Hierarchical heterostructures of two-dimensional (2D) nanomaterials are versatile platforms for nanoscale optoelectronics. Further coupling of these 2D materials with plasmonic nanostructures, especially in non-close-packed morphologies, imparts new metastructural properties such as increased photosensitivity as well as spectral selectivity and range. However, the integration of plasmonic nanoparticles with 2D materials has largely been limited to lithographic patterning and/or undefined deposition of metallic structures. Here we show that colloidally synthesized zero-dimensional (0D) gold nanoparticles of various sizes can be deterministically self-assembled in highly-ordered, anisotropic, non-close-packed, multi-scale morphologies with templates designed from instability-driven, deformed 2D nanomaterials. The anisotropic plasmonic coupling of the particle arrays exhibits emergent polarization-dependent absorbance in the visible to near-IR regions. Additionally, controllable metasurface arrays of nanoparticles by functionalization with varying polymer brushes modulate the plasmonic coupling between polarization dependent and independent assemblies. This self-assembly method shows potential for bottom-up nanomanufacturing of diverse optoelectronic components and can potentially be adapted to a wide array of nanoscale 0D, 1D, and 2D materials. Graphene’s zero-gap band structure with its unique linear energy–momentum relationship near the Dirac points yields a 2.3% wavelength-independent absorbance throughout the visible and near-IR ranges3. Such unique optoelectronic properties, especially for an atomically-thin material, have led to intensive research in graphene photonics including surface-enhanced Raman spectroscopy5, photocatalysis6, and photodetection8. Graphene-based devices such as photodetectors are superlative for their versatility across a wide range of wavelengths9. However, spectral and polarization specificity/sensitivity are often desired for applications in high speed and compact photonic devices11. As monolayer graphene does not inherently possess these properties, there is interest in hybrid photonic systems that introduce multifaceted optical responses to graphene-based metasurfaces.Graphene-based devices have emerged as potentially versatile components for nanoscale optoelectronics applications18. Gold nanostructure integration is a common approach for plasmonically controlling the optical response of host materials. Plasmonic nanostructures form enhanced, highly-localized electromagnetic fields under illumination and have been shown to enhance the photoabsorption of graphene to achieve 20 times higher photoresponsivity12. The geometry of the gold structures can determine the polarization selectivity of metasurfaces15, with the strongest enhancement occurring selectively at wavelengths matching with the SPR frequency of the nanostructures17.One way to increase properties such as efficiency and spectral selectivity of graphene-based metasurfaces is through coupling of the incident excitation with surface plasmon resonance (SPR) structures19. This results in poor control over the nanostructure geometry, with concomitant poor performance of the resulting metastructure and devices.To date, plasmonic nanostructure integration on graphene and other 2D materials has largely been achieved through pattern definition (e.g. lithography) and metallization . While precise, these conventional methods have limitations in scalability and geometric control, respectively. In contrast, plasmonic gold nanoparticles (AuNPs) can be colloidally synthesized with high yield and with a very large diversity in morphology . However, the potential of colloidal nanoparticles for solid state devices has been limited due to their tendency to form arbitrary, non-uniform, and/or close-packed aggregates upon assembly onto surfaces24. Controllable assembly depends on architectured templates with nanoscale features to confine particles of varying shapes and sizes as well as control over the surface coverage and interparticle interactions. Nanoscale trough-like features used for single particle alignment are achievable in 2D materials such as graphene through mechanical deformation. Such alteration of the surface topography of graphene through mechanical deformations has previously been studied to enhance its mechanical26 and optoelectronic27 properties. Buckle delamination on stretchable substrates results in uniaxially aligned crumples with a periodicity on the order of tens of nanometers29. Additionally, graphene can be conformally wrinkled on deformed elastomers with precise control over the wrinkle morphology, wavelength, and amplitude31. Mechanically deformed elastomer templates  have previously been used to assemble colloidal AuNPs32. To date, templated assembly of nanoparticles on deformed or architectured 2D materials has not yet been explored.One way that colloidal AuNPs have been integrated with substrates is through templated assembly, which allows for large-scale bottom-up geometric control of nanoparticle arraysHere we report a bottom-up approach for 2D/0D heterostructures with tunable anisotropic optical properties by using graphene templates which were fabricated via strain induced mechanical surface instabilities to deterministically assemble AuNPs. By controlling processing parameters, we designed deformed graphene templates with uniaxial buckle delaminated wrinkles with a periodicity of a few dozen nanometers, as well as aligned sinusoidal wrinkles spanning up to several hundred nanometers to match the dimensionalities of monodisperse AuNPs. The surface coating of the AuNPs was optimized for colloidal stability and uniformity under the deposition and assembly conditions and could be further altered to increase the steric repulsion between AuNPs and to enable further tunability to the metasurface optical properties. This novel method for self-assembly of nanomaterial heterostructures allows independent control over the various processing factors for each nanomaterial and resulting metastructure properties.28, and is outlined in Fig. Fabrication of buckle delaminated graphene substrates was adapted from previous work33. Several surface functionalizations were tested to optimize the AuNPs for alignment and resistance to aggregation. The as-synthesized citrate ligands on AuNPs served as a precursor for further particle functionalization, due to its high density of charge as well as potential for displacement for subsequent functionalization. The anionic charge was employed for the wrapping of the particles with positively charged poly (PAH) polyelectrolyte. Thiolated poly(ethylene glycol) (PEG) was also used to functionalize AuNPs with hydrophilic, neutrally charged polymer brushes.20 nm citrate capped AuNPs were synthesized by the Turkevich method34. Particles coated with PEG were found to align within the wrinkles and resist aggregation upon drying. The primarily steric stabilization of the PEG molecules also prevented aggregation that is prevalent for the particles stabilized electrostatically. PEG functionalization has the additional advantage of allowing for the removal of excess capping agents through repeated washing in water. Thorough washing of the particles was pivotal to prevent substrate fouling by organic residues which interfere with the uniformity of the self-assembly.As shown in Fig. While graphene crumples resulted in aligned AuNPs due to geometric confinement, there were several factors that presented challenges for large-scale uniform assembly and tunability. The high aspect ratio of the crumples often leads to substrate dewetting during the AuNP assembly process, resulting in striped patterns due to stick–slip behavior as seen in Fig. To access a broader range of characteristic length scales and to assemble AuNPs of various sizes, we adopted a conformal uniaxial wrinkling approach, whereby the 2D material (e.g. graphene) conforms to the corrugations of the underlying polymeric substrate. Since this approach is mechanistic in nature , the self-assembly process is material-agnostic and extendable to assembly onto arbitrary 2D materials and thin film templates.35 as well as the bonding to PDMS, a ~5 μm layer of PDMS elastomer was bonded to the PS by spin coating, followed by treatment with tens of seconds of O2 plasma at low power to create a rigid silica skin layer with a thickness of ~6–11 nm36. Tuning this silica layer thickness by increasing plasma time and power (dosage) allows for fine control of the final wrinkle dimensions due to the relative mismatch in plane-strain moduli between PDMS and plasma-induced silica skin layer37. Graphene is then transferred onto the silica/PDMS surface by standard wet transfer procedures using a thin film gold handle layer as mentioned previously. Conformal uniaxial wrinkling of the graphene was performed through the same shrinking procedure as for buckle delaminated graphene. This resulted in sinusoidal wrinkles with controlled wavelength and amplitude within which AuNPs could be self-assembled.Fabrication of the architectured 2D substrates is outlined in Fig. 38. The AuNPs were self-assembled onto the graphene templates  measurements  of dried particle samples shows an inverse relation between PEG grafting density and particle diameter  (> 99.9%), poly , isopropanol (ACS Plus grade), and 200 proof ethanol (HPLC grade) were purchased from Sigma-Aldrich. Sodium citrate dihydrate (lab grade) was purchased from Fischer Scientific. Methoxy-poly(ethylene glycol)-thiol  was purchased from Creative PEGWorks. Nanopure water (18 MΩ⋅m) was obtained from a Millipore system. All chemicals were used as received without further purification.Monolayer graphene on copper foil was purchased from Grolltex (SC6 × 6). Polystyrene sheets were purchased from Scientific Explorer-Poof Slinky (item number 0SD300-10A). TFA gold etchant was purchased from Transene Company, Inc. Polydimethylsiloxane (Sylgard 184), hydrogen tetrachloroaurate  was applied.Polystyrene (PS) sheets were ultrasonicated in deionized water for 10 min then repeated in isopropanol. For buckle delaminated graphene, the graphene was transferred directly onto the polystyrene. For conformal graphene wrinkling, Sylgard 184 PDMS with a mixing ratio of 7:1 was first spin-coated at 6000 rpm for 90 s onto the polystyrene surface and then cured together at 80 °C for 12 h. To ensure bonding between the PDMS and PS, the PS sheet was treated by oxygen plasma at 250 W for 10 s in a Plasma Therm 700 before spin coating. Depending on the target silica thickness, the PDMS was then subjected to 10–60 s of oxygen plasma at 20 W. Graphene with a 50 nm evaporated gold handle layer was transferred to the substrates (PDMS or PS) by standard wet transfer procedures. The handle layer was etched with a 20 mg/mL KI, 5 mg/mL I26. 500 mL nanopure H2O and 1.25 mL 0.1 M HAuCl4 were brought to a boil while stirring at 600 rpm. 2.5 mL 20% sodium citrate solution (m/m) was added rapidly, and the reaction was stirred for 30 min at just below boiling, after which an additional 0.625 mL of sodium citrate solution was added. After 10 min, the particles were cooled to room temperature, centrifuged at 9000 rcf, and washed once with nanopure water.20 nm AuNPs were prepared by the Turkevich method38. Gold seeds (~ 14 nm) were first prepared by the Turkevich method. For the growth solutions, 475 mL nanopure H2O and 1.27 mL 0.1 M HAuCl4 were combined in a flask followed by addition of gold seeds. The seed concentration used was 0.0619 nM for 50 nm AuNPs, and 0.0138 nM for 80 nm AuNPs. While stirring at 600 rpm, 110 μL of 10% sodium citrate (m/v) was added, followed by 5 mL 0.03 M hydroquinone. Particles were stirred 60 min more, then centrifuged at 3000 rcf and resuspended in nanopure water. AuNPs were characterized by TEM and UV–Vis spectroscopy.AuNPs (50 and 80 nm) were prepared by the seed-mediated method of Chan and coworkers2O at a concentration of 1 nM. 4 mL of 10 mg/mL poly (PAH) polymer and 2 mL of 0.01 M NaCl were mixed, then added to the AuNP solution. The mixture was incubated overnight, before centrifuging at 9000 rcf and discarding the supernatant containing excess polyelectrolyte. Particles were redispersed in 200 proof ethanol before deposition.For polyelectrolyte wrapping, 20 nm AuNPs were dispersed into 10 mL HFor mPEG-SH functionalization, aqueous AuNP solutions at a concentration of 1 nM were incubated in 0.2 mM mPEG-SH MW 5k or 1.6 mM mPEG-SH MW 40k overnight. Particles were transferred to clean centrifuge tubes and washed 6 × with nanopure water to remove all traces of organic residues. Particles were then pelleted once more and dispersed in 200 proof ethanol to the target concentration . To determine the hydrodynamic thickness of PEG layers, dynamic light scattering (DLS) measurements of PEG-coated AuNPs in ethanol were taken with a Malvern Zetasizer Nano ZS and the difference compared to citrate capped AuNPs in water was calculated. PEG grafting density was determined through thermogravimetric analysis of lyophilized particle samples (~ 10 mg each) with a TA instruments QA TGA running in air.2 wrinkled substrates. The deposition was performed to yield uniform wetting of the substrate with menisci pinned at the substrate edges. Samples were dried overnight in a saturated ethanolic atmosphere. Aligned AuNPs on graphene were characterized by SEM in a Hitachi S4800 Field Emission Scanning Electron Microscope (SEM) and Hitachi SU-70 SEM. Polarized UV–Vis measurements were performed with a Glan Thompson calcite polarizer in a Cary-5000 UV–Vis spectrometer.The target substrate was first prewet with 20 μL of 200 proof ethanol followed by drop casting of 20 μL of ethanolic mPEG-SH functionalized AuNP solutions onto 1 cm


Twitter data exhibits several dimensions worth exploring: a network dimension in the form of links between the users, textual content of the tweets posted, and a temporal dimension as the time-stamped sequence of tweets and their retweets. In the paper, we combine analyses along all three dimensions: temporal evolution of retweet networks and communities, contents in terms of hate speech, and discussion topics. We apply the methods to a comprehensive set of all Slovenian tweets collected in the years 2018–2020. We find that politics and ideology are the prevailing topics despite the emergence of the Covid-19 pandemic. These two topics also attract the highest proportion of unacceptable tweets. Through time, the membership of retweet communities changes, but their topic distribution remains remarkably stable. Some retweet communities are strongly linked by external retweet influence and form super-communities. The super-community membership closely corresponds to the topic distribution: communities from the same super-community are very similar by the topic distribution, and communities from different super-communities are quite different in terms of discussion topics. However, we also find that even communities from the same super-community differ considerably in the proportion of unacceptable tweets they post. Social media, and Twitter in particular, are widely used to study various social phenomena, see for example  . We then outline individual research methods applied. Network analysis is used to construct retweet networks, detect communities, and study their evolution through time (subsection Evolving retweet communities). Machine learning is applied to train and evaluate a hate speech classification model (subsection Hate speech classification). Methods of content analysis are used to detect topics discussed in the tweets (subsection Topic detection). In the next section, Results and discussion, we combine the results of individual methods to reveal some interesting insights gained from the collected Twitter data.For this study, we collected a set of almost 13 million Slovenian tweets in the 3 year period, from January 1, 2018 until December 28, 2020. The set represents an exhaustive collection of Twitter activities in Slovenia. The tweets were collected via the public Twitter API, using the TweetCaT tool  and 4,597,865 retweets .A retweet network is a directed graph. The nodes are Twitter users and the edges are retweet links between the users. An edge is directed from the user We form a sequence of network snapshots, with a sliding window of 1 week, to study the evolution of a retweet network. The snapshots are overlapping, where each snapshot comprises an observation window of 24 weeks (about 6 months). We employ an exponential edge weight decay, with half-time of 4 weeks, to eliminate the effects of the trailing end of a moving network snapshot. This provides a relatively high temporal resolution between subsequent networks, but we later select just the most relevant intermediate timepoints.The set of network snapshots thus consists of 133 overlapping observation windows, with temporal delay of 1 week. The snapshots start with a network at Informally, a network community is a subset of nodes more densely linked between themselves than with the nodes outside the community. A standard community detection method is the Louvain algorithm ,Build a new network where a pair of the original nodes is linked if their total Co-membership across all the Louvain trials is above a given threshold (90% by default),Identify the disjoints sets which then represent the detected communities.As a result of using Ensemble Louvain, nodes without a clear community membership  are isolated and excluded from further analyses. The resulting communities are of approximately the same size as produced by individual Louvain trials, but with drastically improved stability and reproducibility . The only non-political community is Sports. All the remaining, smaller communities, are represented as Rest.Hate speech classification is approached as a supervised machine learning problem. Supervised machine learning requires a large set of examples labeled for hate speech, and typically involves a considerable initial effort to produce such labeled examples. The labeled examples are then used to train classification models to distinguish between the examples of hate and normal speech ,Offensive—tweets include offensive generalization, contempt, dehumanization, or indirect offensive remarks,Violent—the author threatens, indulges, desires or calls for physical violence against a target; this also includes tweets calling for, denying or glorifying war crimes and crimes against humanity.During the annotation process, and for training the models, all four classes were considered. However, in this paper we take a more abstract view and distinguish just between the normal, acceptable speech, and the unacceptable speech, i.e., inappropriate, offensive or violent.The hate speech annotation schema is adapted from OLID , between the annotators and the models, and then compare if a model comes close to the inter-annotator agreement.Several machine learning algorithms were used to train hate speech classification models. First, three traditional algorithms were applied: Naïve Bayes, Logistic regression, and Support Vector Machine with a linear kernel. Second, deep neural networks, based on the Transformer language models, were applied. We used two multi-lingual language models, based on the BERT architecture . In subsection Evolution of offensive topics we focus on the three prevailing topics, and show the evolution of acceptable and unacceptable tweets posted by the top communities.The topic detection method we apply requires to set the number of topics in advance. We experimented with different preset values to find an appropriate level of detail where no obvious topics are neither merged nor split across multiple topics. This experiment resulted in six topics, each defined by a probability distribution over constituent words. In general, a tweet discusses several topics with different probabilities. For easier interpretation of the results, we selected just the most probable topic assigned to each tweet.local Ljubljana, year, price, municipality, road, city, Slovenia, car, water, vehicle, center, Maribor, Euro, apartment, shop, house, registration, firefighter, mayor;sports match, year, Slovenia, show, win, season, movie, team, book, city, Ljubljana, league, Maribor, award, interview, concert, weekend, game;health measure, human, mask, virus, government, epidemic, Slovenia, infection, country, coronavirus, doctor, week, health, number, case, work, life, help, school;family child, year, human, school, life, woman, head, hand, parent, world, thank you, man, word, language, end, thing, mother, book, family;politics government, party, state, year, money, Slovenia, minister, media, president, election, work, salary, law, parliament member, human, Janša, Šarec, court, politics;ideology Slovenia, country, human, year, Slovenian, nation, border, migrant, war, communist, government, Europe, Janša, power, army, world, media, justice, leftist.In Table A topic is defined by the probability distribution over words, and we provide the top most probable words for each topic. Each topic is assigned a shorthand label to adequately characterize it and to facilitate further analyses. We assigned the topic labels manually, on the basis of the most probable words, and by inspecting several tweets for each topic. The six detected topics are listed below:Figure In this subsection we turn attention to the topic distribution per community. We focus just on the top four communities, already identified in Fig. Figure P and Q, is defined by the Jensen–Shannon divergence  Lin :\documenC, at timepoint t. We denote by C across the five timepoints C changes over time by computing the distances between subsequent timepoints Ci and Cj by computing the distance between their cumulative distributions Let Results with the differences in topic distributions are in Table The right-hand side of Table Similarities between the communities in terms of topic distributions are consistent with the formation of super-communities. A super-community is a set of communities that are densely linked together by the external influence links, i.e., retweets  tweets posted by the three communities.The three communities are very different in size and in their Twitter activities. Figure Out of the six topics detected, we first consider the two prevailing topics, politics and ideology, taken together. Figure The change of the government in Slovenia in 2020 coincides with the emergence of the Covid-19 pandemic. In Fig. In contrast to the politics and ideology, the health topic draws relatively low number of unacceptable tweets. However, as the pandemic progressed, and increasingly more unpopular public measures were taken, so has the volume of unacceptable tweets increased.This paper concludes a trilogy on the analysis of a comprehensive Slovenian Twitter data corpus, from the 2018–2020 period. In the first part  and in other languages (Zollo et al.


Peer Review Week 2021, the Eurosurveillance editorial team would like to send out a warm thank you to all our Rapid communications reviewers (90 experts in 2021 so far). Rapid communications have been a special feature over the last 25 years, through which the journal has had an impact and served the public health and communicable disease communities with timely and authoritative evidence for rapid public health action. Our peer reviewers are essential in shaping the quality and usefulness of the published articles, especially on short notice and we acknowledge all those who have reviewed rapid communications for us in 2021, despite their high workload during the ongoing COVID-19 pandemic:On the occasion of Cornelia Adlhoch; Marco Ajelli; Franz Allerberger; Erik Alm; Tommi Asikainen; Agoritsa Baka; Ian Barr; Senad Begić; Kaatje Bollaerts; Robert Booy; Maria José Borrego; Udo Buchholz; Saverio Caini; Jesus Castilla Catalan; Marco Cavaleri; Michelle Cole; Vittoria Colizza; Victor Corman; Laura Cornelissen; Benjamin Cowling; Natasha Crowcroft; Fortunato D'Ancona; Nicholas Davies; Brechje de Gier; Walter Demczuk; Aparna Dressler; Robert Dyrdak; Michael Edelstein; Dirk Eggink; Brendan Flannery; Arnaud Fontanet; Luca Freschi; Karthik Gangavarapu; Edward Goldstein; Thorolfur Gudnason; Eric Haas; Walter Haas; Thomas Harder; Kristin Hegstad; Derval Igoe; Michael G Ison; Michael Jackson; Kari Johansen; Annemarjut Jääskeläinen; Taishi Kayano; Marian Killip; Satu Kurkela; Amparo Larrauri; Kathy Leung; Bruno Lina; Michal Mandelboim; Andrea McCollum; Eleanor Mcnamara; Michael McNeil; Angeliki Melidou; Stefano Merler; Benjamin Meyer; Joël Mossong; Judith Mueller; Marcel Müller; Micha Nübling; Takis Panagiotopoulos; Maria Pardos de la Gandara; Pasi Penttinen; John Pettersson; Diamantis Plachouras; Katarina Prosenc; Giovanni Ravasi; Maximilian Riess; Julien Riou; Emmanuel Robesyn; Werner Ruppitsch; Gianpaolo Scalia-Tomba; Emily Scher; Gianfranco Spiteri; Carl Suetens; Jonathan Suk; Sheena Sullivan; Pham Thi Mui; Marianne van der Sande; Esther Van Kleef; Pauline Vetter; Qiang Wang; Matthijs Welkers; Eloise Williams; Thomas Williams; Emma Wiltshire; Shangxin Yang; Dominik Zenner; Walter Zingg.We extend this acknowledgement to everyone who reviewed articles for us during the last 25 years and an excuse to those who reviewed a rapid communication this year and who we may have missed in this list.


Background and purpose:Eucommia ulmoides polysaccharides (EUP) can regulate the immunity of macrophages, but the functional status of macrophages is related to osteoarthritis and synovial inflammation. The purpose of this study is to explore whether EUP has the effect of inhibiting osteoarthritis and its possible mechanism.Methods: MTT test was used to evaluate the appropriate concentration of EUP and real-time quantitative polymerase chain reaction (RT-qPCR) was conducted to detect the effect of EUP on gene expression in RAW 264.7 cells. The osteoarthritis model was constructed by the anterior cruciate ligament transection (ACLT) in the rabbits. These rabbits were divided into three groups, sham operation group, OA group, and EUP group. The changes in articular cartilage were detected by gross observation and histological staining, and Micro-CT tested subchondral bone. Finally, the changes of macrophages in synovial tissue were studied by immunohistochemistry.Results: The results showed that EUP at the concentration of 50ug/mL and 100ug/mL were beneficial to the proliferation of macrophages. The qPCR results indicated that EUP inhibited the expression of inflammation-related genes IL-6, IL-18 and IL-1β, and promoted the expression of osteogenic and cartilage-related genes BMP-6, Arg-1 and transforming growth factor beta (TGF-β). The results of in vivo experiments suggested that the degree of destruction of articular cartilage in the EUP group was significantly reduced, and the Osteoarthritis Research Society International (OARSI) score was significantly reduced. Compared with the OA group, the subchondral cancellous bone density of the EUP group increased, the number and thickness of trabecular bone increased, and the separation of trabecular bone decreased. Synovial macrophage immunohistochemistry results manifested that EUP, on the one hand, reduced M1 polarized macrophages, on the other hand, accumulated M2 polarized macrophages.Conclusion: EUP can promote articular cartilage repair and subchondral bone reconstruction. The regulation of the polarization state of macrophages may be one of its mechanisms to delay the progression of osteoarthritis. Osteoarthritis is a degenerative joint disease, which is mainly manifested as progressive degeneration of articular cartilage, including narrowing of the joint space, changes in subchondral bone structure, osteophyte formation, synovitis etc., . Now morLow-grade inflammation is related to the occurrence and development of osteoarthritis . Ayral evia anti-CD14 conjugated magnetic beads. Eucommia ulmoides is one of the most popular Chinese herbal medicines currently studied. Its main effects are anti-inflammatory, immune regulation and anti-osteoporosis  was obtained from Shanghai Yeyuan biotech Co., Ltd., . The RAW 264.7 cell line was purchased from Procell Life Science Technology Co., Ltd. . RAW264.7 cells were seeded in 96-well plates with a cell concentration of 2 × 103 cells per well. The culture medium of macrophages is HyClone Dulbecco’s Modified Eagle Medium  + 5% fetal bovine serum . The RAW264.7 cells were added to DMEM (control group) and DMEM containing different concentrations of EUP  and were cultured and recorded under a light microscope. On days 1, 3, and 5, cell proliferation experiments were performed with MTT. The absorbance was measured at 490 nm using a microplate reader .−ddct method with reference to the control group. The primers used for genetic testing are shown in Macrophages were cultured in DMEM and DMEM + EUP  medium for 5 days, and use RNAiso plus (TAKARA) to extract total macrophage RNA according to the instructions provided by the reagent supplier. NanoDrop2000 software was used to determine the total RNA concentration. Then a reverse transcription kit (TAKARA) was used to synthesize cDNA from the obtained total RNA sample. Simply put, 1 μg of total RNA was reverse transcribed at 37°C for 15 min, and then heated at 85°C for 5 s. In the qPCR process, 2 μl of cDNA was used to amplify the target gene. The reactions were run for 40 cycles using a Step one Plus real-time PCR System (Applied Biosystems). Using β-actin as a reference gene, the expression of IL-6, IL18, IL1β, BMP-6, Arg-1, TGF-β was detected. The relative expression of the target genes was calculated using the 2th week after surgery. The sham operation group and OA group were injected with normal saline, and the EUP group was injected with 100 μg/ml EUP. The injection volume into the joint cavity is 0.1 ml/kg, once a week for five consecutive weeks. At the 9th week, the animals were sacrificed by isoflurane inhalation anesthesia combined with potassium chloride intravenous injection. During the experiment, using vernier caliper and weight scale to measure the width of the knee joints (measure the widest part of the knee) and weight weekly as shown in 18 adult male New Zealand white rabbits ) were purchased from Experimental Animal Center of Guangdong Medical University  and divided into three groups, namely sham operation group, OA group and EUP group. ACLT was performed to establish an OA model. Simply put, after isoflurane inhalation anesthesia, the inside of the knee joint was cut to expose the patella and patellar tendon, and the nodular sac was cut. The anterior cruciate ligament (ACL) was exposed as shown in After rabbit knee articular cartilage was obtained, the degenerative changes of the femoral condyle and tibial plateau cartilage were observed under a dissecting microscope and the severity of OA was scored. The higher the score, the more severe is the cartilage degeneration. The cartilage tissue was fixed with 4% paraformaldehyde for 48 h, and decalcified with 10% EDTA solution for 4 weeks, embedded in paraffin after dehydration. A continuous section with a thickness of 5 μm was prepared, stained with the Safranin O/Fast Green. The histological stained specimens were scored. The above gross and histological scores were based on the methods described in the previous literature .3) of the two hydroxyapatite (HA) models.The reconstruction of the femoral condylar bone was observed by micro-CT. Scanning was performed on a micro-CT imaging system of small animals . Each sample was scanned layer by layer , and the scanned fault image was obtained. And the quantitative analysis of bone mineral density (BMD) and trabecular thickness, bone trabecular spacing and trabecular number was carried out. BMD was determined by directly calibrating the decay coefficients  method was used and SP immunohistochemistry kit was purchased from Beijing boason Biotechnology Co., Ltd., . In short, after conventional dewaxing, the tissue sections were incubated in 3% hydrogen peroxide in 37°C for 10 min, put into citric acid buffer (pH6.0), boiled , and cooled for 20 min. The normal goat serum working fluid was closed in 37°C for 10 min. The anti F4/80 , iNOS  and CD206  were all purchased from Abcam company. The secondary antibodies labeled with biotin were incubated overnight at 4°C and incubated at room temperature for 1 h. Then, the streptavidin labeled with horseradish peroxidase was added and incubated at 37°C for 30 min. Finally, DAB was stained for 10 min.t-test. A one-way analysis of variance (ANOVA) was used in the comparison among the groups, p < 0.05 was considered to be significant.SPSS 19.0 software package was used for statistical analysis. All results were expressed as mean ± SD. The difference between the two groups was tested by Generally, macrophages are round or oval, and they can be multi-process when they are active. p < 0.01) (The procedure was shown in  < 0.01) . The res < 0.01) . Comparep < 0.01), but the BMD of the EUP group was higher than that in the OA group (P< 0.05) In order to further test the effect of EUP on subchondral bone. We used Micro-CT to scan the subchondral bone and perform data analysis. As demonstrated in The possible mechanisms were explored by detecting the immunophenotype of macrophages in synovial tissues. F4/80 is a marker of macrophages, iNOS is a marker of M1-like macrophages, and CD206 is a marker of M2-like macrophages. As shown in The monosaccharide components of EUP include glucose, fructose, mannose, fucose, galactose, and arabinose among others . Sugars Through PCR assay, we found that EUP significantly inhibited the expression of IL-6, IL-18 and IL1β. Studies have shown that the cytokine IL-6, IL-1β and IL-18 can promote inflammation and apoptosis of chondrocytes, they inhibit the expression of aggrecan and stimulate the expression of MMPs, thus contribute to the pathogenesis of OA . TherefoIn this study, we found that the rabbit knee joint widens after cutting the ACL. This may be related to inflammation of the joint cavity and increased joint cavity effusion . CompareOur results indicated that the BMD of trabecular bone significantly increased, the separation of trabecular bone became smaller and the number of trabecular bone increased in EUP group. According to qPCR results as previously described that EUP promoted Arg-1 and BMP-6 expression in macrophages. The results of immunohistochemistry demonstrated that EUP inhibited the expression of macrophages and M1-like macrophages, and increased the expression of M2-like macrophages. As mentioned earlier, in our experiment, EUP promotes the expression of Arg-1 in macrophages. The transformation of macrophages from pro-inflammatory M1 to M2 may be related to EUP promoting the expression of Arg-1. In vivo detection found that the cartilage regeneration was significantly improved, and micro-CT scanning found that EUP was beneficial to subchondral bone reconstruction. Examination of synovial macrophages revealed that the number of macrophages and M1 type macrophages decreased and the number of M2 type macrophages increased in the EUP group compared with the OA group. Through our experimental results, we believe that EUP can delay the progression of osteoarthritis and the effect of regulating the immunity of macrophages partly explains the underlying mechanism.In this study, we found that EUP exhibited no obvious toxicity to cells at the concentrations of 50μg/ml and 100μg/ml, but favored cell proliferation. qPCR assay indicated that EUP significantly inhibited the expression of macrophage inflammation related genes IL-6, IL-18 and IL-1β and promoted the expression of osteogenesis and chondrogenesis related genes BMP-6, Arg-1 and TGF-β.


We postulated that FOXC2 regulates its own expression in sepsis, and its transcriptional autoregulation directs lymphatic EC cell-fate decision. Bioinformatic analysis identified potential FOXC2 binding sites in the FOXC2 promoter. In human lung EC, we verified using chromatin immunoprecipitation (ChIP) and luciferase assays that FOXC2 bound to its own promoter and stimulated its expression after LPS stimulation. Chemical inhibition of histone acetylation by garcinol repressed LPS-induced histone acetylation in the FOXC2 promoter region, and disrupted LPS-mediated FOXC2 binding and transcriptional activation. CRISPR/dCas9/gRNA directed against FOXC2-binding-element (FBE) suppressed LPS-stimulated FOXC2 binding and autoregulation by blocking FBEs in the FOXC2 promoter, and repressed expression of lymphatic EC markers. In a neonatal mouse model of sterile sepsis, LPS-induced FOXC2 binding to FBE and FOXC2 expression in lung EC was attenuated with garcinol treatment. These data reveal a new mechanism of LPS-induced histone acetylation-dependent FOXC2 autoregulation.The innate immune response of pulmonary endothelial cells (EC) to lipopolysaccharide (LPS) induces Forkhead box protein C2 (FOXC2) activation through Toll Like Receptor 4 (TLR4). The mechanisms by which FOXC Dll4 expression through TLR4-ERK-FOXC2 axis to program endothelial cell (EC) specification and induce inflammatory angiogenesis in the neonatal mouse lung (FOXC2 is a member of the forkhead box (FOX) transcription factor family . It playuse lung .FOXC2 in humans causes hereditary lymphedema distichiasis (LD) syndrome and primary valve failure in veins of lower extremities   is a chronic lung disease that develops in premature babies exposed to hyperoxia and sepsis. The aberrant vasculature and alveolar structure were observed in the lungs in which BPD developed . Sriniva (HPMEC) , but how2 at 37°C. HPMEC-Im were transfected overnight with the indicated plasmids or empty plasmids (mock) with Lipofectamine 3000 (Thermo-Fisher) as per the manufacturer’s protocol. Ultrapure LPS was purchased commercially from Invivogen. Garcinol was purchased from Santa Cruse. For experiments with inhibitors, primary HPMEC and HPMEC-Im were pre-treated with 25 μM Garcinol for 45 min prior to the addition of LPS (0.5 μg/ml).Immortalized HPMEC (HPMEC-Im) was generated as described before . BrieflyCare of mice before and during the experimental procedures was conducted in accordance with the policies at the University of Missouri- Kansas City Lab Animal Resource Center (Protocol 1510) and the National Institutes of Health guidelines for the care and use of laboratory animals. All protocols had prior approval from the University of Missouri- Kansas City Institutional Animal Care and Use Committee. Wildtype C57BL/6 strain was obtained commercially from Charles River . LPS injections (2 mg/kg) to the mice were given intraperitoneally (i.p), and sterile saline used for controls  with or without 25 μmol/kg Garcinol pretreatment. Mice were then euthanized using a 100 μl i.p injection pentobarbital, exsanguinated with the cessation of a heartbeat, and the lungs were harvested and utilized as described below.FOXC2 gene was amplified with 5′-tgagctagccca ccatgcaggcgcgctactccgtgtccga-3′ and 5′-agtctcgagtcagtatttcgtgc agtcgtaggagtaggg-3′ from HPMEC genomic DNA and cloned into pIRES-Puro-EGFP (Addgene) to generate pIRES-Puro-EGFP-FOXC2 WT. Oligos 5′-caccgtccgggattcctagaggga-3′ and 5′-aaactccctctaggaatcccggac-3′; 5′-caccgcgagggaaactcagtttgt-3′ and 5′-aaacacaaactgagtttccctcgc-3′; 5′-caccgattggctcaaagttccggg-3′ and 5′-aaaccccggaactttgagccaatc-3′ were annealed to generate double-strand gRNA templates next to FBEs about −1.7, −0.9, and −0.45 kb in FOXC2 promoter and then cloned into phU6-gRNA plasmid, respectively, to generate phU6-gRNA targeting FBEs in FOXC2 promoter for FBE dCas9/gRNA assay. 0.45 and 2 kb upstream DNA of FOXC2 promoter regions were amplified with 5′-actgctagccgctttcagcaagaagacttttgaaacttttcc-3′ and 5′-tgagagcgagagagcgcgagaga-3′; 5′-tattggaaataagtggcacgcc-3′ and 5′-tgagagcgagagagcgcgagaga-3′ from HPMEC genomic DNA and cloned into pGL4.10 to generate pGL4.10-FOXC2-Pomoter respectively for luciferase assay.The FOXC2 CA, generated by cloning the FOXC2 DBD in-frame with the VP16 transcriptional activation domain , was a gLentiviral vector containing the dCas9  and pack® Luciferase Assay System (Promega) according to the manufacturer’s instructions.pGL4.10 -FOXC2-Promoter, pGL4.75 Renilla and pIRES-Puro-EGFP-FOXC2 WT (or CA) plasmids were co-transfected into HPMEC-Im cultured in 96-well plate. Next day, luciferase assay was applied with Dual-GloFOXC2 promoter primers: −1.7 kb, 5′-cccgtgtttagccttgttaaag-3′ and 5′-ctaggaatcccggacagtttg-3′; −0.9 kb, 5′-cctcgataggttatccttgacg-3′ and 5′-tggattggaatggcaggg-3′; −0.5 kb, 5′-tgattggctcaaagttccgg-3′ and 5′-aagaggccaagtcccttttag-3′.HPMEC-Im were treated with LPS for 0.5 h, with or without garcinol pretreatment, and then ChIP was applied according to the manufacturer’s instructions (Thermo Fisher Scientific). Rabbit anti FOXC2 antibody  was used to pull down FOXC2 binding DNA and rabbit anti acetylated H3K27antibody  was used to pull down H3K27ac binding DNA. qPCR was performed to quantify pulled-down DNA with Foxc2 promoter primers: 5′-cgactggagatgttgaaggaa-3′ and 5′-attttatgccaaccttgacgc-3′Mice were treated with LPS for 3 h, with or without garcinol pretreatment, and then lung EC were isolated. ChIP was applied according to the manufacturer’s instructions . Anti-FOXC2 antibody  was used to pull down FOXC2 binding DNA. qPCR was performed to quantify pulled-down DNA with mouse HPMEC-Im was transduced by lentivirus containing dCas9 and then screened with 200 mg/L hygromycin to generate dCas9 stable cell line HPMEC-Im/dCas9. Mixed FBE gRNAs were transfected into dCas9 stable HPMEC.For endothelial cell isolation, all lobes of the lung from 2 neonatal C57BL/6 pups (4–7 days old) were pooled per condition. The isolation of mouse lung endothelial cells was done as described previously .Actb and human 18S were purchased commercially from Sigma. mouse Actb and human 18S were used as the housekeeping gene. The relative gene expression was calculated using the Pfaffl method.Total RNA was extracted from HPMEC and mouse lung EC using the PureLink RNA Mini Kit (Life Technologies) following the manufacturer’s instruction and cDNA was synthesized from 1 μg of RNA using an iScript cDNA synthesis kit  according to the manufacturer’s instruction. qRT-PCR was run on a Bio-Rad IQ5 with SYBR green master mix (Bio-Rad). The primers for mouse and human target genes, and mouse HPMEC-Im grown to the 90% confluence in 60-mm dishes had various treatments, and lysates were used for immunoprecipitation studies. It was done as described previously .HPMEC-Im and mouse lung tissue were homogenized in RIPA lysis buffer containing commercially available protease and phosphatase inhibitors (Sigma) with after LPS treatment, with the clarified lysates used for immunoblotting. Immunoblotting was done following standard protocol. The primary antibodies used were: goat anti-FOXC2 , mouse anti- CRISPR-Cas9 , mouse anti-phospho-Serine [(p)Ser], mouse anti-phospho-Threonine [(p)Thr] and mouse anti-ACTB (Sigma). Densitometry was performed using ImageJ Software  and changes were normalized to ACTB.n = 3–4 per group.IF was done as in our previous study . The lunP < 0.05 was considered significant. For cell culture experiments, data are from a minimum of three independent experiments with adequate technical replicates used for quantification. All animal data were obtained in littermate controls. For animal experiments, a minimum of 3 animals were used for each experimental group. RNA quantification and PCR results had 2–3 technical replicates. For all data, we initially examined whether distribution of data was Gaussian using the D’Agostino-Pearson omnibus normality test. If data were normally distributed, then ANOVA with a post hoc Tukey test was used for analysis. If data did not meet Gaussian assumptions, a Mann-Whitney U-test was used for analysis. For most analysis, fold-changes were calculated related to expression/changes in untreated controls. Statistical analysis was done using Graphpad Prism 7.0 .Data are presented as mean ± SD or median with interquartile range. Lab experiments were reviewed and approved under the University of Missouri-Kansas City IBC, protocol number 18–28. Animal experiments were reviewed and approved under the University of Missouri-Kansas City IACUC, protocol number 1510-02.FOXC2 expression in HPMEC-Im, we compared FOXC2 mRNA and protein expression with or without LPS stimulation. qRT-PCR showed FOXC2 mRNA expression increased 6 h after LPS treatment  predicted 17 potential FBEs  and one FBE at −456 in the upstream of FOXC2 gene utilizing CRISPR/dCas9/gRNAs technologies. We identify that dCas9/gRNAs specifically target those FBEs and repress LPS-stimulated  hotspot  within 2erial EC . Both ChFOXC2 autoregulation influences EC cell fate, we transfected mixed FBE-targeted gRNAs into dCas9 stable expressed HPMEC-Im and analyzed expression of lymphatic markers. After blocking FOXC2 autoregulation, EC had decreased expression of lymphatic EC specification markers, which suggests that FOXC2 autoregulation may play an important role in EC programing and differentiation during lung development. CRISPR/dCas9 strategy is broadly used to study DNA-protein interaction and gene expression regulation, but dCas9 footprint covers 78.1 bp ± 37.9 bp  and the National Institutes of Health guidelines for the care and use of laboratory animals. All protocols had prior approval from the University of Missouri- Kansas City Institutional Animal Care and Use Committee.VS and SX: conception and design. SX, WY, and HM: data collection. VS, SX, WY, HM, and SY: analysis and interpretation, drafting and editing the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A timely, confirmed diagnosis of Idiopathic Pulmonary Fibrosis (IPF) has a significant impact on the evolution of the disease. The current model of care in the Lazio region  was assessed on the basis of real-world data provided by the four reference centers responsible for diagnosing and treating IPF. The 5-year, population-based, retrospective longitudinal study provided the data that is at the basis of the current proposal for a new clinical and therapeutic pathway (DTCP) and has been shared with regional decision makers. A DTCP must be defined and based on four pillars: GPs, pulmonologists, IPF centers, and telemedicine. Each must play a role within a sort of hub-and-spoke model. IPF centers remain the hubs, while spokes are identified in trained GPs and pulmonologists. Care pathways are able to impact on clinical outcomes. Translating clinical practice guideline recommendations into clinical processes is a critical aspect that should be constantly monitored. In the case of a progressive life-limiting disease, such as idiopathic pulmonary fibrosis (IPF), a timely and accurate diagnosis is essential. The patient pathway, through which a confirmed diagnosis of IPF is reached, could have a significant impact both in terms of access to available treatments and progression of the disease.IPF is a specific and aggressive form of chronic fibrosing interstitial pneumonia. The diagnostic process is complex, and a definitive diagnosis must be based on clinical, laboratory, radiologic, and/or pathologic data. Late diagnoses could still occur due to difficulties in differentiating IPF vs. other interstitial lung diseases (ILD), and also due to a lack of knowledge of the disease , 2. EarlIn fact, access to antifibrotic therapies, such as nintedanib  and pirfIn 2015, Italian Ministerial Decree n.70, dated 2 April 2015 , capable of offering a full array of services, and with affiliated centers (spokes), which offer a more limited number of services but that are still able to direct patients to more intensive services provided by the hubs.Since 2018, four reference centers in the Lazio region  have been responsible for the diagnosis and treatment of IPF. These centers are: Pulmonology Unit at the Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Fondazione PTV “Policlinico Tor Vergata,” Azienda Ospedaliera San Camillo Forlanini, and Azienda Ospedaliera-Universitaria Sant'Andrea (since 2018). All four IPF centers are located in Rome.No regional clinical and therapeutic pathway (DTCP) has yet been defined for IPF. Nevertheless, only these centers with their multidisciplinary team (MDT) are able to confirm a diagnosis of IPF, perform high-resolution computed tomographies (HRCT), and prescribe antifibrotic therapies. Therefore, the lack of formalized referral criteria for IPF centers could have an impact on the possibility of accessing available treatments.A collaborative study was conducted by all four IPF reference centers. All of them defined and contributed to the research. A 5-year, population-based, retrospective longitudinal study was designed to assess how this hub-based model is effective in early diagnosis and therapeutic management.The primary goal was to collect aggregate data on pre-diagnosis IPF patient pathways and patient characteristics. Evidence was then used to describe the current model of care. Then a SWOT analysis was conducted to identify strengths, weaknesses, opportunities, and threats associated with the current model of care. Results were shared and discussed during an in-person, multi-stakeholder meeting. Finally, opportunities for improvement were identified to promote and support the definition of a regional DTCP.Similarly to other studies, we chose to conduct a survey that aimed at describing a real-world model of care for IPF –10.A survey was then created to collect data on patients diagnosed with IPF through the four above mentioned IPF centers between 2014 and 2018. Previously published analyses for Italy refer to cases recorded in 2009 [in Lazio ] and in The survey was conducted from June to August 2019. Aggregated data was collected on demographic , clinical (treatments before confirmed diagnosis), and organizational variables .The survey was only submitted to the four IPF centers participating in the study. Only IPF centers were involved, since all patients with suspected IPF had to refer to them for a confirmed diagnosis. Furthermore, the diagnostic criteria that were adopted for IPF were compared among centers at the beginning of the study and assessed as being comparable, also given the presence of a multidisciplinary team (MDT) in each of them. This guaranteed for homogeneity in case retrieval and comparativeness of data. The Pulmonology Unit of the Fondazione Policlinico Universitario Agostino Gemelli IRCCS collected and analyzed the data.Collected data showed that the number of patients with a newly confirmed diagnosis of IPF in the Lazio region increased from 82 in 2014 to 344 in 2018 , with a During the study period, the number of patients eligible for treatment increased both in absolute (from 67 to 302) and relative values . This means patients received a confirmed IPF diagnosis in the early stages of the disease. For this study, we chose to adopt the same criteria as AIFA  to identify reasons of ineligibility for treatment. In particular, a significant reduction in the proportion of ineligible patients due to DLCO levels emerged  and that incidence of IPF increases with age. Diagnosis before age 45 was rare. The majority of cases were in the age range between 70 and 79 years. Patients > 80 years of age increased in relative terms from 3.7 to 21.8% during the study period. The incidence rate was 2.82 per 100,000 residents (age ≥ 45 years) in 2014 and reached 11.30 per 100,000 residents in 2018. Almost all cases of confirmed diagnosis resided in the Lazio region (91% in 2018) and in the province of Rome (79.9% in 2018). Rates per 100,000 residents confirmed a major incidence in the province of Rome , even if the difference compared to Frosinone , and Rieti  was small. The increasing trend is confirmed by the incidence rate at a provincial level.In addition, most of the treatable patients had previously visited with a pulmonologist (67.9% in 2018) and/or a GP (16.2% in 2018). Transfer between IPF centers appeared low (only 4.3% of patients moved from one center to another in 2018).A multidisciplinary team was already active in all of the IPF centers. The structure of these teams varied slightly. Six additional pulmonologist (in three centers) and three nurses were the major changes applied, in order to respond to the significant increase in the patient population. Overall, eleven additional staff members were reported.The average number of open days increased from 1.3 to 3.7% per week. None of the IPF dedicated outpatient services was open on Saturday. Since 2017, two pulmonology outpatient services have remained open 5 days per week.The healthcare services provided were quite homogenous among the three active centers during the entire study period. Two (out of three) IPF centers were able to offer echocardiograms. While lung biopsies were always available, only one center was able to provide respiratory physiotherapy, mobility rehabilitation, and psychological consultations.de facto network for diagnosing and managing IPF patients has been operative in the Lazio region. The four IPF centers can count on multidisciplinary teams for patient management. There is, however, room for improvement because, in the absence of an official and shared definition of a regional DTCP, the process the patient must follow is quite erratic when attempting to reach a qualified center. In addition, all four IPF centers are located in Rome, while a not so irrelevant number of patients live in other areas of the region. To prevent this additional burden on patients, several activities  could be delegated to spoke centers. During the study period, a key potential role emerged that had already been played by pulmonologists and GPs.As demonstrated by the data collected , 2, a deOpportunities for improvement were identified in the definition of criteria that justify the decisions of GPs to refer a patient with suspected symptoms to a specialized pulmonologist and then to an IPF center. In addition, the selection and definition of indicators which can adequately monitor the key phases of the diagnostic and therapeutic pathway were proposed. Furthermore, multidisciplinary teams should be formalized both in terms of structure and size  in relation to workload .Moreover, the aging of the population, as well as the evolution of natural disease history –15, are Our study demonstrates the potentialities of a collaborative study that aims at collecting real-world data, in order to prove the need to optimize the current model of care. The dissemination phase was accurately planned. Results were shared with regional decision makers . An in-pDuring the multi-stakeholder meeting, results were further discussed in terms of patient population evolution, workload, and available resources for IPF centers. The SWOT analysis  was the Telemedicine is the fourth pillar. This is essential in allowing for and maintaining communication among GPs, pulmonologists, IPF centers, and patients. Positive experiences regarding the use of e-health solutions for IPF patients have already been reported –20. We pThe positive impact of telemedicine on the management of IPF cases has been already demonstrated both in studies conducted during the pandemic and in trials. It has been demonstrated the ability of telemedicine to both reduce exacerbations and hospitalizations . In addiA regional clinical and therapeutic pathway (DTCP) is requested for defining and managing the multi-pillar system. Communication and training should be guaranteed, and an integrated telemedicine solution should be defined and managed at a regional level. Coordination in the diffusion of telemedicine is crucial. For instance, single initiatives at healthcare provider level can have only a limited impact on the patient pathway. An integrated platform or system is the only way to respond to both decision makers and the needs of patients. In addition, a coordinated diffusion of telemedicine has positive externalities on clinical research, because it will allow to have a unique real word database based on uniform criteria of data collection for IPF. As a consequence, it will be possible both to monitor real time the evolution of the disease, and to have real world evidence (RWE) on the effectiveness of available treatments and on the impact of DPCP.In Italy, two regions  have already defined a DTCP for IPF. Both are transparent regarding the structure of multidisciplinary teams. Only the Lombardia region defined the role of different clinicians in relation to the phases of the pathway  , while tThe proposal elaborated (and described here) for the Lazio region will represent the first case of a regional DTCP explicitly involving various health professionals, even external to IPF centers, and will give relevance to digital solutions. It can be interpreted as a hub-and-spoke model. IPF centers remain the hubs, while spokes are identified in trained GPs and pulmonologists.During the meeting, all stakeholders agreed on the need for a better defined clinical pathway. Policy makers supported the proposal to involve other players, external to the IPF centers, in order to guarantee a continuity in adequate care. Patient representatives positively reacted to the multi-stakeholder approach suggested for developing a new DTCP. In addition, the implementation of telemedicine was welcomed. Finally, an increase in awareness and competencies in terms of IPF diagnosis emerged as crucial points for an efficient new DTCP.In the end, both regional and hospital-specific databases can contribute to a more complete analysis of patient pathways. A larger collaborative study would be a relevant further development.The approach proposed for IFP could guide the definition or refinement of DTCP of other interstitial lung diseases (ILSs). In particular, the same four pillars approach could be quite similar in pre-the diagnosis phase for ILSs that share common symptoms and required the involvement of the same health professionals. Then, the role of telemedicine can be adapted to the specific disease pathway.A 5-year, population-level, retrospective longitudinal study was not only able to demonstrate a significant increase in the ability to detect IPF cases, but also to identify the key players of the current model of care based on four qualified IPF centers. A SWOT analysis allowed to elaborate a proposal that was introduced and discussed during a multi-stakeholder meeting in which patient representatives and also regional decision makers participated. The proposal of a three-pillar system  supported by telemedicine was well accepted. This would represent the first case of a regional DTCP explicitly involving different health professionals, even external to IPF centers, and would give relevance to digital solutions. To further elaborate and implement this, a multi-stakeholder approach should be maintained.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The study was communicated to the Ethical Commitee of each IPF center. Written informed consent was not required to participate in this study in accordance with the national legislation and the institutional requirements.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors received unrestricted financial support by Boehringer Ingelheim.All authors has received a research grants from ALTEMS to participate to the study. For other studies, LR has received consulting fees from Boehringer Ingelheim, Roche, FibroGen, Celgene, RespiVant, Nitto, Bristol Myers Squibb, Pliant Therapeutics, Veracyte, CSL Behring, Novartis, and speaker honorarium from Boehringer Ingelheim, Roche, Zambon, CSL Behring and participated in Data Safety Monitoring Board or Advisory Boards for Boehringer Ingelheim, Roche, FibroGen, Veracyte. Departement of Experimental Medicine - Respiratory Medicine (Università di Roma “Tor Vergata”) was funded by Almirall, Boehringer Ingelheim, Chiesi Farmaceutici, Novartis, and Zambon. PR participated as a lecturer in scientific meetings and courses under the sponsorship of Almirall, AstraZeneca, Biofutura, Boehringer Ingelheim, Chiesi Farmaceutici, GlaxoSmithKline, Menarini Group, Mundipharma, and Novartis and as an advisor in scientific meetings and courses under the sponsorship of Almirall, AstraZeneca, Biofutura, Boehringer Ingelheim, Chiesi Farmaceutici, GlaxoSmithKline, Menarini Group, Mundipharma, and Novartis. AS has received consulting fee from Boehringer Ingelheim, Roche and participated in Data Safety Monitoring Board or Advisory Boards for Roche. Then received support for attending meetings and/or travel from Roche. FV has received a speaker honorarium from from Boehringer Ingelheim, Roche. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.