 μM/ml) , and TNF − α (20 ng/ml) + mangiferin (500 μM/ml).HNPCs were seeded in a 6-well plate (1 × 10n = 7) or TNF-α  in the presence or absence of mangiferin  for 7 days [All surgical procedures were performed under sterile conditions. The specimens of mouse spines were obtained from 10-week-old mice. After complete dissection of the spine and removal of the surrounding soft tissues, the lumbar spine was cut to a length of 5 mm while maintaining the integrity of the IVD. The lumbar spines of the mice were soaked in 100 U/ml penicillin/streptomycin for 30 minutes. The IVD tissues were incubated and stimulated with PBS (r 7 days . All IVDμl of PBS (n = 5) with or without 0.2 μg of mangiferin  was delivered into the punctured disc through an intradiscal injection via a 33-gauge Hamilton syringe  3 days after puncture.Three-month-old Sprague-Dawley rats were purchased from the Animal Center of Shandong University. All animals were housed under controlled identical, standard specific pathogen-free (SPF) environmental conditions  with free access to food and allowed to move freely. Local administration of mangiferin into the rat caudal disc via puncture was performed as previously reported . Brieflyα and different concentrations of mangiferin. A cDNA synthesis kit  was used to reverse transcribe identical amounts of RNA into cDNAs according to the manufacturer's instructions. Real-time PCR was performed with SYBR Green PCR Master Mix  on a thermal cycler ; the results were normalized to GAPDH. The specific PCR products for each gene were confirmed by performing a melting curve analysis. We used the mean from the control group as value 1. The relative gene expression levels were calculated using the formula 2ΔΔCt−. The sequence-specific primers used in the present study are listed in HNPCs were cultured for 1 or 24 h using the methods described above to detect different indicators . TRIzol κBα , rabbit anti-phosphorylated IκBα (pIκBα) , rabbit anti-p65 , and rabbit anti-phosphorylated p65 (pp65) . PVDF membranes were also incubated with a rabbit anti-GAPDH antibody  to ensure equal protein loading. After washes with Tris-buffered saline- (TBS-) Tween 20, the membranes were then incubated with the secondary antibody  for 1 h, and the immunolabeled bands were detected using a Fluor Chem E Chemiluminescent Western Blot Imaging System . The data were analyzed using the ImageJ software .HNPCs were cultured for 1 or 48 h using the methods described above to detect different indicators by Western blotting . Protein2; slice thickness: 1.4 mm) in the coronal plane. All the radiographic images were saved in a Neusoft PACS/RIS DICOM 3.0 medical imaging system . The IVD height and the adjacent upper and lower vertebral body heights were measured using Neusoft PACS/RIS measuring tools, and the disc height index (DHI) was calculated from these values.Seven days after the initial puncture, each group of rats was randomly selected to undergo X-ray and MRI scans before sacrifice . The ratμm sections. Trypsin  was used for antigen retrieval, and 3% hydrogen peroxide was used to eliminate endogenous peroxidase activity. All primary antibodies were diluted in 1× antibody dilution buffer  and 0.01% Proclean ), and the serial sections were incubated with primary antibodies  at 4°C for 12 h. Then, the sections were incubated with goat anti-rabbit and anti-mouse immunoglobulin- (IgG-) horseradish peroxidase (HRP) secondary antibodies  for 60 minutes at room temperature. HE staining was performed according to the manufacturer's protocol. An IX71-SIF microscope  was used to obtain the images.The IVD tissues collected from mice and rats in each treatment group were immersed in 4% paraformaldehyde for 72 h and then subjected to decalcification, after which they were embedded in paraffin and cut into 5 α (20 ng/ml), TNF − α (20 ng/ml) + mangiferin (100 μM/ml), or TNF − α (20 ng/ml) + mangiferin (500 μM/ml) for 48 h [α (20 ng/ml) + mangiferin (100 μM/ml), or TNF − α (20 ng/ml) + mangiferin (500 μM/ml) for 1 h, and the level of the NF-κB pp65 protein was detected. The cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.2% Triton X-100 for 15 minutes, and blocked with 1% BSA for 30 minutes at room temperature. Then, the cells were incubated with rabbit anti-iNOS , rabbit anti-COX-2, rabbit anti-MMP-13, rabbit anti-collagen-II , rabbit anti-NF-κB p65 , and rabbit anti-NF-κB pp65  primary antibodies for 12 h at 4°C. Afterwards, the cells were incubated with a fluorescently labeled goat anti-rabbit immunoglobulin (IgG) secondary antibody  for 1 h at room temperature.HNPCs were seeded on cell coverslips and cultured with PBS, TNF-for 48 h  before tTrypsin  was used for antigen repair of IVD tissues in mice, and 3% hydrogen peroxide was used to eliminate endogenous peroxidase activity. All primary antibodies were diluted in 1× antibody dilution buffer  and 0.01% Proclean ), and the mouse IVD tissues were incubated with primary antibodies  at 4°C for 12 h. Then, the tissues were incubated with a fluorescently labeled goat anti-rabbit IgG secondary antibody  for 1 h at room temperature. The images were captured using a fluorescence microscope .Safranin O and fast green staining were performed to determine changes in proteoglycans. The mouse and rat IVD sections were stained with a Safranin O kit  according to the manufacturer's recommended procedure. An IX71-SIF microscope  was used to capture the images.TUNEL staining of mouse IVD tissues from each treatment group cultured as described above for 7 days and HNPCs from each treatment group cultured as described above for 24 h  was perfHNPCs were cultured as described above for 24 h . A JC-1 μM DCFDA at 37°C for 20 minutes in the dark and mixed every 15 minutes. Then, the HNPCs were washed three times with serum-free culture medium to reduce interference from excess DCFDA. The images were captured using a fluorescence microscope .HNPCs were cultured as described above for 24 h . We usedHNPCs were cultured according to the above method for 24 h , and MitHNPCs were dehydrated in a graded series of ethanol solutions  for 15 minutes per solution and infiltrated with propylene oxide embedding medium overnight. Ultrathin sections (50 nm) were obtained using an EMUC7 ultramicrotome (Leica), poststained with uranyl acetate and lead citrate and visualized using a transmission electron microscope .P < 0.05 was considered statistically significant.All data are presented as the mean values ± standard deviations (SD) and were analyzed using the GraphPad Prism v.5.0 software . Comparisons of various treatment groups were performed using analysis of variance (ANOVA) with Tukey's post hoc test. α (20 ng/ml), TNF − α (20 ng/ml) + mangiferin (100 μM/ml), or TNF − α (20 ng/ml) + mangiferin (500 μM/ml) for 24 h to detect the mRNA level and 48 h to detect the protein level. After treatment, mRNA was extracted from the HNPCs, followed by real-time PCR. As indicated in Figures α stimulation, the expression levels of COX-2 and iNOS were increased by 2.74 and 29.31 times compared with the PBS group. Coculture with different concentrations of mangiferin (100 μM/ml and 500 μM/ml), the expression of these cytokines decreased by 82.16% and 83.75% and 97.19% and 97.52%, respectively, compared with the TNF-α group. Next, total protein was extracted from treated HNPCs, and Western blotting was performed to detect the expression levels of COX-2 and iNOS. As shown in Figures α group, and the protein levels of COX-2 and iNOS in the TNF-α group were 3.91 and 2.62 times higher than those in the PBS group. After treatment with different concentrations of mangiferin, the expression levels of these inflammatory cytokines decreased in a dose-dependent manner, the levels of COX-2 and iNOS were reduced by 51.58% and 90.94% and 35.74% and 84.75%, respectively. As shown in Figures α group were 11.38 and 13.57 times higher than those in the PBS group, respectively. The mean fluorescence intensities of COX-2 and iNOS in the mangiferin (100 μM/ml) and mangiferin (500 μM/ml) groups decreased by 57.52% and 65.72% and 54.78% and 78.20%, respectively, compared with those in the TNF-α group. Therefore, mangiferin reduced the TNF-α-induced increases in the expression levels of COX-2 and iNOS, which alleviates the inflammatory response during the process of IVDD.Previous research has shown that increased expression of inflammatory cytokines is an important factor leading to IVDD. Therefore, we investigated the role of mangiferin in the progression of IVDD by culturing HNPCs with PBS, TNF-α promotes the expression of ADAMTSs and MMPs, leading to changes in the synthesis of ECM molecules and increased ECM degradation, the increased expression of MMP-13 and ADAMTS-5 in degenerative IVD tissues results in decreases in Col-2 and aggrecan levels [α (20 ng/ml), TNF − α (20 ng/ml) + mangiferin (100 μM/ml), or TNF − α (20 ng/ml) + mangiferin (500 μM/ml) 24 h to detect the mRNA level and 48 h to detect the protein level. As shown in Figures α group than those in PBS group, while the production of Col-2 and aggrecan gradually increased by 1.48 and 1.87 and 1.51 and 3.47 times after treatment with different concentrations of mangiferin. As shown in Figures α stimulation were 4.07 and 4.25 times higher than those in PBS group, respectively. Compared with the TNF-α group, the expression levels of these cytokines gradually decreased by 63.94% and 89.44% and 39.64% and 64.83% after treatment with 100 μM/ml and 500 μM/ml mangiferin. We extracted total proteins from HNPCs after cultivation with TNF-α and mangiferin to further explore the role of mangiferin in inhibiting the degeneration of HNPCs. As shown in Figures α-stimulated HNPCs than in HNPCs treated with PBS alone, while lower expression levels of aggrecan and Col-2 were observed in TNF-α-stimulated HNPCs than in PBS-treated HNPCs. The expression levels of MMP-13 and ADAMTS-5 were significantly decreased in response to the mangiferin treatment compared with those in the TNF-α group, while the expression levels of aggrecan gradually increased. Compared with TNF-α group, the expression of Col-2 was not increased after 100 μM/ml of mangiferin treatment, while the expression of Col-2 was increased after 500 μM/ml of mangiferin treatment. These results were verified by IF staining, as shown in Figures α group were 2.73 times higher than those in the PBS group, and the expression levels of Col-2 in the TNF-α group decreased by 63% than those in the PBS group. The mean fluorescence intensities of MMP-13 in the mangiferin (100 μM/ml) and mangiferin (500 μM/ml) groups decreased by 59.19% and 60.10% compared with those in the TNF-α group, and the expression levels of Col-2 in the mangiferin (100 μM/ml) and mangiferin (500 μM/ml) groups were 1.02 and 2.05 times higher than those in the TNF-α group. Taken together, the experimental results shown in Figures As TNF-n levels , 29. Theα (20 ng/ml), TNF − α (20 ng/ml) + mangiferin (100 μM/ml), or TNF − α (20 ng/ml) + mangiferin (500 μM/ml) to verify whether mangiferin inhibits mitochondrial dysfunction caused by TNF-α-induced oxidative stress in HNPCs. We used TEM to observe the mitochondrial morphology of HNPCs. As shown in α. However, treatment with different concentrations of mangiferin gradually reversed this phenomenon, which may prove that mangiferin plays a role in protecting against mitochondrial damage in HNPCs. OPA1, Drp1, and TFAM are biomarkers of mitochondrial morphology and function [α can be alleviated after treatment with mangiferin. As shown in Figures α-induced mitochondrial damage in HNPCs. According to previous studies, ROS production increases in degenerative IVD tissue and forms a positive feedback loop that further promotes the production of ROS. In addition, mitochondria are the main target of ROS attack, and inhibiting the production of ROS may become an important part of IVDD treatment. As shown in Figures α group were 11.38 times higher than those in the PBS group. The DCFDA indicated that the expression levels of ROS in the mangiferin (100 μM/ml) and mangiferin (500 μM/ml) groups decreased by 80.52% and 91.66%, respectively, compared with those in the TNF-α group.We cultured HNPCs with PBS, TNF-function , 31, as α group, after treatment with mangiferin at different concentrations, the mRNA expression levels of Bax and c-caspase-3 decreased by 72.30% and 78.53% and 82.16% and 83.75%, respectively, and increased the expression of Bcl-2 by 1.25 and 1.82 times, respectively. Consistent with the trend of mRNA, mangiferin can significantly decrease the protein levels of Bax and c-caspase-3, increase the protein level of Bcl-2, and play a protective role in the inhibition of apoptosis. As shown in Figures α increased the number of TUNEL-positive HNPCs compared with the number detected in HNPCs treated with PBS alone, but different concentrations of mangiferin prevented the TNF-α-induced increase in the number of TUNEL-positive HNPCs; furthermore, Hoechst 33342 staining also proved that mangiferin inhibited the apoptosis of HNPCs caused by TNF-α.HNPC apoptosis has been shown to play a pivotal role in the degeneration of the IVD, over the past few years, and apoptosis in the IVD has become a focus area of research . Relevanα (20 ng/ml), TNF − α (20 ng/ml) + mangiferin (100 μM/ml), or TNF − α (20 ng/ml) + mangiferin (500 μM/ml) for 7 days. Total protein was extracted from the cultured mouse IVD tissues for Western blot analysis, as shown in Figures α group. After treatment with different concentrations of mangiferin, the expression of COX-2, iNOS, ADAMTS-5, and MMP-13 decreased by 28.22%, 18.16%, 24.84%, and 18.49% and 42.47%, 46.49%, 33.16%, and 29.61, respectively. As shown in Figures α-stimulated IVDs compared with IVDs treated with PBS alone. After treatment with two different concentrations of mangiferin, the levels of these inflammatory cytokines decreased in a dose-dependent pattern, and mangiferin mitigated the TNF-α-induced loss of aggrecan and Col-2. Safranin O staining is a method used to detect proteoglycans and glycosaminoglycans. We used Safranin O staining to evaluate the degree of IVDD in mice and verify the role of mangiferin in inhibiting IVDD. As shown in α group compared with the control group. After treatment with different concentrations of mangiferin, the proteoglycans in the IVD group were gradually increased compared with the TNF-α group. In Figures α group. Compared with TNF-α group, the expression of MMP-13 and COX-2 decreased by 49.46% and 48.04% and 66.68% and 74.61%, respectively, after treatment with different concentrations of mangiferin. As illustrated in Figures α group. After treatment with 100 μM/ml and 500 μM/ml mangiferin, the level of Bcl-2 was increased by 1.41 and 1.74 times, and the levels of Bax and c-caspase-3 were decreased by 25.33% and 33.63% and 22.92% and 27.80, respectively. As illustrated in Figures α group, and the number of TUNEL-positive cells gradually decreased in response to the mangiferin treatment. Based on these results, mangiferin might protect against IVDD by reducing the apoptosis of HNPCs.We cultured mouse IVDs from the upper and lower vertebral bodies in vitro with PBS, TNF-We established a rat IVD needle puncture model and injected mangiferin into the rat IVD to verify that mangiferin inhibits IVDD in rats. As shown in Figures κB signaling pathway is known to play an important role in the development of IVDD. We stimulated HNPCs with PBS, TNF-α (20 ng/ml), TNF − α (20 ng/ml) + mangiferin (100 μM/ml), and TNF − α (20 ng/ml) + mangiferin (500 μM/ml) for 1 h and extracted total mRNA and total protein for analysis to explore whether mangiferin protects against IVDD by inhibiting the NF-κB signaling pathway. Notably, pIκBα and p65 are important parameters used to measure the activation of the NF-κB signaling pathway [κB1 increased 1.70 times after stimulation with TNF-α for 1 h, whereas the expression level of NF-κB1 decreased by 42.15% and 51.12% after treatment with different concentrations of mangiferin. As shown in Figures κBα/IκBα and pp65/P65 increased after treatment with TNF-α, whereas their levels were decreased after treatment with different concentrations of mangiferin. In addition, the IF staining shown in Figures α-induced increase in the level of pp65 was abolished by treatment with mangiferin. As shown in Figures α-induced p65 nuclear translocation.Activation of the NF- pathway . As showα has been widely studied and shown to facilitate the development of IVDD. As mentioned above, TNF-α promotes the production of various inflammatory cytokines and causes ECM degradation, leading to increased catabolism of the IVD [α-induced inflammatory response model of HNPCs to explore the role of mangiferin in inhibiting IVDD and to investigate the mechanism of this effect. The expression levels of COX-2 and iNOS secreted by HNPCs were significantly elevated after treatment with TNF-α compared with those in the PBS group; consistent with previous studies, it was demonstrated that TNF-α caused the inflammatory response of HNPCs and resulted in the production of important inflammatory cytokines that contribute to IVDD. Intriguingly, after treatment with mangiferin, the expression of these inflammatory cytokines decreased compared with the TNF-α group. Furthermore, TNF-α increased the expression of MMPs and ADAMTSs, which leads to the destruction of the ECM, an important aspect of the pathological process of IVDD [α markedly increased the expression of MMP-13 and ADAMTS-5, leading to a significant decrease in aggrecan and Col-2 expression, which are the main components of the IVD, implying a certain level of anabolism [α enhanced the expression level of the catabolic markers MMP-13 and ADAMTS-5 and diminished the expression levels of the anabolic markers aggrecan and Col-2 compared with the control group. The addition of mangiferin at different concentrations alleviated the loss of Col-2 and aggrecan induced by TNF-α and decreased the expression of MMP-13 and ADAMTS-5, indicating that mangiferin might protect against disorganization of anabolism in IVDD.Previous studies have confirmed that IVDD is mediated by the abnormal production of proinflammatory molecules secreted by various cell types –37; vari the IVD ; therefo of IVDD , 43. In nabolism . Previounabolism . In our α treatment, and the expression level of ROS was increased, which in turn led to inflammatory response of HNPCs and the damage of mitochondrial function, and mangiferin reduced these changes in mitochondria and ROS production. Moreover, mangiferin plays a protective role in mitochondrial function by affecting the expression of mitochondrial morphology and function-related indicators, thereby mitigating the process of IVDD by reducing mitochondrial dysfunction and ROS production. Apoptosis causes a significant decrease in the number of living and functional IVD cells, which results in increased expression of proinflammatory cytokines and exacerbates IVDD [α induces the apoptosis of NP cells, which significantly increases the apoptotic rate and senescence [α stimulation of HNPCs, we observed increased levels of Bax and c-caspase-3 and decreased levels of Bcl-2, and these results suggest that TNF-α could increase the expression of proapoptotic parameters and reduce the expression of antiapoptotic parameters and may be involved in mitochondrial apoptosis pathway. After treatment with mangiferin, the levels of Bax and c-caspase-3 decreased, and the expression of Bcl-2 increased. These results imply that mangiferin reduces the TNF-α-induced increase in the apoptosis rate of HNPCs, thus plays a role in relieving IVDD. To further validate our study, we further observed the effect of mangiferin on TNF-α-induced apoptosis of HNPCs by TUNEL and Hoechst 33342 staining. Compared with TNF-α stimulation group, mangiferin can alleviate the expression of inflammatory cytokines, reduce the loss of Col-2 and aggrecan, and alleviate the apoptosis of IVD in mice. Moreover, in vitro experiments further suggested that mangiferin can alleviate multiple pathological processes in IVDD.Previous studies reported the abnormal morphology and dysfunction of mitochondria in aging NP cells , and mittes IVDD . A largenescence , 44, 45.nescence . In our In the in vivo experiment using rats, compared with the needle puncture group, intradiscal injection of mangiferin can alleviate the loss of IVD height and IVD major contents and reduce the expression of inflammatory cytokines. Therefore, the results suggest that mangiferin might play a protective role in IVDD in vivo and in vitro.κB signaling pathway is involved in IVDD, activation of NF-κB signaling pathway can accelerate IVDD, while antagonism of NF-κB signaling pathway can alleviate the aging process [α is involved in various pathological processes related to IVDD, mainly by activating the NF-κB signaling pathway [κB1 expression caused by TNF-α. Levels of the pIκB and pp65 proteins, which are critical parameters for investigating the activity of the NF-κB signaling pathway [α. The levels of pIκB and pp65 were significantly reduced by mangiferin treatment. In addition, nuclear translocation of p65 determines the activity of NF-κB signaling pathway [κB signaling pathway.In addition, previous studies have reported that activation of the NF- process . TNF-α i pathway . The det pathway , also in pathway . MoreoveIn conclusion, mangiferin exerts a positive effect on alleviating IVDD by inhibiting the inflammatory response of HNPCs, increasing the loss of major components that contributes to the progression of IVDD, and preserving mitochondrial function by reducing ROS production, which reduces the expression of apoptosis-related proteins. In our future studies, we expect to develop mangiferin as a clinical treatment for IVDD.