With the development of nanotechnology, nanomaterials have been used in dental fields over the past years. Among them, graphene and its derivatives have attracted great attentions, owing to their excellent physicochemical property, morphology, biocompatibility, multi-differentiation activity, and antimicrobial activity. In our review, we summarized the recent progress about their applications on the dentistry. The synthesis methods, structures, and properties of graphene-based materials are discussed. Then, the dental applications of graphene-based materials are emphatically collected and described. Finally, the challenges and outlooks of graphene-based nanomaterials on the dental applications are discussed in this paper, aiming at inspiring more excellent studies. Oral health is quite important because it can deeply affect human health and quality of life . HoweverTissue defects  caused by traumas, infections, or tumors are one of the most common diseases in dental field . CurrentIn the field of dentistry, dental implants have been widely applied to restore the missing teeth due to their various advantages. It is well known that osseointegration is the gold standard for successful dental implantation. Titanium and its alloy have been applied as dental implant materials because of its good biocompatibility, mechanical properties, and so on. Except for all its merits, titanium implants also have failures due to the poor osseointegration. Therefore, it is important to improve the performance of titanium dental implants, and modifications of dental implant surfaces played an important role . VariousNanomaterials have showed wonderful performances in improving the strength and resist wear of tooth fillers and sealants. Moreover, nanomaterials also performed excellent antimicrobial properties in the application of restorative materials . Owing tAmong various nanomaterials, graphene, as a promising two-dimensional (2D) carbon-based nanomaterial, is the thinnest and strongest material. In 2004, it was first isolated by Novoselov and Geim using mechanical exfoliation with a sticky tape and they won the Nobel Prize in 2010 . GraphenDuring the past 20 years, significant advances have been achieved in regulating properties of graphene and its derivatives, elucidating their underlying mechanisms, and broadening potential applications. Up to now, there are more than 2,942 studies related to graphene-based materials for dental applications. Although numerous excellent reviews have been published, most of those reviews were mainly focused on certain specific aspect. Therefore, a comprehensive review is needed to summarize and analyze all the progress, especially the achievements from more than 2,271 research papers published in the past 5 years . Such anGraphene, as a promising one-atom thick, 2D carbon-based nanomaterial, is currently the thinnest and strongest material. GO and rGO are the two main derivatives of graphene. Graphene and its derivatives have similar structures but different functional groups, which could lead to the differences in the physical and chemical properties. In this part, the synthesis and structure of graphene and its derivatives will be discussed.2 hybridized carbon atoms arranged in honeycombed lattice, and the structure of graphene is composed of six-membered rings stacked in parallel shape with no chemical groups on the surface of graphene  located on the basal plane and the edges of GO, which are beneficial to combine different biomolecules such as proteins and drugs. The function of the above groups aims at interacting with proteins in the form of covalent, electrostatic, and hydrogen bonding. Considering the structure of GO, the amount of carbon atoms in GO has declined to 40%–60%, which are replaced with oxygen atoms. Therefore, GO displays the hydrophilic property. Although GO has shown more hydrophilic than graphene, GO displays defective structure, poor insulating property, and mechanical property .3 and KMnO4, as oxidants, were dissolved in concentrated H2SO4 to oxidize graphite. However, the Hummers method has the drawbacks of generation of toxic gas such as NO2 and N2O4, residual nitrate , and thermally reduced GO (TRGO) on human dental follicle stem cells and analyzed the involved specific mechanism . The resWhen the biomaterials were implanted to the tissue, the inflammation would be provoked by the protein interactions. During this process, many factors such surface charge, topography, and chemical compositions were involved and affected the resorption of protein. Besides, many molecules also played an important role in the process of inflammation such as betaines . MoreoveTherefore, when using graphene-based nanomaterials as the coatings of dental implants and so on, we should also focused on its effect on the surrounding tissue inflammation. In the study of Rosa et al., they confirmed that friction during the dental implants emplaces when the loads is over 400 mN . Howeverin vitro studies have shown that graphene and its derivatives showed the multi-differentiation ability such as osteogenic differentiation and regeneration of dental pulp.Ideal biomaterials in the tissue engineering show the ability to induce the adherence, proliferation, and differentiation of cells. Many Many studies have proved that graphene-based materials have the potential in promoting the osteogenic differentiation of various cells including MC3T3-E1, bone marrow mesenchymal stem cells (BMSCs), periodontal ligament stem cells (PDLCs), dental pulp stem cells (DPSCs), etc.  with electrospinning technique by incorporated with rGO and polycaprolactone (PCL) and investigated their enhanced neurogenesis of DPSCs . The resin vivo , and GO-Car/hydroxyapatite (HAp) can decrease by 67%, 86.4%, and 78.2%, respectively  .Many studies have reported that graphene could stimulate different types of stem cells to form osteoblasts. Fan et al. reported that graphene/HAp composite sheet displayed good biomimetic mineralization . The simTo confirm the osteogenic differentiation of rGO, some researchers had fabricated rGO coatings and composites . Kim et in vivo also confirmed the above results. Then, Azadian et al. also successfully fabricated the polyvinylidene fluoride (PVDF)–polyvinylalcohol (PVA)–GO scaffold and evaluated the ALP activity, calcium contents assays, and expression of osteogenic gene markers  . A good To intensify the neural differentiation of graphene-based materials, Seonwoo et al. prepared a NFs incorporated with rGO and PCL by electrospinning technique and investigated the enhanced neurogenesis of DPSCs . The resin vivo experiment, more new alveolar bone was found and filled the furcation defect. Even more interestingly, periodontal ligament-like, cementum-like tissue was also observed in the GO group  were impaired significantly, indicating that the addition of GNPs may improve the physico-mechanical properties of materials but not ideal for all materials in terms of bonding properties . Unlike s mutans .2, silane primers formed adhesive layer show the poorest mechanical properties   have been used to improve the antimicrobial-adhesive effects of PMMA resin by Lee and his co-workers . The imm(G-AgNp) . The resTitanium and its alloys have been widely used in dental implants, owing to their various advantages such as good biocompatibility, high mechanical property, and corrosion resistance . HoweverIt is well known that the osseointegration is the gold standard of the success of dental implants. Therefore, the new bone formation between dental implants and bone tissues is of great significance. In regard to the superior osteogenic differentiation of graphene in bone tissue engineering, they also attracted much attention in the modification of dental implant. Park et al. reviewed that the strategies of graphene-based modifications could be mainly divided into four categories: 1) a layer-by-layer assembly; 2) PMMA-mediated method; 3) electrophoretic deposition; and 4) APTES-induced method .in vitro and osseointegration in vivo (in vitro. All the results indicated that GO coating showed a positive effect on improving the bioactivity of PDLCs. Jung and his colleges prepared a dexamethasone (Dex)–loaded rGO coating on Ti13Nb13Zr (MPCR-TNZ) multipass rolled titanium alloy surface by using the spin coating technology after pretreatment with APTES, showing a stable long-term release behaviors of Dex  on titanium . Accordi methods . In addi2/GNs) composites, which were coated on the surface of zirconia by an atmosphere plasma spray technique  has been widely utilized for in-office whitening for a long time. The H2O2 molecules can penetrate deep the teeth and carry out the bleaching process. However, the relative high concentrations of H2O2 caused some side effects such as tooth sensitivity and gingival irritation –ICG composite to enhance the efficiency of ICG  is known as an alternative method for treatment of periodontitis and peri-implantitis. Pourhajibagher et al. investigated the effect of graphene quantum dot (GQD)–curcumin (Cur) on the perio-pathogen biofilms combined with PDT . The probiofilms . Comparey of ICG . The resS. aureus and S. mutans, showing that the highest antibacterial ability of FG (4 wt%) for S. aureus and S. mutans was 88.1% and 85.3%, respectively  and five times (TiGV) to evaluate the inhibition properties of Candida albicans biofilms , showing the high stability and storage performance  is one of the most common side effects of orthodontic treatment, which is caused by enamel surface demineralization . Therefo2, and GO-CaF2 all exhibited superior prevention of decalcification. Besides, GO-Ag and GO-Ag-CaF2 group showed better antibacterial activity compared with other groups, which may be related to the synergic effect of GO and Ag. Moreover, the color variation of f-GO coatings on the dentin surface is negligible, showing that GO is a promising dentin anti-demineralization resistant material.Dental caries and dental erosion were associated with the demineralization of dentin, which caused by acids from bacteria, food, and environments, leading to dentin hypersensitivity and pain . Nizami via a non-covalent functionalization by the interaction between oxygenated carbon functional parts and COL through hydrogen bonding  and guided tissue regeneration (GTR) as barrier membrane to hinder the soft tissue invasion of new bone . Althoug bonding . The mem bonding . MeanwhiThere is close interaction between bacteria and dental caries, and endodontic and periodontal diseases. Several groups of bacteria that require a common antibacterial strategy are usually involved. Amoxicillin (AMOX) is a kind of broad-spectrum antibiotic that is the first-choice antibiotic in the treatment of endodontic infection in Asian and European countries. In the conditional paste, the dose is not accurately controlled . Drug caGraphene-based materials, the promising candidate for dentistry materials, have been widely used in dentistry research, owing to its cell differentiation and antibacterial property. This review summarized recent advances in expanding the types of graphene-based materials and the studies about dentistry-related properties, deepening the understanding of categories of graphene-based materials. Compared with other related studies, it displayed a quite comprehensive and detailed review about the great achievements in dental applications such as bone tissue engineering, coatings for dental implants, antibacterial properties, and COL membranes. Except for the usage in the tissue engineering, dental implant coatings, COL membrane, and adhesive, we also focused on some new fields such as drug delivery, prevention of enamel and dentin from demineralization, biosensor for oral biomarker detection, and inhibition of the growth of fungal. For the application of graphene nanomaterials in the biosensor, it could be used to detect bacterial and viral markers, drugs, and cancer markers. With the development of the application of graphene in dentistry, there are still some challenges remaining to be tackled until the final commercialized .in vitro and in vivo. Because of the limited understanding of graphene and its derivatives, a major challenge in clinical applications is the uncertainty of its cytotoxicity in vitro and in vivo and its potential mechanisms. Up to date, there is not an agreement on the cytotoxicity and potential hazards of graphene-based materials according to various studies. The factors that involved in the cytotoxicity include concentrations, surface functionalization, types of graphene family and synthetic methods, and the layer numbers. Many studies have focused on the dose dependent effect on the cytotoxicity, yet there is still no agreement for the upper limit concentration (in vivo and in vitro.The excellent biomaterials should have a good biocompatibility without long-term cytotoxicity ntration . When itAnother challenge of graphene-based materials is biodegradation, especially in the tissue engineering. With the formation of new tissue, the ideal biomaterial should be free of any toxic products. Up to date, there are limited literatures related to the biodegradation of graphene-based materials. Therefore, graphene-based material as an ideal biomaterial should be considered and explored to solve this problem.Although the graphene used in the current study is a controlled and uncontaminated defect-free sample, the synthesis quality still should be highly examined when it was used in the clinic. Actually, the formed variety of unpredictable defects is mainly due to the differences in the synthesis methods. Once the defects occurred, the properties such as susceptibility and electronic structure will be changed . Therefo2/M phase in the GO groups. Thus, the effect of GO on the cell cycle should be carefully considered and explored before the final clinical use.Little of the current studies are focused on the effect of GO on the cell cycle. Nowadays, Hashemi et al. innovatively focused on the effect of GO on the cell cycle . In the Although graphene has many advantages in the dental field, its clinical translation still needs to be carefully considered. Graphene-based materials had been used as coatings mostly in tissue engineering and dental implants surfaces. When used as the dental implant coatings, friction could cause the delamination of carbon-based coatings on the titanium with the loads over 400 mN . Rosa etWhen used as a biosensor, the large surface of graphene-based nanomaterial provided good adhesive conformability and the functionalization of graphene could be achieved by AMP-graphene peptide for biological recognition. However, there are different kinds of bacteria in the mouth. Therefore, more strategies should be made to recognize more bacteria, and the methods for the functionalization of graphene biosensor are quite important.GO had been successfully used as a drug carrier for AMOX, which is a broad-spectrum antibiotic and could be used to control dental infections. However, the limitation is that whether two or more different drugs could be carried simultaneously is not sure and more studies are needed.Eventually, graphene and its derivatives will be of great interest for a long time in the dental field. Although there are some limitations in the real clinical usage of dentistry, graphene, as a more reliable and more friendly biomaterial, can prompt more effective dental treatments in the near future.Although there are many studies focused on the antibacterial effect of graphene-based materials on the single strain of bacteria or monoclonal biofilm, yet no study revealed the antibacterial effect on the mature polymicrobial biofilms. On the basis of the above limitations, there is still a long way before the final clinical usage of graphene-based materials in the dental fields.All acronyms and abbreviation used in the text are explained in


N = 67) completed a collaborative food preparation task. The qualitative analysis enabled us to identify three functions of questions: anticipation questions, exploration questions, and confirmation questions. We have discussed in this study how the types of questions identified are associated with: (i) the accomplishment of interactional goals and (ii) complementary temporalities in the collaborative activities.Researchers have been interested in the investigation of the interactive functions of questions in conversational contexts. However, limited research has been conducted on the interactive functions of questions in embodied collaborative work, that is, work that involves the manipulation of physical objects. This study aimed to identify the interactive functions of questions in embodied collaborative work. To do so, we conducted a systematic qualitative analysis of a dataset of 1,751 question-answer sequences collected from an experimental study where pairs of participants ( Questions are one of the most important linguistic and embodied resources giving structure to social interactions, from human interaction in every day  and instQuestions embedded in embodied collaborative work  rely on the coordination of bodily resources such as manual gestures, gaze, posture, and facial expressions to achieve shared goals. The joint accomplishment of shared goals goes beyond the coordination of verbal and non-verbal behaviors in a synchronized manner over time. Collaboration influences the action and planning of interacting partners and shapes interactive outcomes, such as when partner A asks “What should we do with this (while pointing to a bag of flour)?” B replies: “we must put half of that in this bowl (while touching the bowl),” and A acknowledges B: “Alright, I see it now.” This short question-answer sequence illustrates how the intentions, plans, and goals of each interacting partner come into play in embodied collaborative work.N = 67,134 participants) completed a collaborative food preparation task. Case studies have been used to investigate the interactive functions of questions in embodied collaborative work . The data collected were in French. The only constraint involved in the collaborative ravioli-making task was time duration (refer description of Task below). Hence, group members could freely interact while making the ravioli, as it would occur in a comparable real-world situation.A total of 134 participants  were recruited from the student population of the University of Neuchâtel, Switzerland . They were fluent speakers of French and reported having limited previous cooking experience. They had previous practice of simple skills like combining and heating ingredients but had not mastered more complex skills . Participants received 25 CHF compensation each for half an hour of their time along with an incentive of 0.25 CHF per pair for each produced ravioli of good quality.The task consisted of two kinds of sessions, namely, performance sessions and transmission sessions. The question-response sequences analyzed in this study were collected from performance sessions only where participants prepared ravioli together in pairs. Their goal was to produce as many good-quality ravioli as possible in 10 min. Each pair had at their disposal a ball of 150 g of dough, 200 g of filling made of ricotta cheese and concentrated tomato paste , a 24-hole ravioli mold with zigzag sealing for easy release, a pasta maker, a rolling pin, a cutting board, 2 pizza cutters, 2 knives, 4 teaspoons, 2 kitchen cloths and kitchen paper, 250 g of flour, and a stopwatch. The collaborative food preparation task had 10 consecutive phases listed as follows: (1) divide the dough in two, (2) add flour to the dough, (3) use rolling pin to flatten the dough, (4) use pasta maker to flatten the dough, (5) cut dough in half, (6) put the first layer of dough over the mold, (7) add the filling, (8) cover with second layer of dough, (9) flip over the mold, and (10) cut ravioli. Immediately after the time was up, the ravioli was assessed by the experimenter.The identification of questions followed the coding scheme for question-response sequences in conversation developed by In French, content (Q-word) questions include interrogative pronouns such as qui/who, que/what, lequel/which, où/where, quand/when, and combien/how much. They can be followed by noun phrases, which form a syntactic constituent. Polar questions can be accompanied by a finite verb, a negative adverb, adverb of frequency, a clitic, or a subject. In alternative questions, each of the alternatives in the question is stressed. Any positive polar question can be turned into an alternative question by adding ou/or and the interrogative final particle hein/not. Declarative syntax ending with an interrogative intonation is the more frequent way in which questions are composed in French .TM for later synchronization with multimodal annotations in ChronoViz  reported in the literature. Data were transcribed in InqScribehronoViz . TranscrhronoViz . The synn = 1,521) of the 1,751 question-response sequences initially identified were task-related. New rounds of coding and revision of codes led to the categorization of the questions opening the 1,521 question-response sequences into three distinguishable themes. The interactive functions of questions in the collaborative food preparation task were as follows: (1) anticipation questions, (2) exploration questions, and (3) confirmation questions. Anticipation questions were requests for information about embodied actions that participants should perform in the next phases of the collaborative task while completing the present phase. Exploration questions were requests for information about embodied actions that the participant posing the question should execute to successfully progress in the ongoing phase of the task. Confirmation questions were requests for confirmation about embodied actions that were just performed or about to be performed in the ongoing phase of the task. Anticipation questions corresponded for 26% (n = 394), exploration questions corresponded for 41% (n = 618), and confirmation questions corresponded for 32% (n = 491) of all the task-related questions that initiated the 1,521 question-response sequences that we analyzed. Twenty percent of the 1,521 question-response sequences (n = 304) were double-coded. Inter-rater agreement for the three types of questions was very good (kappas ≥ 0.79). Disagreements across coders were resolved through discussion.The familiarization with the data and the initial coding resulted in the differentiation between questions related to the collaborative task and questions that were not. We found that 84% (Below we present an example and description of each type of question-response sequence taken from the dataset to illustrate how they operated in practice.Example 1 shows the coordination of an anticipation question with embodied actions and transition phases in the collaborative task (refer Methods for description of task phases).P1 made a polar question (1) acting as an anticipation question. This is the first element of the adjacency pair. He made it while pressing the first layer of the already flatten dough against the ravioli mold with his hand palms . The resExample 2 presents an occurrence of an exploration question that one participant posed while performing an embodied exploration of the pasta maker. Dyads were instructed to carefully pass the dough through the pasta maker, which had two rollers that could be adjusted using the knob on the side. This allowed making the dough gradually thinner. Each time before passing the dough through the pasta maker, dyads had to turn the knob to switch levels . The change in gaze direction of P3 toward the pasta maker directed the attention of P4 toward the same object . DirectiExample 3 illustrates a question-response sequence about joint decision-making that was initiated by a confirmation question. The sequence shows participants deciding where they should trim the excess dough so it could successfully cover the entire surface of the ravioli mold.The polar question of P5 (1) acted as a request for confirmation that opened the question-response sequence. P6 confirmed the decision of P5 to trim the excess dough where she had previously planned. However, he did it quietly using reduced speech volume (2) which did not seem to be very convincing for P5. As a result, P5 made a second confirmation question (3) as she had to be completely sure about how much excess dough she had to trim off. Having removed too much dough may have resulted in a shorter layer of dough that did not cover the complete surface of the mold. After a short silence, she lifted her head a looked at P6 . The chaWe investigated the interactive functions of content (Q-word) questions, polar questions, and alternative questions in a dataset of 1,751 question-response sequences collected in an experimental study in which 67 pairs of participants completed an embodied collaborative task in the laboratory. We did it using a collaborative food preparation task. The kind of task chosen aimed at increasing the ecological validity of the experimental design , 2019. FWe made a distinction between task-related and non-task-related questions  and observed that a significant proportion of questions were task-related. Then, we identified the interactive functions of task-related questions using the coding scheme developed by The three illustrative examples we presented showed the multiple ways in which the three types of questions were linked to the accomplishment of interactional goals: (i) planification of future collaborative activities (example 1), (ii) learning how an essential tool for the successful completion of the collaborative task works (example 2), and (iii) decision-making about an embodied action that would negatively affect group performance if it were done incorrectly. Interestingly, such goals referred to complementary temporalities in the activity. While example 1  referreda priori hypotheses to test. For example, to have tested whether anticipation questions predicted higher performance in the number of good quality ravioli produced in comparison with exploration and confirmation questions would not have been appropriate considering that we did not have a condition for each type of question to calculate how each affected the performance individually. Another limitation of this study was the fact that we did not analyze the temporal distribution of questions in relation to their interactive functions throughout the embodied collaborative task. For example, it may have been the case that exploration questions were more frequent at the beginning of the task when participants had to figure out the functions of the cooking utensils that they had available. These are the kind of hypotheses we plan to test in further studies.The exploratory nature of the study did not allow us to define Future experimental studies will enable researchers to make predictions about what types of questions may lead to an increase in the quality of embodied collaborative work  across tasks and large samples. This is important if we want to propose interventions to improve collaborative work involving the manipulation of physical and digital objects.https://osf.io/5wdgt/.The data that support the findings of this study are openly available at the Open Science Framework (OSF): The studies involving human participants were reviewed and approved by Ethics Committee, University of Neuchâtel, Neuchâtel, Switzerland. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.LB: conceptualization, data curation, formal analysis, investigation, methodology, project administration, and writing original draft. FB: investigation, formal analysis, and writing original draft. Both 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.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.


Cumulative and synergistic impacts from environmental pressures, particularly in low-lying tropical coastal regions, present challenges for the governance of ecosystems, which provide natural resource-based livelihoods for communities. Here, we seek to understand the relationship between responses to the impacts of El Niño and La Niña events and the vulnerability of mangrove-dependent communities in the Caribbean region of Colombia. Using two case study sites, we show how communities are impacted by, and undertake reactive short-term responses to, El Niño and La Niña events, and how such responses can affect their adaptive capacity to progressive environmental deterioration. We show that certain coping measures to climate variability currently deliver maladaptive outcomes, resulting in circumstances that could contribute to system ‘lock-in’ and engender undesirable ecological states, exacerbating future livelihood vulnerabilities. We highlight the significant role of social barriers on vulnerabilities within the region, including perceptions of state abandonment, mistrust and conflicts with authorities. Opportunities to reduce vulnerability include enhancing the communities’ capacity to adopt more positive and preventative responses based on demonstrable experiential learning capacity. However, these will require close cooperation between formal and informal organisations at different levels, and the development of shared coherent adaptation strategies to manage the complexity of multiple interacting environmental and climatic pressures. Ecosystems and human communities on low-lying coasts  characterizes channel margins and river mouths, fronting mangrove communities dominated by Avicennia germinans (black mangrove) with scattered Laguncularia racemosa (white mangrove). Conocarpus erectus (buttonwood), Pelliciera rhizophorae (tea mangrove) and the fern Acrostichum aureum are found at the boundaries of the mangrove forest communities, with areas of shrubby Batis maritima  and Sesuvium portulacastrum (shoreline purslane)  Andrade . There i−1 HIMAT . The sed−1 HIMAT . Resulti2 coastal lagoon with a maximum water depth of 2 m and separated from the Caribbean Sea by a 400–800-m wide sand bar  to collect information on the degree of community dependency on the mangrove ecosystem, how periods of heavy rain or drought affect this dependency, and how the community copes with, and responds to, these hydrological and other environmental pressures. Following established techniques  the misuse of the mangrove and lagoon for disposal of waste from an increasing peri-urban population, particularly in La Boquilla where waste accumulation is a key cause in the reduction in mangrove extent in the Ciénaga de La Virgen  populations due to changing rates and patterns of sedimentation during the period 1995–1996, following works to improve water connectivity of the lagoon with the Magdalena River. The harvest of oysters declined and the income of fishers was reduced by 18% as a consequence , and expressed an overwhelming feeling of abandonment by the state. As an example, according to the accounts given to the authors by local community members, the last Colombian President who visited the CGSM was Ernesto Samper Pizano (President 1994–1998) in 1996/1997. Communities in CGSM face shortages of basic services, such as water supply, sewerage and electricity  during extreme floods in La Niña episodes, responses included moving to a dry neighbouring house, sleeping in their canoes or temporarily relocating to a nearby city ; or (b) for those living in the stilt house villages at the CGSM, building a second (or even third) floor (tambo) onto their homes, which they then dismantle once the water level decreases or, in the same way, (c) those making a living from harvesting clams cope with rise in water level by lengthening the stilts (zanco) that they use to move around in the water. Second, there are responses that devise alternative livelihood practices based on existing knowledge and experiences. Hence, responses are dominated by an individual’s ability to be flexible and diversify their source of livelihood: ‘one cannot let himself starve, we find another activity. We had to abandon all the work in agriculture and survive making crafts’. Third, communities’ responses also included the acquisition of new knowledge: ‘those of us who did not know how to fish, we learned, and those who did not know about crafts also learned, because it was a way to survive in the time of that flood’. For example, fishers learn to harvest alternative fish species, use different fishing gear and change the areas where they fish according to environmental and climatic conditions.Crassostrea rhizophorae) was previously harvested as a primary resource, but the species declined sharply in the late 1990s following a combination of El Niño in 1995–1996, La Niña between 1998 and 2000, changes in salinity and an increase in sedimentation  has also been encouraged by the establishment, since 2000, of three crab processing plants in Puebloviejo on the north-east edge of the CGSM . It also highlighted the underpinning role of the ecosystem in the interactions between environmental and climatic risks and coping responses (Elwell et al. Our study provides an example of how proactive attitudes, in-depth ecosystem knowledge and adaptation learning processes assist coastal poor communities to cope with difficult times. However, the communities’ responses are mostly shaped by short-term gains, and weak institutional support in mitigating, preparing and helping these communities to respond to, and recover from, natural and climatic hazards. As the planet warms, and sea-level rises, understanding how the interactions of physical, climatic, environmental and institutional mechanisms re-organize behavioural responses to these threats needs to complement the information available from quantitative and qualitative models of social vulnerability, to offer predictions of potential thresholds that can ‘lock-in’ communities in undesirable socio-ecological states. Future development of governance for climate change adaptation needs to tackle all these dimensions of vulnerability.


Non-invasive diagnostic and prognostic biomarkers for urinary bladder cancer (BC) are eagerly awaited, since these could improve patient’s quality of life and reduce resources and costs for the health care system. The PD-1/PD-L1 axis is an important immune escape mechanism for tumors, and both high tissue expression and soluble levels of PD-L1 have been shown to be associated with worse prognosis for patients with various tumor. The knowledge about soluble PD-L1 in body fluids such as serum and urine in patients with BC is sparse, and more studies are needed to investigate its role as a biomarker for the disease. In the present study, we show that high serum PD-L1 levels are associated with aggressive disease and development of metastatic disease in BC patients.p = 0.038 and p = 0.61, respectively). Significantly higher serum sPD-L1 levels were found in patients with muscle invasive disease and metastatic disease, compared to patients with non-muscle invasive BC and non-metastatic disease (p < 0.05). There was also a trend for higher urine sPD-L1 levels in patients with metastatic disease, compared to patients with non-metastatic disease (p = 0.05). The results from this study suggest that sPD-L1 in serum, but not in urine, could be a potential prognostic biomarker for patients with BC.Soluble PD-L1 (sPD-L1) levels have been identified as a potential biomarker for various cancers, but its diagnostic and prognostic value in urinary bladder cancer (BC) remains to be fully elucidated. In this study, we investigated sPD-L1 levels in serum and urine samples from 132 patients with BC and compared them to 51 patients with hematuria (controls). The levels of sPD-L1 in serum and urine were determined using ELISA. Soluble PD-L1 could be detected in 99.5% of the serum samples and 34.4% of the urine samples. Patients diagnosed with BC had significantly higher urinary levels of sPD-L1, compared to controls, however no difference were found in serum sPD-L1 levels ( Urothelial bladder cancer (BC) is the ninth most common cancer worldwide with an estimation of 549,000 new diagnoses in 2018, and each year 200,000 people die due to the disease . PatientIn order to survive and progress, tumor cells needs to evade destruction by the immune system. One mechanism for this is to deregulate immune checkpoints, which are involved in a plethora of pathways crucial for maintaining self-tolerance and for modulating the duration and amplitude of immune responses. One such immune checkpoint is the programmed cell death ligand 1 (PD-L1), which is a ligand to the co-inhibitory receptor programmed cell death 1 (PD-1). Binding of PD-L1 to PD-1 results in inhibition of the T-cell mediated immune response, which could result in tumor cells escaping immune surveillance ,7. The eBoth PD-1 and PD-L1 exist in soluble forms, namely soluble PD-1 (sPD-1) and soluble PD-L1 (sPD-L1) . SolublePatients were recruited from the BLAdder cancer Blood and Urine Study (BLABUS), a prospective cohort study consisting of patients with macroscopic hematuria who were referred to the Department of Urology at the University hospital of Örebro for suspicion of bladder cancer. The vast majority of the patients underwent computed tomography urography, and an outpatient cystoscopy. Patients with visual suspicion of a bladder tumor at cystoscopy or urography thereafter underwent a transurethral resection of the bladder tumor (TUR-B) under general anesthesia. The diagnosis of BC was confirmed by a histopathological examination of the resected tissue samples. Tumor stage was defined according to the 2009 TNM classification of BC, and tumor grade was designated according to the WHO 2004 and WHO 1999 grading schemes. Patients without a history of BC and with a normal cystoscopy and urography were included as controls. Clinical data were extracted from medical records until 1 May 2019. Cause of death was defined as cancer specific if the patient had metastatic BC at time of death, and unrelated to BC if no signs of metastatic BC were detected at time of death. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the ethics committee in Uppsala/Örebro region .g for 10 min at 4 °C. Both urine and serum samples were stored at −80 °C until analyses were performed. All samples were analyzed retrospectively, and samples from cases and controls were processed simultaneously. At inclusion, blood and urine samples were obtained from all patients, and were collected prior to any cystoscopy or treatment of the patient. Urine samples were centrifuged at 2000 rpm for 10 min, and the serum was obtained by centrifugation at 3000× Soluble PD-L1 in serum and urine were measured by a commercially available ELISA for PD-L1, according to the manufacturer´s instructions . Control samples with known concentrations of PD-L1  were included in each ELISA . The minimum detectable level of sPD-L1 in the ELISA was 4.52 pg/mL, and the detection range was 25.0–1600 pg/mL. The optical density was measured using a Multiskan Ascent plate reader  at 450 and 540 nm. 2-values above 0.9 were considered as acceptable. Samples were measured in duplicates, and for each sample the coefficient of variation  was calculated. The acceptable range of CV was 0–10%, samples with higher CV values were analyzed one more time. Blanks and standards were assayed according to the manufacturer´s instructions. The mean values of Absorbance vs. Concentrations were plotted and a 4-Parameter Logistic (4PL) non-linear regression model fit was applied, and Rp < 0.05.In order to test the data for normality, a Shapiro-Wilks test was used. A Mann-Whitney U test or Kruskal-Wallis test were subsequently used to compare sPD-L1 levels between groups of patients. The urinary levels of sPD-L1 were dichotomized into absent or present, based on the detection level of sPD-L1 in the analysis method, and a Chi-square test was performed to test for association between dichotomized urinary sPD-L1 and cancer. Chi-square or Fisher´s exact tests were performed to test for associations between categorical variables. A Spearman correlation test was used to test for correlation between continuous data variables. Levels of sPD-L1 were subsequently divided into low or high, based on the median level of sPD-L1 among cases. The Kaplan-Meier method was used to estimate survival probabilities and the log-rank test was used to compare groups. A logistic regression model was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of the association between sPD-L1 levels and metastatic disease. All analyses were performed in SPSS version 22.0 . A statistical significance was considered at A total of 132 BC patients and 51 controls were recruited between 1 October 2016 and 31 March 2019. Compared to controls, patients diagnosed with BC were significantly older at inclusion, they were more commonly current or former smokers, and of male sex. No difference in body mass index (BMI) at the time of inclusion were seen between cases and controls . Of the p = 0.002). No correlation between sPD-L1 in serum and BMI, age, or smoking status were found. Soluble PD-L1 could be detected in 182 of 183 (99.5%) of the available serum samples and the median concentration was 95.2 pg/mL (65.2–214.4 pg/mL). Men had significantly higher levels of sPD-L1, compared to women . No significant difference in serum levels of sPD-L1 between cases and controls were found . When adjusting the model for tumor grade (low vs. high) and stage, this association was diminished, and no longer statistically significant . Patients that died during follow-up  had significantly higher serum sPD-L1 levels compared to patients who survived . A survival analysis showed that patients with sPD-L1 levels above median (94.0 pg/mL) had shorter overall survival time (19.6 months), compared to patients with sPD-L1 levels below the median value . Furthermore, no correlations were found between sPD-L1 in urine and BMI, age, or smoking status. Soluble PD-L1 could be detected in 56 of 163 (34.4%) of the available urine samples, and the median concentration was 74.2 pg/mL (57.5–669.2 pg/mL). No correlation between serum sPD-L1 and urinary sPD-L1 levels were found . No associations between sPD-L1 levels in urine and pT-stage and grade (low vs. high) were evident , however patients who were diagnosed with metastatic disease or who developed distant metastases during follow-up had higher levels of urinary sPD-L1, compared to patients without metastatic disease (p = 0.05). No association between sPD-L1 levels in urine and all-cause mortality was found (p = 0.09).Patients with BC had higher sPD-L1 levels in urine compared to controls  compared to patients with low stage disease (T0–T2), although they found no association between sPD-L1 and lymphatic or visceral metastases . SupportEven though there have been therapeutic breakthroughs for BC with the use of antibodies targeting PD-1 or PD-L1, there are still many questions that needs to be answered. One of the most important questions is why only a portion of the patients responds to immunotherapy. Thus, there is a demand for biomarkers that can identify responders versus non-responders prior to applying immunotherapy. Levels of sPD-L1 have previously been associated to treatment response to the immune checkpoint inhibitor Nivolumab (anti-PD-1) in patients with e.g., lung cancer . FurtherA strength with the current study is the choice of controls, which reflects a clinical setting where the major aim is to separate between patients presenting with hematuria due to BC or other causes. We have also investigated the levels of sPD-L1 in both serum and urine samples from these patients. The study also has some limitations, mainly the small cohort size and the short follow-up time of the patients . Another limitation of this study is that the tissue expression of PD-L1 were not measured in these patients, thus making it impossible to compare mPD-L1 expression to sPD-L1 levels in serum and urine. Even though the study was underpowered for some of the analyses, the results suggest a potential value of serum sPD-L1 as a prognostic biomarker for BC, which motivates further investigations using larger study cohorts with longer follow-up time. Our results showed that sPD-L1 can be detected in both serum and urine samples in BC. Patients with BC had higher urinary sPD-L1 levels than controls, but since only a third of the samples had detectable levels it appears like urinary sPD-L1 is unsuitable as a biomarker for BC. High sPD-L1 levels, mainly in serum, were found to be associated with a more aggressive BC, as indicated by pT-stage and metastatic disease, and could thus be a potential prognostic biomarker for the disease. Further prospective and longitudinal studies are needed to assess serum sPD-L1 as a biomarker for monitoring of BC patients.


In view of their high surface area and fast charge transport provided by their 3D porous structure, graphene-cellulose aerogels have great potential as sustainable supercapacitor electrodes.With increasing energy demand driving the need for eco-friendly and efficient energy storage technology, supercapacitors are becoming increasingly prevalent in wearable devices because of their portability and stability. The performance of these supercapacitors is highly dependent on the choice of electrode material. The high capacitance and mechanical properties needed for these materials can be achieved by combining graphene’s stable electrical properties with renewable cellulose’s excellent mechanical properties into porous aerogels. In this study, graphene-cellulose hydrogels were prepared by a one-step hydrothermal method, with porous, ultra-light, and mechanically strong graphene-cellulose aerogels then prepared by freeze-drying. These composite aerogels possess excellent mechanical strength and high specific capacitance, capable of bearing about 1095 times the pressure of their own weight. Electrochemical tests show the specific capacitance of these composite aerogels can reach 202 F/g at a scanning rate of 5 mA/cm Supercapacitors are an increasingly prevalent type of energy storage device, characterized by fast charge/discharge rates, long cycle life, high power density, and wide operating temperature range. They have established applications in numerous fields, including national defense, railways, new energy vehicles, electronics, communications, and aerospace ,2,3. WheGraphene is an ideal electrode material for capacitor because of its large specific surface area, more energy storage per unit mass, fast charge-discharge, and theoretical capacitance of 550 F/g ,7,8,9. HThere are two main ways to solve graphene agglomeration and expand the application of graphene materials in supercapacitors. One is to prepare three-dimensional structures such as graphene hydrogel, graphene aerogel, and graphene foam ,14. The 2 scanning rate. Lightweight composites can also withstand about 1095 times their weight without breaking. Effective spacer selection and simple and fast preparation methods make this porous, light, mechanically strong, high capacitance and environmentally friendly three-dimensional aerogel structure have great potential for supercapacitor electrode materials.In order to effectively solve the problem of graphene agglomeration, the electrical properties of graphene are maximized to prepare electrode materials for supercapacitors with excellent quality. In this work, a new preparation method of graphene-cellulose composite aerogel was proposed. It is the combination of the three-dimensional structure of graphene prepared by hydrothermal reduction method and the insertion of spacers between graphene sheets. Firstly, the graphene-cellulose composite hydrogel was prepared by hydrothermal method in the mixed solution of GO and cellulose, and then, the graphene-cellulose composite aerogel was prepared by freeze drying. The specific capacitance of the composite aerogel can reach 202 F/g at 5 mA/cmAs shown by SEM, all samples exhibit uniform three-dimensional porous structure. From From −1, 1719 cm−1, 1660 cm−1, and 1385 cm−1 correspond to O-H, C=O, C=C, and C-OH vibrations indicating the presence of hydroxyl, carbonyl, and benzene carboxyl groups bounded to the basal and edges of GO, respectively. In the FTIR spectra of GA aerogels and GA-MCC aerogels, these bands weakened. Even at 3338 cm−1 and 1660 cm−1 positions, there is almost no band. This indicates that GO has been reduced to GA [−1 and 1428 cm−1 vibration bands represent O-H and C-H bonds, respectively, which are characteristic bands of cellulose [−1 and C-H bond at 1428 cm−1 increase in turn. It can be concluded that with the increase of cellulose content in raw materials, the number of O-H and C-H bonds in the composites increases gradually. O-H bond and C-H bond can form hydrogen bond, so that the mechanical strength of the composites increases gradually, which also provides the basis for the mechanical properties test results later.ed to GA ,42,43,44ellulose . From thThe specific surface area and pore structure of GA aerogel and graphene-cellulose composite aerogel were determined by nitrogen adsorption-desorption test. As shown in As shown in 2/g). For GA-MCC composite aerogels, the cellulose in mesoporous can not only effectively improve the accessible surface area of GA and electrolyte but also significantly reduce the ion diffusion distance from electrolyte nanoreservoir to GA nanosheets. Therefore, the capacitance performance of GA-MCC-4 aerogel is slightly increased compared with GA aerogel. According to the CV curve and specific capacitance calculation formula, the specific capacitance of GA-MCC-4 aerogel can reach 202 F/g at the scanning rate of 5 mA/cm2. At the scanning rate of 10 mA/cm2, the specific capacitances of GA aerogel and GA-MCC-4 aerogel were 91 F/g and 147 F/g, respectively. According to the previous literature, Lamberti, A et al. also used hydrothermal reduction method to prepare graphene and copper composite aerogel. The highest specific capacitance was 62.3 F/g at 5 mA/cm2 scanning rate [2 [2 [In order to evaluate the electrochemical behavior of the samples, electrochemical tests were carried out on GA aerogel and GA-MCC-4 aerogel. The electrical properties were evaluated by cyclic voltammetry and AC impedance method. ing rate . The red rate [2 . The redte [2 [2 .3) was purchased from Shanghai Yixin Chemical Co., Ltd. . Potassium permanganate powder (KMnO4) was purchased from Qufu Xinxin Chemical Co., Ltd. . Sulfuric acid (H2SO4 98 wt.%) and hydrogen peroxide (H2O2 30 wt.%) were purchased from Anshan Anji Chemical Co., Ltd. . Deionized water was purchased from Sigma-Aldrich . Microcrystalline cellulose (MCC) powder (particle size: 50 μm) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. .Graphite crystal (99 wt.%) was purchased from Jiangsu Changde High-tech Carbon Materials Co., Ltd. . Sodium nitrate  was dissolved in 20 mL deionized water to prepare 10 mg/mL GO solution. Six groups of 10 mg/mL GO solution were added 0, 20, 50, 100, 150, and 200 mg cellulose, stirring at room temperature for 2 h, until the solution mixed evenly. Pour six groups of solution into the hydrothermal reactor, and then put the hydrothermal reactor into the oven. Graphene hydrogel and five groups of graphene-cellulose hydrogel were obtained by heating at 180 °C for 10 h. All samples were frozen rapidly in liquid nitrogen and then put into the freeze dryer. After 48 h, graphene aerogel and graphene-cellulose aerogel were obtained. They were named GA, GA-MCC-1, GA-MCC-2, GA-MCC3, GA-MCC-4, and GA-MCC-5, respectively, and further characterization experiments were carried out.−1. X-ray diffraction (XRD) analysis was performed using XRD-6100 in the scanning range of 5–60° and the scanning speed of 5°/min. Brunner–Emmet–Teller (BET) measurement is to obtain the specific surface area and pore structure of aerogels by nitrogen adsorption. The dynamic thermal analyzer (DMA) with the model of DMA242E Artemis was used. The sample was cut into a cylinder with a height of 0.5 cm, and the compression test was carried out at room temperature. Electrochemical measurement was performed on an electrochemical workstation (1470 E), using 6 M potassium hydroxide solution as electrolyte, silver chloride electrode as reference electrode, graphite rod as counter electrode, platinum sheet electrode clip as working electrode. The aerogels were cut into small wafers with a height of 0.25 cm and a diameter of 1.8 cm, and one quarter was taken as the experimental material. The electrical properties were evaluated by cyclic voltammetry and AC impedance. The scan rate of CV curve was 5, 10, 20, 50, and 100 mA/s, and the potential window was −0.2–0.2 V. The electrochemical impedance spectroscopy (EIS) curves were collected from 10 mHz to 100 kHz.The scanning electron microscope (SEM) images were obtained by using QUANTA200 instrument. Transmission electron microscopy (TEM) was measured by transmission electron microscopy (JEM-2100). Fourier transform infrared spectroscopy (FT-IR) was performed on Spectrum 400 instrument, and the scanning range was 550–4000 cm2. This lightweight, solid, high capacitance, green three-dimensional aerogel structure has great potential value as electrode material for supercapacitors.In this work, graphene-cellulose hydrogels were prepared by one-step hydrothermal method, and then freeze-dried to prepare graphene-cellulose aerogels. The surface morphology, internal composition, mechanical and electrical properties of the composite aerogels were obtained by SEM, TEM, XRD, FTIR, BET, mechanical property test, and electrical property test. SEM, TEM, and BET tests showed that the composite had uniform mesoporous structure and large specific surface area, and the cellulose and graphene had been mixed evenly. The results of XRD and FTIR confirmed that there was a close hydrogen bond between cellulose and graphene, and cellulose had been successfully inserted between graphene sheets to slow down its π–π stacking. Through the pressure test of composite aerogels, it was found that the light composite aerogels could withstand the weight of about 1095 times of their own weight without breaking. Moreover, with the increase of cellulose content, the pressure value of composite aerogels increased. It shows that cellulose plays a supporting role in the composite. When the electrical properties of the composite were tested, the specific capacitance of the composite aerogel could reach 202 F/g at the scanning rate of 5 mA/cm


We used the NHATS COVID-19 module to examine whether individuals in long-term care communities were lonelier than individuals in the community during the pandemic. Additionally, we examined whether individuals in long-term care communities with more restrictive policies concerning visitors and communal activities were more likely to experience loneliness than individuals in communities with less restrictive policies. Approximately 45% of individuals in long-term care communities (n=134) felt at least a moderate amount of loneliness during COVID-19 when compared to 34% of individuals in the community  (p<.05). However, the association was no longer statistically significant after adjusting for age, race, and sex. Among individuals in long-term care communities with the most restrictive policies, 48% experienced loneliness compared to 44% individuals in less restrictive communities. However, this finding was not statistically significant. Discussion will focus on similarities and differences within these populations that could have led to these results.


Sarcoidosis is a systemic granulomatous disease affecting various organs, and the lungs are the most commonly involved. According to guidelines, diagnosis relies on a consistent clinical picture, histological demonstration of non-caseating granulomas, and exclusion of other diseases with similar histological or clinical picture. Nevertheless, chest imaging plays an important role in both diagnostic assessment, allowing to avoid biopsy in some situations, and prognostic evaluation. Despite the demonstrated lower sensitivity of chest X-ray (CXR) in the evaluation of chest findings compared to high-resolution computed tomography (HRCT), CXR still retains a pivotal role in both diagnostic and prognostic assessment in sarcoidosis. Moreover, despite the huge progress made in the field of radiation dose reduction, chest magnetic resonance (MR), and quantitative imaging, very little research has focused on their application in sarcoidosis. In this review, we aim to describe the latest novelties in diagnostic and prognostic assessment of thoracic sarcoidosis and to identify the fields of research that require investigation. Sarcoidosis is a systemic inflammatory disease characterized by widespread development of non-caseating epithelioid cell granulomas, and the lungs are the most affected organs .According to the statement on sarcoidosis released by the American Thoracic Society (ATS), the World Association for Sarcoidosis and Other Granulomatous Disorders (WASOG), and the European Respiratory Society (ERS), the diagnosis relies on a consistent clinical picture, histological demonstration of non-caseating granulomas and exclusion of other disease with similar histological or clinical picture. In this context, chest X-ray (CXR) plays a crucial role obviating the need for biopsy in patients with typical clinical presentation and typical radiographic findings, the so called Löfgren syndrome, characterized by bilateral hilar lymphadenopathies, erythema nodosum, and/or bilateral ankle arthritis .Computed Tomography (CT) is more sensitive than CXR in identifying lymphadenopathies and in depicting subtle parenchymal abnormalities , and it Prognostic assessment in sarcoidosis still relies on a four-level staging system based on radiographic appearance, conceived by Scadding in 1961, nearly sixty years ago, that goes from stage 0—normal findings—to stage IV—lung fibrosis with no chance of clinical and radiographic resolution if untreated ,5.Therefore, despite the advances in chest imaging modalities including the wider use of radiation-saving techniques, the ever-increasing applications of chest magnetic resonance (MR), and the introduction of objective and quantitative techniques, according to guidelines, diagnostic and prognostic assessment in sarcoidosis seems to be stuck on CXR ,4. In thImaging plays a central role in both diagnosis and monitoring of thoracic sarcoidosis .Typical and atypical radiographic and CT findings in sarcoidosis have been widely described in literature, with high-resolution computed tomography (HRCT) being far superior in detecting and characterizing lung disease compared to CXR . LymphadLymphadenopathies are the most common intrathoracic manifestation of sarcoidosis (occurring in 75–80% of patients at some point in their disease) and are typically symmetrical and non-necrotic, with extremely variable degree of enlargement. Lymph node calcification is not uncommon and, when present, may be punctate, amorphous, eggshell, and popcorn-like in appearance . The chaTypical parenchymal abnormality consists of small nodules, occurring with a perilymphatic distribution and with variable degree of involvement among individual patients, typically visible along the peribronchovascular interstitium, the pleural surfaces, including fissures, and the centrilobular regions; sarcoid granulomas frequently cause nodular thickening of the axial perihilar and peribronchovascular interstitium .Nodules are usually small (diameter 1–4 mm), with irregular borders and often have a bilateral and symmetrical distribution involving mainly upper and middle lung zones. Less commonly, they may show an asymmetric involvement of the lung parenchyma or a lower lung zone predominance; exceptionally a miliary pattern may be seen .alveolar sarcoidosis); occasionally they contain air bronchograms. Cavitary lesions are rare (3.4–6.8% of cases), usually indicating severe and active sarcoidosis, resulting from either ischemic necrosis or vasculitis . As . As 4]. Current guidelines in management of sarcoidosis recommend serial chest radiography—evaluating possible shifts in the Scadding staging—and PFTs (pulmonary function tests) in order to detect changes in disease severity ,4. NeverOver the past years, many studies have tried to overcome the aforementioned limitations of chest radiography in prognostic assessment of sarcoidosis.not in Scadding stage—significantly correlated with pulmonary function trends  [First attempts were made by combining a semiquantitative radiographic scoring system—yet subjective—with clinical or physiologic data ,36. ZappC, DLCO) .p < 0.0001) [HRCT has allowed an additional improvement in prognostic assessment of sarcoid patients. Some studies have combined HRCT findings and physiological variables, which independently may predict survival, presuming that a more powerful prognostic discrimination may come from an integration of both ,38. WalsHowever, these studies share a common limit, which is the underlying subjectivity in their semi-quantitative methods and poor inter-observer agreement. Over the last few years, quantitative CT (QCT), defined as the use of computerized tools to quantify disease on CT, and radiomics, with their objective and reproducible results, have been tested in the assessment of several lung disease, including lung cancer, emphysema and interstitial lung disease ,39,40. Tp < 0.0001 for all comparisons), that, currently, guide the management of this disease [LR (standard deviation of lung radiodensity) was significantly higher in patients with lung fibrosis. Moreover, CT-QI correlated with FVC, FEV1, and TLC  [Erdal et al. conceived a two-point correlation function (TPCF) based approach to assess the severity of lung involvement in sarcoid patients using CT. They developed a program to produce a lung texture score (LTS) that objectively quantified the disease extension both in terms of interstitial and alveolar processes. LTS strongly correlated with PFT  ( disease . In the apacity) .Despite the potential detection of subclinical lung disease and the excellent correlation with functional tests, the prognostic value of the proposed approaches was not assessed.Other QCT tools have been developed, providing good results in terms of prognostic assessment and correlation with PFT, and have been tested in ILD, mainly IPF, but not in sarcoidosis. CALIPER , a QCT tool based on both 3D histogram features within a regional voxel and morphological analysis, has been tested in several fibrosing lung disease showing good agreement with PFT and prognosis. CALIPER has shown the ability to quantify the vessel-related structures (VRS), corresponding to the volume of pulmonary vessels and associated structures, such as perivascular fibrosis, which can not be quantified visually. The prognostic value of VRS has been shown in several fibrosing lung disease, including IPF, hypersensitivity pneumonitis, CTD-ILD, and unclassifiable ILD ,43. Evenp < 0.001 for skewness, kurtosis, fractal dimension, and Geary’s C), with sarcoid ones showing lower skewness and kurtosis—likely caused by increased opacification due to parenchymal abnormalities that make distribution of Hounsfield units in HRCT more normally distributed—and lower fractal dimension, higher Moran’s I and lower Geary’s C—indicating more similar adjacent pixels likely due to nodule conglomeration and/or fibrosis. Additionally, these measures significantly correlated with PFT, with significant association between Geary’s C and FVC (p = 0.008) and proved to be a better predictor of functional impairment compared to the Scadding stage system [Finally, only one study assessed the utility of Radiomics in sarcoidosis. Ryan et al. applied radiomic measures, obtained from HRCT, in 73 sarcoid patients and compared them with PFT. They found that global radiomic measures differed significantly between sarcoid patients and healthy controls (e system . AlthougImaging of sarcoidosis has been the subject of a wide number of publications over the last 60 years providing a detailed description of all the possible radiographic and CT findings associated with this disease. Nevertheless, despite the advances in thoracic imaging modalities and the introduction of new computerized tools for the analysis of chest imaging examination, diagnostic and prognostic assessment of sarcoidosis, according to guidelines, still relies on chest radiography. Some well-known techniques, including low dose CT and MRI, have not been explored enough in sarcoid patient. Other more recent techniques, including quantitative CT tools and Radiomics, are showing their potential in diagnostic and prognostic assessment of several ILD but have only marginally been tested in sarcoidosis. Future research assessing and validating these new modalities and techniques in sarcoid patients might lead to a different management of these patients allowing to obtain more information with a reduced or absent radiation exposure and to improve prognostic assessment.


OBJECTIVES/GOALS: Studying IPF associated health care utilization (HRU) in real world settings, provides the opportunity to produce generalizable results that can directly inform models of care delivery. The objective of this study was to examine real-world differences in the natural history of annual HRU and treatment trends associated with IPF in a large, community-based population of patients with IPF, compared to matched non-IPF controls. METHODS/STUDY POPULATION: Cases of IPF were identified using case validated algorithms in the Kaiser Permanente Northern California adult population from 2000 to 2014. Each case was matched to at least one and no more than five non-IPF controls by age, sex, race/ethnicity and length of enrollment. The date of the first occurrence of the IPF-specific diagnostic code was considered the index date for cases and matched controls. Comorbidity burden and HRU was assessed in the five years pre- and post-index date, including hospitalizations, outpatient visits, use of diagnostic and monitoring studies and medications. Poisson generalized estimating equations models with robust standard errors were used to estimate adjusted case-control differences in HRU, accounting for clustering within matched sets. RESULTS/ANTICIPATED RESULTS: 691 patients were identified with incident IPF during the study period and matched to 3,452 control subjects. Demographics were well balanced between cases and controls due to matching. Patients with IPF had a higher burden of all selected co-morbidities and higher HRU compared to controls. In the pre-index period, IPF members had significantly higher rates of all diagnostic procedures (p < 0.001) and health care visits, with the exception of skilled nursing facility care (p < 0.001). The greatest relative difference was observed with use of Chest CT . In the post-index period compared to controls, patients with IPF had higher rates of serial testing (p < 0.001) and inpatient and outpatient care including, all-cause hospitalization (1.55), emergency room visits (1.19), outpatient visits (1.18), and skilled nursing facility stay (1.35). DISCUSSION/SIGNIFICANCE OF IMPACT: Patients with idiopathic pulmonary fibrosis experience increased co-morbidity and healthcare resource utilization compared to controls. This increased burden extends beyond the index-period as previously documented and is true for a large real-world cohort. CONFLICT OF INTEREST DESCRIPTION: NA


Ink4a and p21Waf1/Cip1. However, the expression of alpha smooth muscle actin (ACTA2) and proteoglycan decorin (DCN) increased in response to IPF-derived ECM. Production of the proinflammatory cytokines C-X-C Motif Chemokine Ligand 8 (CXCL8) by lung fibroblasts was upregulated in response to senescent and profibrotic-derived ECM. Finally, the profibrotic cytokines transforming growth factor β1 (TGF-β1) and connective tissue growth factor (CTGF) were upregulated in response to both senescent- and profibrotic-derived ECM. In summary, ECM deposited by IPF fibroblasts does not induce cellular senescence, while there is upregulation of proinflammatory and profibrotic cytokines and differentiation into a myofibroblast phenotype in response to senescent- and profibrotic-derived ECM, which may contribute to progression of fibrosis in IPF.Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with poor survival. Age is a major risk factor, and both alveolar epithelial cells and lung fibroblasts in this disease exhibit features of cellular senescence, a hallmark of ageing. Accumulation of fibrotic extracellular matrix (ECM) is a core feature of IPF and is likely to affect cell function. We hypothesize that aberrant ECM deposition augments fibroblast senescence, creating a perpetuating cycle favouring disease progression. In this study, primary lung fibroblasts were cultured on control and IPF-derived ECM from fibroblasts pretreated with or without profibrotic and prosenescent stimuli, and markers of senescence, fibrosis-associated gene expression and secretion of cytokines were measured. Untreated ECM derived from control or IPF fibroblasts had no effect on the main marker of senescence p16 Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease of unknown aetiology that is characterised by exaggerated deposition of extracellular matrix (ECM) leading to an irreversible decline in lung function and ultimately death . GeneticIn IPF, the accumulation of fibroblasts and ECM leads to formation of fibroblastic foci, the active regions of fibrosis in which myofibroblasts are responsible for excessive matrix deposition and that invade morphologically healthy lung . The chaWe hypothesised that the altered composition of fibrotic ECM negatively impacts fibroblast function and leads to cellular senescence in IPF. Here, we aimed to investigate whether IPF-derived ECM was able to induce cellular senescence by culturing primary control fibroblasts on Ctrl– and IPF–fibroblast or senescent and profibrotic-derived decellularized ECM and, subsequently, analyse gene expression of markers of senescence and fibrosis and secreted factors that are both part of the SASP and the fibrotic response.https://www.umcg.nl/EN/Research/Researchers/General/ResearchCode/Paginas/default.aspx; last accessed 28 May 2021) and national ethical and professional guidelines . Additional lung fibroblasts were obtained from the Woolcock Institute of Medical Research, Australia. Approval was provided by the Human Ethics Committee of the University of Sydney and the Sydney South West Area Health Service. Fibroblasts were isolated from macroscopically and histologically normal tissue as described before [Primary human lung fibroblasts were obtained from lung tissue of patients undergoing lung transplantation or tumour resection surgery at the University Medical Center Groningen (UMCG). The protocol was consistent with the Research Code   supplemented with 10% foetal bovine serum , 100 U/mL penicillin, 100 µg/mL streptomycin  and 1% GlutaMAX (Gibco), referred to as complete growth medium, and used between passage 4 and 6. Fibroblasts were routinely checked for mycoplasma infection using a PCR assay and used only when certified negative . To quie2) were seeded per well in complete growth media and allowed to attach for 24 h. Cells were then maintained in incomplete growth media for another 24 h to quiesce the fibroblasts before treatment. For the generation of profibrotic ECM, fibroblasts were stimulated with 1 ng/mL transforming growth factor β1  for 72 h. To induce cellular senescence and senescent ECM, fibroblasts were exposed to 150 µM hydrogen peroxide  for 2 hrs, washed twice with an equal amount of phosphate buffered saline  and incubated in complete growth media containing 5% FBS for 72 h [Fibroblasts  were seeded on cover slips in complete growth medium and underwent the same treatment as described above. Coverslips were decellularized and fixed in 2% PFA and 0.2% glutaraldehyde and prepared for SEM. Cover slips were washed with 0.1 M cacodylate buffer and incubated with 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h at room temperature. Then, cover slipes were washed with water and dehydrated with 30, 50 and 75% ethanol (EtOH), 15 min per each step, respectively, followed by dehydration in 100% EtOH for 30 min. To dry the samples, cover slips were first incubated in a 1:1 mixture of 100% EtOH and tetramethyl silane (TMS) for 10 min followed by 15 min incubation with 100% TMS after which the cover slips were air dried. Afterwards cover slips were gold coated using a sputter coater and cover slips were imaged using a Zeiss Supra55 at 3 KV with SE2 detector .Fibroblasts  back on the ECM substrate in complete growth media and cultured for 72 h. After 72 h, culture supernatants were collected, pooled and centrifugated for 5 min at 300× g to remove debris and dead cells, after which cell-free supernatant was stored at −80 °C for further analysis. Fibroblasts were incubated using 0.5% trypsin/EDTA (Sigma–Aldrich) until all cells were detached. The trypsin/EDTA mixture was inactivated using Hank’s Balanced Salt Solution  containing 5mM ethylenediaminetetraacetic acid (EDTA) and 2.5% v/v FBS. Cells were stained with trypan blue (Sigma–Aldrich) and manually counted using a haemocytometer. In addition, cells were lysed in RNA lysis buffer  and stored at −80 °C until RNA isolation. Cells to be used for SA-β-Gal staining were fixed with 2% paraformaldehyde  and 0.2% glutaraldehyde in PBS for 5 min and directly stained using the protocol described below.Culture plates with ECM substrate were thawed at 37 °C and rinsed once with sterile PBS. PBS was aspirated, and two different fibroblast donors  were seeTotal RNA was isolated using a Nucleospin RNA isolation kit (Machery-Nagel) according to manufacturer’s instructions, after which the samples were stored at −80 °C. Total RNA was quantified using the Nanodrop ND−2000 . RNA was reversed transcribed into cDNA using the ReverseAid First Strand cDNA Synthesis Kit (Thermo Scientific). DNA was amplified using the GoTaq Probe qPCR Master Mix  and TaqMan Gene Expression Assay (Thermo Scientific) on a ViiA7 Real-Time PCR System , with the relevant predeveloped primers listed in Cellular senescence was assessed using a previously described protocol for senescence-associated β-galactosidase  staining . Fixed f5 cells counted.Secreted IL-6, CXCL8, DCN, TGF-β1, Osteoprotegerin (OPG), receptor activator of nuclear factor kappa-B ligand (RANKL) and CTGF were measured using Human DuoSet ELISA kits  according to the manufacturer’s instructions. Colour development was obtained through the reaction between 3,3′, 5,5″-tetramethylbenzidine (TMB) and horseradish peroxidase (HRP). As all extracellular RANKL, excreted by cells, was complexed to OPG and not detectable with a direct RANKL ELISA, a RANKL/OPG complex sandwich ELISA was developed by using a human capture RANKL antibody  and a human detection OPG antibody  [p < 0.05. Significance was either expressed as p-value or * p < 0.05, ** p < 0.01 and *** p < 0.001 unless stated otherwise.RT-qPCR data were analysed for outliers using the following approach. First, the coefficient of variability (CV) was calculated for 18S and GOI. Then, technical triplicates with a %CV above 1 were inspected and excluded if 18S was >0.2 Ct and >0.5 Ct for GOI as technical errors. SA-β-Gal outliers were detected using the following approach. The variation between duplicate samples and the number of positive cells in ImageJ did not match with the TissueFAXS images. Statistical analyses were performed using GraphPad Prism 8 . Data were tested for normal distribution using the D’Agostino and Pearson test. Data were log-transformed if distribution was not normally distributed, after which all data was normally distributed; then, we used a repeated measures one-way ANOVA, or, if data points were missing, a mixed-effects analysis (REML) was used. ANOVA tables can be found in the To investigate the effect of deposited ECM by control or IPF lung fibroblasts on the senescence induction, we first wanted to confirm that our decellularization protocol resulted in cell-free matrix deposition. Waf1/Cip1 and p16Ink4a confirmed this finding, as there was no change in expression in response between Ctrl- and IPF-derived ECM in both fibroblast donors  on IPF-derived ECM (p = 0.046) in fibroblasts cultured on IPF-derived ECM when compared to Ctrl-derived ECM  on senescent-derived Ctrl-ECM compared to control  on senescent derived Ctrl-ECM, while fibrotic-derived IPF–ECM demonstrated a decrease  in fibroblasts after three days of culture (Fibrotic- and senescence-derived ECM modulated the SASP cytokines IL-6 and CXCL8 in fibroblasts. To further investigate the response to stimulated ECM, we characterised fibrosis-associated gene expression and cytokine production in fibroblasts. The expression of ACTA2, COL1A1, FN1 and DCN did not demonstrate a difference in response to fibrotic- or senescence-derived ECM A–D. Anal control H. CTGF s culture I. Donor  culture .Ink4a, p21Waf1/Cip1, or a series of secreted factors, associated both senescence and fibrotic responses. However, when fibroblasts were cultured on ECM derived from TGF-β1- or H2O2-stimulated Ctrl- or IPF-fibroblasts, we detected upregulation in CXCL8, CTGF and TGF-β1. Collectively, the data presented in this study suggest that exposure of fibroblasts to Ctrl- or IPF-derived ECM under basal conditions does not induce cellular senescence nor induce cytokine production. Importantly, fibroblasts exposed to stimulated active environment differentially secrete factors that are likely to impact further fibrotic activities.In IPF, both cellular senescence and excessive/altered ECM deposition are identified as major drivers of fibrosis. These changes impact cell function and contribute to disease progression by creating a positive feedback loop favouring fibrosis. In the present study, we cultured fibroblasts on Ctrl- or IPF-fibroblast-derived ECM for three days and examined the cellular response, focusing on cellular senescence and expression of fibrotic markers. We report that unstimulated Ctrl- or IPF-derived ECM did not affect markers of senescence, such as p16Ctrl- and IPF-derived ECM generated from fibroblasts under basal and stimulated conditions which were decellularized and ECM deposition was confirmed with SEM. These ECMs displayed differences in structure between the basal and treatment groups. However, owing to technical limitations with the SEM, the images were captured of ECMs deposited on glass cover slips, which may reflect an underestimation of the amount of ECM deposited upon tissue culture plastic, as used for our cell seeding experiments. Furthermore, the SEM images in this study of cell-free ECM are comparable to those of Philp and colleagues . HoweverDecorin secretion was increased from fibroblasts exposed to IPF-derived ECM, compared to Ctrl-derived ECM. DCN has been implicated in IPF and plays a role in wound repair by limiting the function of both TGF-β1 and CTGF . The fibInk4a and p21Waf1/Cip1, the main markers of senescence; instead, it resulted in upregulation of proinflammatory cytokine CXCL8 compared to control ECM. These data indicate that ECM generated under stimulated conditions mimicking senescence or profibrotic environment leads to a cellular response, which is in line with what has been reported previously [The current study was designed to examine the effect of ECM on the induction of a senescent phenotype in fibroblasts. When the ECM was generated under basal conditions, both the control- and IPF-derived ECMs failed to induce changes in the senescence status of the fibroblasts, clearly demonstrating the assuaging effects of the ECM deposited when the fibroblasts are in a nonstimulatory environment. To further investigate the response of fibroblasts, we generated ECM under senescent or fibrotic conditions to mimic active fibrosis in control– and IPF–fibroblasts. Next, we characterised cellular senescence and the fibrotic response. Fibroblasts cultured on senescent- or fibrotic-derived ECM did not show an induction of p16eviously . Increaseviously . TogetheTo further expand on the finding that senescent- or profibrotic-derived ECM leads to activation of proinflammatory cytokines, we characterised fibrosis target genes and secretion profile. Fibroblasts cultured on IPF-derived ECM under stimulated conditions led to increased TGF-β1 and CTGF secretion. Surprisingly, we did not measure higher secretion levels of OPG in response to IPF–ECM. OPG is essential for ECM homeostasis by osteoclasts in the bone. Moreover, OPG is induced by TGF-β1, and it is suggested to bind CTGF, potentially contributing to fibrosis . On the Waf1/Cip1 and p16Ink4a in senescence, as reflected in this study [The experimental setup for this study was designed to measure the impact of ECM on the senescence phenotype after three days of culture. We did not observe any modulation in senescence markers after three days. This could implicate, in the context of senescence, that fibroblasts need prolonged exposure to an altered ECM before they make this adaptation. There is evidence suggesting that the upregulation of proinflammatory cytokines may precede the induction of cell-cycle inhibitors p21is study . Moreoveis study . It is nis study .In conclusion, we have shown that fibroblast exposed to Ctrl- or IPF-derived ECM under nonstimulated conditions does not induce cellular senescence. Instead, we observed upregulation of proinflammatory cytokines and profibrotic cytokines in response to senescent or profibrotic ECM. These data suggest that, to induce a strong response to their environment, not only is composition important in IPF but a secondary stimulant that induces DNA damage such as ROS, or pathological stiffness might be required to create a positive feedback look that perpetuates fibrosis in IPF.