Picha. pastoris strain X33. This FCP promotes cell migration and adhesion and maintains rBMSC stemness by binding integrin β3. Its effects were blocked by both integrin β3 siRNA and the integrin β3 inhibitor Cilengitide. A template-independent ab initio prediction modeling approach is the best approach to construct a stable FCP protein model, which predicts the binding sites between FCP and integrin β3. FCP may be used in the in vitro culture and clinical regeneration of stem cells that highly express integrin β3, such as hematopoietic stem cells. The study provides information on the molecular structure of FCP and its bioactivity, which can be used to design new compounds.Constructing bionic extracellular matrix (ECM) is an attractive proposition for tissue engineering and clinical regeneration therapy involving the stemness of stem cells. Here, a novel recombinant protein fibronectin-collagen peptide (FCP) was designed to modulate the function of ECM expressed by Design a novel recombinant fibronectin-collagen peptide biomimetic ECM.• FCP promotes cell adhesion, migration, and proliferation.• Predicted and verified FCP structure and affinity with integrin β3.• FCP binds integrin β3 to maintain rBMSC stemness.• The online version contains supplementary material available at 10.1007/s00253-022-11965-4. Stem cells play an important role in tissue engineering and clinical regeneration therapy due to their ability to self-replicate and potential to differentiate into multiple cell lines, called stemness Keating . The cliExtracellular matrix (ECM), a noncellular three-dimensional macromolecular network composed of collagens, fibronectin, elastin, and several other glycoproteins  and P. pastoris strain X33  were used for cloning and heterologous expression. PCR purification kits, gel extraction kits, and micro preparation kits were purchased from Tiangen . CC  was artificially synthesized by GL Biochem . FN10 was recombinantly expressed in our Lab  and ECV304-eGFP  cells were purchased from the Chinese Academy of Sciences . They were cultured in Roswell Park Memorial Institute 1640 medium  containing 10% fetal bovine serum, penicillin (100 I.U./mL), and streptomycin (100 μg/mL) . Rat bone marrow mesenchymal stem cells (rBMSCs) were extracted in our laboratory approved by the Animal Care Committee of Jinan University (JNU20200826-11) and were performed in accordance with animal ethics guidelines of Agricultural Animals for Research and Teaching at Jinan University. rBMSCs were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum, penicillin (100 I.U./mL), and streptomycin (100 μg/mL).pPICZαΑ vector to give a recombinant plasmid named pPICZαΑ-FCP; then, the pPICZαΑ-FCP was transformed into P. pastoris strain X33. Zeocin-resistant clones were selected on YPD plates containing 0.1 mg/mL of Zeocin to obtain different copies of the integrated DNA of FCP. All strains were cultivated overnight at 30 °C in 5 mL of YPD medium, and 1% of the culture was transferred to 25 mL of YPG in a 250 mL baffled flask. The cells were grown at 30 °C in a shaking incubator until the OD600 reached 1.5. The cell pellets were then induced in YPM. At the same time, methanol  was added to the culture, and 1-mL samples were collected at 0, 24, 48, and 72 h after induction. The expressions of proteins were monitored by SDS-PAGE. FCP proteins were purified using the Ni Sepharose 6 Fast Flow column combined with gel filtration Sephadex G-25. Polymerase chain reaction , agarose gel electrophoresis, and western blot , etc., were used to verify the authenticity of FCP. Expanded culture of highly expressing P. pastoris strain X33 in 2L YPD media was performed. Additionally, the circular dichroism (CD) spectrum of FCP was measured with a Chirascan plus Circular Dichroism Spectrophotometer .The cDNA encoding FCP was inserted into the An in vitro scratch wound healing model was used to assess the cell migration induced by FCP. Briefly, ECV304 cells were plated in 12‐well plates and cultured until approximately 95% confluence before the scratch study was undertaken. A scratch was created with a 1 ml sterile pipette tip. After scratching, cells were washed using PBS to remove cell debris caused by the scratch. Another 1 mL treatment solution  was added to cells. Three images of the scratch area were photographed using an inverted microscope  at 0, 18, and 36 h. The ImageJ software was used to determine percentage closure (%).In another separate experiment, ECV304 cells were treated with or without FCP, integrin β3 siRNA, and NC group (negative control: cells transfected with scrambled siRNA), respectively. All operations are performed as above.5 cells/mL) were seeded on tissue culture plates (96-well) and allowed to attach for 4 h at 37 °C. After incubation, non-attached cells were removed by three rinses with PBS. The remaining cells were fixed with 2% paraformaldehyde for 20 min and stained with 1% crystal violet  for 20 min.Crystal violet staining was performed to assess the adhesion of FCP. Briefly, culture plates were coated with 250 nmol/L of FCP or Cilengitide overnight at 4 °C. Next, the plates were washed three times with PBS. Subsequently, ECV304 (1.0 × 105 cells/mL) were seeded on plates (24-well) and calculated approximately 95% confluence. Then, after being treated with different test substances for 24 h, we discarded culture supernates and washed ECV304 cells using PBS 3 times, following fixed them in 4% paraformaldehyde. In the next moment, washed with PBS and then permeabilized with ice-cold 0.5% Triton X-100. The cells were blocked with PBS containing 2% bovine serum albumin, then incubated with primary antibody of vinculin  overnight at 4 °C; after that, Alexa Fluor 488 goat anti-rabbit IgG  were co-incubated at room temperature for 30 min. Next, TRITC Phalloidin  was incubated at room temperature for 10 min. Finally, the sections were stained with 4′,6-diamidino-2- phenylindole (DAPI)  and imaged with a confocal laser scanning fluorescence microscope .After adhesion, cell spread areas were characterized by vinculin and TRITC Phalloidin and detected by immunofluorescence staining assays via a confocal microscope. Briefly, ECV304 . ECV304 cells were treated with FCP, integrin β3 siRNA, or scramble siRNA for 24 h separately in the second separated assays. In the last separate experiment, ECV304 cells were treated with FCP, Cilengitide, and FCP combined with Cilengitide (250 nmol/L) for 24 h. All test corresponding control groups were given.4) were added to each well and incubated at 37 °C and 5% CO2 for 12 h. Cells were respectively treated with or without FCP, FN10, and CC (250 nmol/L) for 4 h. Then, tube formation was observed and captured with an inverted microscope .The tube formation assay was performed with ECV304-eGFP. Briefly, 96-well plates were coated with 50 μL/well matrigel, then polymerized at 37 °C for 30 min. Then, ECV304-eGFP (3 × 10https://swissmodel.expasy.org/)  (Yang and Zhang https://robetta.bakerlab.org/) .The structural modeling of FCP was performed using bioinformatic webservers such as SWISS-MODEL (http://bioinfo3d.cs.tau.ac.il/PatchDock/php.php)  to screen the docking results from PatchDock. Finally, 3D and 2D plot analyses of the protein interaction surface through PyMoL v.2.5.1 and Ligplot+v.2.2 respectively.Utilizing the FireDOCK server   three different target sequences of the siRNA as presented in Table 5 cells per well) in the logarithmic growth phase were seeded in 6-well plates and cultured overnight, then transfected with integrin β3 siRNA or scramble siRNA, respectively according to the manufacturer’s protocol of Ribio. Transfected cells were incubated for another 24 h and treated with FCP; then, scratch wound assay and immunofluorescence staining assay were used to examining the effects of FCP.Shortly, the ECV304 cells (2 × 103 cells per well) were inoculated into a 6-well plate precoated with FCP, Cilengitide, and FCP combined with Cilengitide (250 nmol/L), respectively; the group that did not precoat with anything as the blank control. The cells were cultured continuing for 14 days. After that, the cells were washed three times with PBS and incubated with 1 mL of crystal violet staining solution  for 30 min. After being washed three times, the cells were observed under an optical microscope  and photographed. At the same time, cells on another independent 6-well plates were collected to detect stemness-related genes: NANOG; REX1; PPAR-γ2; ALP; SOX9, and integrin β3.rBMSCs . To obtain cDNA, 1 μg of RNA was reverse transcribed by using the reverse transcription kit . An equal volume of cDNA was then used for RT-qPCR using the SYBR-Green Quantitative PCR kit  and the CFX96 Touch Real-Time PCR Detection System . The rat-specific primers were as follows: NANOG; REX1; PPAR-γ2; ALP; SOX9; and integrin β3. All primers were purchased from BGI . Relative expression levels of genes were examined by 2in Table .SD) of at least three independent experiments. Statistical analyses were performed using the GraphPad Prism 6 software . Differences between more than two groups were analyzed using one-way ANOVA followed by Tukey s HSD comparison test. Statistical significance was set at P < 0.05.All data are expressed as mean ± standard deviation . A similar trend was observed in the number of attached cells . After 36 h of FCP treatment, the wound was almost completely closed in the wound healing assay, while others were still open  of wound closure was observed in the FCP groups (99.32% ± 0.69%) than in the control groups (50.68% ± 2.31%), , Fig. . Similar%), Fig. .Fig. 2CeDifferent prediction models of FCP were validated using Ramachandran Plot, Whatcheck, and Verify3D by the SAVES 6.0 online server. Validation statistics showed that RoseTTAFold best predicted the structure of FCP compared with the other methods, evidenced by the presence of the maximum residues (83.6%) in the most favored regions of the Ramachandran plot, 100% of the residues with an average 3D-ID score ≥ 0.2, and the largest proportion of green regions (68.1%) in Whatcheck Fig. . The besBased on these results, the RoseTTAFold model was used for molecular docking. After protein–protein docking via PatchDock and filtering the docking results by FireDock, the stable docking complex of FCP-integrin β3, with the highest binding energy of − 15.68 kcal/mol, was obtained. The 3D plot of the docking complex showed that the C-terminal FN10 region of FCP formed a relatively stable complex with integrin β3 Fig. . More sp–8 M . Gene expression levels of both SOX and ALP, gene markers of chondrogenic and osteogenic differentiation abilities of rBMSCs, were higher than others, especially compared to gene expression levels in the control group (P < 0.05). However, gene expression of PPAR-γ2, an adipogenic gene, was similar in FCP-treated and control groups (P > 0.05). When Cilengitide was used to inhibit integrin β3, the expression of all genes was significantly downregulated. After treatment with both FCP and Cilengitide simultaneously, the related genes were upregulated compared with those in the Cilengitide groups . The SPR technique is a powerful tool in the study of target molecule interaction and has the advantages of low cost and direct and quantifiable results. However, because SPR is sensitive to interfering factors within the sample, it represents an indirect interaction between proteins.The high affinity of FCP for integrin β3 was verified by SPR assay. Integrin β3 protein, captured on an NTA chip, bound FCP with an affinity constant of 5.37 × 10P > 0.05). This finding is difficult to explain, but we speculate that it is related to the function of integrin β3. Further study of these results was outside the scope of the current study due to laboratory constraints. For a sample, we are unable to test karyotypic stability after transfer generation.Collectively, these data suggest that integrin β3, an important receptor on the surface of ECV304 cells, may be a key target for FCP to exert its biological effects. By knocking down integrin β3 expression in ECV304 cells and competitively blocking integrin β3 binding sites with Cilengitide, FCP-induced angiogenesis, migration, and adhesion of ECV304 cells were reduced. rBMSCs were chosen as the model system in the present studies due to ethical constraints and other difficulties in obtaining hHSCs. Considering that rBMSCs are one of the most widely used stem cells in the field of tissue engineering  and CC (a kind of human-like collagen) proteins, which have an ideal domain to provide mechanical support for cells. The FCP bionic ECM may provide a microenvironment to support stem cell localization, proliferation, differentiation, and stemness maintenance. In the future, FCP may be used in the in vitro culture of stem cells, such as hematopoietic stem cells and other stem cells with high expression of integrin β3, or even in the field of tissue regeneration engineering.Supplementary file1 (PDF 564 KB)Below is the link to the electronic supplementary material.


We quantified both viral stocks using droplet digital PCR (ddPCR), which revealed that the Omicron stock had equivalent copies of the N gene to Delta at a one log lower infectious virus. The Abbott BinaxNow and Orasure InteliSwab had the highest analytical sensitivity for Omicron while the Orasure InteliSwab and the Quidel QuickVue had the highest analytical sensitivity for Delta. When 14 SARS-CoV-2 real-time PCR positive nasal/nasopharyngeal swab samples , were tested by the four assays, only the QuickVue detected all samples. Antigen test positivity correlated with recovery of infectious virus on cell culture in 9 out of 13 tested specimens from symptomatic, asymptomatic, unvaccinated, and vaccinated individuals. Although our study confirms the reduced analytical sensitivity of antigen testing compared to molecular methods, the Omicron variant was detectable by the four evaluated rapid antigen tests.Ensuring SARS-CoV-2 diagnostics that can reliably detect emerging variants has been an ongoing challenge. Due to the rapid spread of the Omicron variant, point-of-care (POC) antigen tests have become more widely used. This study aimed at (i) comparing the analytical sensitivity (LOD) of 4 POC antigen assays, BD Veritor, Abbott BinaxNow, Orasure InteliSwab and Quidel QuickVue, for the Omicron versus the Delta variant and (ii) verifying the reproducible detection of Omicron by the 4 antigen assays. The LOD for all four assays were evaluated using Omicron and Delta virus stocks quantified for infectivity and genome copies. The four assays detected all replicates of Omicron and Delta dilutions at 10IMPORTANCE In the manuscript, we report an evaluation of the capability of 4 point of care (POC) antigen assays, the BD Veritor, Abbott BinaxNow, Orasure InteliSwab and Quidel QuickVue to detect the Omicron variant of SARS-CoV-2, and we compared their analytical sensitivity for Omicron versus Delta. In this analysis we found that all four assays detected Omicron and Delta at 104 and 105 TCID50/mL, respectively. We further quantified the viral stocks used by droplet digital (ddPCR) and found that the Omicron stock had equivalent copies of the N gene to Delta at a one log lower infectious virus titer and that an increased RNA to infectious virus ratio may be contributing to discrepancies in limit of detection in Omicron compared to Delta. We evaluated 14 SARS-CoV-2 real-time PCR positive nasal/nasopharyngeal swab samples (12 Omicron and 2 Delta), with an average cycle threshold value of 19.1, and only the QuickVue showed 100% agreement. Since the beginning of the SARS-CoV-2 pandemic, the importance of accurate diagnostic assays has been highlighted . As new 2–6–5 TCID50/mL. For the Omicron variant, all assays were able to detect all replicates at 1 × 104 TCID50/mL , BinaxNow and InteliSwab detected 92% (11/12), and QuickVue detected 100% (12/12) of Omicron positives . The 12 Our data indicate that the lower clinical sensitivity of some POC tests is not associated with the recovery of infectious virus, or antibody levels in the upper respiratory samples and are likely related to the analytical sensitivity of the assays for these particular variants. Our LOD results were similar to other studies , 16 illuFor our clinical sample evaluation, our results are similar to other studies , 4 partiThis study has limitations, notably the small clinical samples size evaluated (N = 14), and the relatively high concentration of virus within the clinical samples evaluated (mean N Ct =19.12). However, for our study, lower Ct value clinical samples were intentionally chosen to assess the performance of each assay for the detection of Omicron without the added variable of assay sensitivity associated with lower viral load. Additional limitations include the use of simulated anterior nasal swab samples for testing spiked and clinical specimens; even though this is a nonvalidated approach for sample collection and testing, most of our tested samples were positive and all the runs were valid  endorsing the validity of this approach. Lastly, LOD comparison studies were performed using relatively limited number of replicates for each tested concentration.Our data confirm that antigen tests have lower sensitivity than nucleic-acid amplification tests. The four antigen tests that we evaluated were similar in their ability to detect Omicron variant. Antigen assays had a lower LOD with the Omicron variant which correlated with the higher number of nucleocapsid genomic copies per infectious virus concentration compared to Delta. Evaluations of new variants should include quantification of infectious virus and genomic viral RNA copies for comparing sensitivities of diagnostic tests.We performed a limit of detection (LOD) study for each assay with Delta and Omicron SARS-CoV-2 viral stocks that were quantified for infectious virus concentration by a tissue culture infectious dose (TCID50) assay and viral genome copy number ddPCR . Delta aNasal/nasopharyngeal (N = 14) samples with Ct values < 20 were selected from SARS-CoV-2 positive samples identified as Omicron at the Johns Hopkins Virology Laboratory as a part of whole-genome sequencing for surveillance in December 2021. The clinical diagnosis was performed by the NeuMoDx SARS-CoV-2 assay  and the 23–Viral stock dilutions and clinical specimens were tested by each POC antigen test as per manufacturer instructions with a modification where the provided swab in the assay was immersed in each spiked or clinical specimen.Serology was performed with the EUROIMMUN Anti-SARS-CoV-2 ELISA (IgG) following the package insert  on undilThe Delta  and Omicron  variants used in this study were isolated from clinical specimen as previously described .


In the abnormal circumstances caused by the COVID-19 pandemic, patient portals have supported patient empowerment and engagement by providing patients with access to their health care documents and medical information. However, the potential benefits of patient portals cannot be utilized unless the patients accept and use the services. Disparities in the use of patient portals may exacerbate the already existing inequalities in health care access and health outcomes, possibly increasing the digital inequality in societies.The aim of this study is to examine the factors associated with nonuse of and dissatisfaction with the Finnish nationwide patient portal My Kanta Pages among the users of health care services during the COVID-19 outbreak. Several factors related to sociodemographic characteristics, health, and the use of health care services; experiences of guidance concerning electronic services; and digital skills and attitudes were evaluated.A national population survey was sent using stratified sampling to 13,200 Finnish residents who had reached the age of 20 years. Data were collected from September 2020 to February 2021 during the COVID-19 pandemic. Respondents who had used health care services and the internet for transactions or for searching for information in the past 12 months were included in the analyses. Bivariate logistic regression analyses were used to examine the adjusted associations of respondent characteristics with the nonuse of My Kanta Pages and dissatisfaction with the service. The inverse probability weighting (IPW) method was applied in all statistical analyses to correct for bias.In total, 3919 (64.9%) of 6034 respondents were included in the study. Most respondents  used My Kanta Pages, and 2841 (85.3%) of them were satisfied. Nonusers  were a minority among all respondents, and only 489 (14.7%) of the 3330 users were dissatisfied with the service. Especially patients without a long-term illness , those who were not referred to electronic health care services by a professional , and those in need of guidance using online social and health care services  were more likely nonusers of the patient portal. Perceptions of poor health  and security concerns  were associated with dissatisfaction with the service.Patients without long-term illnesses, those not referred to electronic health care services, and those in need of guidance on the use of online social and health care services seemed to be more likely nonusers of the Finnish nationwide patient portal. Moreover, poor health and security concerns appeared to be associated with dissatisfaction with the service. Interventions to promote referral to electronic health care services by professionals are needed. Attention should be targeted to information security of the service and promotion of the public’s confidence in the protection of their confidential data. The worldwide COVID-19 pandemic limited the provision of nonurgent health care services . During Patient portals offer transparent information about the patients’ health and well-being  and enhaThe potential benefits of patient portals cannot be utilized unless the patients accept and adopt the service ,19. AccoPrevious research has examined differences in patient portal use in different contexts and patient populations. Several studies have reported an association between portal use and sociodemographic background ,23,25,26Factors associated with the patients’ satisfaction with patient portals have been less studied. Mainly descriptive research on the portal users’ experiences exists, and only little research has been conducted with quantitative methods about factors associated with satisfaction . Kong etThe aim of this study is to examine factors associated with the nonuse of and dissatisfaction with the Finnish nationwide patient portal My Kanta Pages (My Kanta) during the COVID-19 pandemic. Only respondents who had used the internet in the past 12 months were included to examine nonuse beyond the first-level digital divide  caused bFinland is a sparsely populated country with 5.5 million residents. The health care system is decentralized, and until the end of 2022, municipalities n=311) are responsible for organizing health care services, which are funded by taxes, state transfers, and user fees 1 are res. One of My Kanta was launched step-by-step starting from 2010  to promoThe number of My Kanta users has grown steadily since its launch , and theThis study was conducted in Finland as part of the FinSote 2020 National Survey of Health, Wellbeing, and Service Use . The queAltogether, 6034 Finnish residents  responded to the questionnaire (response rate 46.5%). In total, 3919 (65.0%) respondents were included in the study sample as they had used health care services and the internet in the past 12 months. The sample was weighted using inverse probability weighting (IPW) correction . The weiParticipation in the study was completely voluntary. Ethical approval was obtained from the Ethics Committee of the Finnish Institute for Health and Welfare (THL/637/6.02.01/2017).nonuse of My Kanta was evaluated with the question “Have you used My Kanta in the past 12 months?” Respondents were asked to respond (1) no or (2) yes. For the analyses, the measure was binary-coded , and the users of My Kanta were set as the reference group.The dissatisfaction with My Kanta was evaluated with a question concerning satisfaction: “If you have used the service, assess the quality of the service using a school grade (4-10).” In the Finnish education system, grades 8-10 represent grades from good to excellent and grades 4-7 from fail to satisfactory [The sfactory . For thesfactory  between Independent variables included characteristics concerning (1) sociodemographic background, (2) health and the use of health care services, (3) experiences of guidance concerning electronic services, and (4) digital skills and attitudes. All the used variables are presented in degree of urbanization was determined according to the municipal classification and divided into 3 categories according to the proportion of people living in urban settlements and the population of the largest urban settlement: urban, semiurban, and rural municipalities [educational level was first divided into 10-year age groups by gender. Each group was divided into 3 categories based on their years of education, with approximately one-third of the respondents in each category: low, median, and high. Hence, the education-level variable had hardly any interaction with age and gender.The respondents’ sociodemographic characteristics included their age, gender, education, and degree of urbanization. Age was used as a categorical variable in the descriptive statistics and as a continuous variable in all analyses. The palities . BecauseSelf-rated health was evaluated with a widely used, single-item measure of self-perceived health status. A subjective assessment of own health has been reported as a more sensitive measure in health monitoring than external measures of health, since it includes biological, psychological, and social dimensions. [good and (2) fairly good were combined to represent good health, and the remaining options represented average or poor health. Long-term illness was binary-coded as (1) yes and (2) no. The use of health care services was binary-coded according to the number of annual outpatient appointments with a physician; 8 or more annual appointments were considered a high use of health care services, and less than 8 were counted as low or average use [Variables concerning the respondents’ health and the use of health care services included self-rated health, long-term illness, and the use of health care services. ensions. . A scalerage use .referral to electronic services was evaluated with the question “If you have used social or health care services in the traditional way  in the past 12 months, were you referred to electronic services ?” For the analyses, option (1) yes, I was referred represented the respondents who were referred to electronic health care services. Option (2) was for those who were not referred to electronic health care services.Variables concerning the experiences of guidance concerning electronic services included referrals to electronic services and the need for guidance on how to use online social or health care services. The need for guidance on using online social and health care services was evaluated with the statement “I need help with using online social and health care services.” In the analyses, the options (1) completely agree and (2) somewhat agree were combined as (1) yes and the remaining options as (2) no or no opinion.The Digital skills were evaluated with 6 validated statements [good skills (mean≤2.5) and (2) poor skills (mean>2.6). The same coding has previously been used in national research [Variables related to digital skills and attitudes included digital skills, perceived benefits of electronic social and health care services, and security concerns. atements . Based oatements . A 5-poiresearch .perceived benefits of electronic social and health care services were measured with 8 statements. A 5-point Likert scale was used to answer the statements (1=completely agree to 5=strongly disagree). Cronbach α for the statements was .91. In the analyses, missing values were coded as neither agree nor disagree. A mean variable from 1 to 5 was calculated for each respondent, and the measure was binary-coded as (1) beneficial (mean≤2.5) and (2) unbeneficial (mean>2.6). The same coding has previously been used in national research [The research .Security concerns were evaluated with the statement “I am concerned about information security when it comes to my personal details”. In the analyses, options (1) completely agree and (2) somewhat agree were combined as (1) yes and the remaining options as (2) no.In all statistical analyses, the IPW method  was applP value of <.10. This cut-off for the P value was used for including the variables in the multivariable model, because the purpose was to identify potential independent variables rather than to test a hypothesis [P value of <.05 was considered statistically significant. Statistical methods suitable for weighted data were used, and SPSS Statistics version 27 was applied for the analyses.Bivariate logistic regression analyses were used to examine the adjusted associations of respondent characteristics with the nonuse of My Kanta and dissatisfaction with the service . First, univariate analyses, adjusted for age, gender, and education, were conducted at a time to examine the association of the dependent variable with each independent variable. Second, a multivariable model was formed, including only those independent variables with a pothesis . In the The weighted majority  of the respondents had used My Kanta in the past 12 months. Most of the My Kanta users  were satisfied with the service. A minority of respondents  had not used My Kanta in the past 12 months.The IPW weighted characteristics of the respondents representative of the Finnish population are presented in Based on the results of age-, gender-, and education-adjusted univariate logistic regression analysis , the folThe results of the fully adjusted logistic regression analysis regarding the nonuse of My Kanta are presented in Based on the results of the age-, gender-, and education-adjusted univariate analyses , the folIn the fully adjusted multivariable model, respondents who were younger, were male, and had a high level of education were more likely to be dissatisfied with My Kanta compared to their counterparts. Respondents with average or poor self-rated health were over 2 times more likely to be dissatisfied with My Kanta compared to respondents with a good perception of their own health. In addition, respondents who perceived electronic services as unbeneficial, who needed guidance, and who had security concerns were more likely to be dissatisfied with My Kanta compared to their counterparts.Most respondents of this nationally representative survey study had used the nationwide Finnish patient portal My Kanta in the previous 12 months and were satisfied with the service. However, more than every 10th user of health care services and the internet were nonusers of the national patient portal, and approximately the same number of users were dissatisfied with the service. Males and those in a need of guidance were more likely to be nonusers of the patient portal and dissatisfied with the service compared to women and those not needing guidance. Not having a long-term illness and low or average use of health care services were associated with the increased likelihood of nonuse of the My Kanta portal. In addition, respondents who were not referred to electronic services and who had poor digital skills were more likely to be nonusers of My Kanta compared to their counterparts. A younger age, higher education, and poor self-rated health were associated with an increased likelihood of dissatisfaction with the service. In addition, respondents who did not perceive electronic health care services to be beneficial and who had security concerns were more likely to be dissatisfied with the service compared to their counterparts.Finland is 1 of the forerunners of digitalization and ranked highest in information exchange and patient-centered information processing in an international comparative study . By presA nationally presentative sample of Finnish residents was included in the analysis. The applied IPW method has previously been reported to improve the accuracy and generalizability of results . HoweverSince only respondents who had used the internet for transactions or for searching for information were included in the study, the results are only applicable when considering the nonuse of patient portals beyond the first-level digital divide caused by the lack of necessary devices and access to the internet. Some respondents who did not use My Kanta used additional patient portals provided by private or public providers of health care services. It is also noteworthy that some respondents might not be referred to electronic health care services, because their transactions in health care do not require further action or electronic services cannot provide support in their situation. The research concerning the users and nonusers of nationwide patient portals is sparse, and comparison is difficult as the properties provided in the portals vary, in addition to the differing patient populations and adjustments in the analyses.The use of nationwide patient portals varies by country and portal ,10. ThisThe results of this study suggested that younger and more educated respondents were more likely to be dissatisfied with My Kanta compared to older and less educated respondents. The findings of this study were supported by the fact that younger generations have grown up with technology and were thus more comfortable using electronic services , which mThis study found that respondents without any long-term illness and with low or average use of health care services are more likely to be nonusers of My Kanta, which is consistent with previous research ,25,27,64Over half of the respondents were not referred to electronic health care services by their care providers, and the nonreferred respondents were less likely to use the nationwide patient portal My Kanta compared to referred respondents. Sääskilahti et al  and KongAlthough patients have prior experience in the use of electronic services and information technology tools, the ability to review and manage medical records on patient portals should not be assumed . ResultsThe previous literature has suggested different ways in which the use of patient portals could be promoted, including ease of entry ,30,72, eIn addition to easy accessibility of the service, assistance on the use should be available at a low threshold. Because patients have previously been reported to seldom seek help from family members, friends, service support, or health care providers , electroDigital skills are necessary for wider patient adoption and use of patient portals ,37,81,82Attitudes about the usefulness, appropriateness, and potential downsides of electronic services may encourage or impede the use . In thisAccording to this study, respondents who had security concerns were more likely to be dissatisfied with My Kanta compared to respondents who felt more secure. Similar to the results of Woods et al , securitAccording to the results of this population-based cross-sectional survey study in the era of COVID-19, patients without long-term illnesses, those not referred to electronic health care services, and those in need of guidance on the use of online social and health care services seem to be more likely nonusers of the Finnish nationwide patient portal My Kanta. Moreover, poor health and security concerns seem to be associated with dissatisfaction with the service. Interventions to promote referral to electronic health care services by professionals are needed. Attention must be paid to information security of the service as well as the alleviation of the patients’ privacy concerns.


BMPR2 (bone morphogenetic protein (BMP) receptor type II) cause pulmonary arterial hypertension. BMPRII is a receptor for over 15 BMP ligands, but why BMPR2 mutations cause lung-specific pathology is unknown. To elucidate the molecular basis of BMP:BMPRII interactions, we report crystal structures of binary and ternary BMPRII receptor complexes with BMP10, which contain an ensemble of seven different BMP10:BMPRII 1:1 complexes. BMPRII binds BMP10 at the knuckle epitope, with the A-loop and β4 strand making BMPRII-specific interactions. The BMPRII binding surface on BMP10 is dynamic, and the affinity is weaker in the ternary complex than in the binary complex. Hydrophobic core and A-loop interactions are important in BMPRII-mediated signalling. Our data reveal how BMPRII is a low affinity receptor, implying that forming a signalling complex requires high concentrations of BMPRII, hence mutations will impact on tissues with highest BMPR2 expression such as the lung vasculature.Heterozygous mutations in  Mutations in BMPR2 is the major genetic cause for pulmonary arterial hypertension (PAH). Here by solving crystal structures of BMPRII in binary and ternary receptor complexes with BMP10, the authors report the molecular recognition between BMPRII and BMP10, and its implication in PAH. Upon signalling complex formation, the constitutively active type II receptor phosphorylates and activates the type I receptor; subsequently the type I receptor phosphorylates Smad1/5 or Smad2/3 to regulate transcriptional responses.Transforming growth factor β (TGF-β) family cytokines control many fundamental biological processes, from the establishment of body patterning during embryonic development, to the maintenance of adult homeostasis in vasculature, wound repair, and immune response. The TGF-β ligands are dimers and initiate signal transduction by forming a signalling complex with two copies of a type I receptor and two copies of a type II receptor; all are single-pass transmembrane proteins containing a small 3. Although BMPRII binds BMP10 with the highest affinity among different ligands1, its affinity is still at least 10-fold weaker than other high-affinity cognate receptor:ligand interactions in the TGF-β family1, and around 10-fold weaker than ActRIIB binding to BMP102. How BMPRII regulates BMP signalling through low-affinity interactions is not known.There are more than 30 genes encoding TGF-β ligands which can be broadly divided into three subfamilies, the TGF-βs, the activins, and the bone morphogenetic proteins (BMPs). Their signalling is mediated by only 7 type I receptors (Activin receptor-like kinase 1-7 (ALK1-7)) and 5 type II receptors (TGF-β receptor type II (TGFβRII), Activin receptor type 2 A and 2B (ActRIIA and ActRIIB), BMP receptor type II (BMPRII) and Anti-Mullerian hormone (AMH) receptor type II (AMHRII)). Although some ligand:receptor interactions are of high affinity and specificity, such as TGFβRII for TGF-β1 and TGF-β3, ALK1 for BMP9 and BMP10, many are promiscuous. For example, ActRIIA/B bind to and mediate the signalling from both activin ligands and some BMP ligands, whereas BMPRII is the low-affinity type II receptor for over 15 BMP ligandsBMPR2 are the major genetic cause for pulmonary arterial hypertension (PAH)5, the consequences of which include remodelling of the pulmonary arteries, elevated right ventricular pressure, right ventricle hypertrophy and eventually heart failure. There is currently no cure for PAH and it represents a significant unmet medical need. Of note, no mutations in genes encoding ActRIIA or ActRIIB have been reported in PAH patients.BMPRII is unique among TGF-β family receptors because it possesses a long carboxy-terminal tail of more than 500 amino acids after the kinase domain. Although both ActRIIA/B and BMPRII can mediate signalling from different BMPs, the phenotypes of knockout mice, tissue distribution, and the effects of mutations in human diseases are very different among these three type II receptors. Loss of function mutations in BMPR2 mutations have been identified in PAH cohorts to date6. Most mutations result in haploinsufficiency, but around 25% are missense mutations in the ECD and ICD. Some missense mutations, including many cysteine substitutions, cause protein misfolding and retention in the endoplasmic reticulum8. Other mutations in the ECD do not affect cell surface localisation and their impact on ligand binding and signalling activity remains unclear.Around 668 BMPR2 mutations cause PAH despite the receptor being rather ubiquitously expressed in the body is intriguing, suggesting a pivotal role for BMPRII in the lung vasculature. Of note, BMPRII is particularly highly expressed in lung vascular endothelial cells, where it mediates the signalling from circulating BMP9 and BMP10. The unique features of these two ligands include binding with very high affinity and specificity to the endothelial-specific type I receptor ALK1, and the co-receptor endoglin (ENG). The importance of ALK1 and ENG in the vasculature is underpinned by human genetics showing that autosomal dominant loss-of-function mutations in these two genes cause Hereditary Haemorrhagic Telangiectasia (HHT)10, a vascular abnormality characterised by telangiectases (broken capillaries) in the nasal mucosa, gastrointestinal tract and skin, and larger arteriovenous malformations in brain, lungs and liver, which can be life-threatening. Mutations in ALK1, ENG, GDF2 (encoding BMP9) and BMP10 have also been reported in PAH patients11. Thus, human genetics strongly supports a role for BMPRII and BMPRII/ALK1-mediated BMP9 and 10 signalling in lung endothelial cells in the pathogenesis of PAH. In accordance with this, we have recently shown that endogenous BMP9 plays a critical role in the maintenance of endothelial integrity, particularly in the pulmonary vasculature12.Why BMPR2 mutations in PAH, crystal structures of BMPRII signalling complexes are yet to be solved, perhaps due to the difficulty of obtaining crystals from the low affinity receptor:ligand complexes. Here we report crystal structures of BMP10:BMPRII complex in two crystal forms, and the ALK1:BMP10:BMPRII signalling complex. We show that although BMPRII utilises the same hydrophobic core as ActRIIA/B and binds to BMP10 at the knuckle epitope, the longer A-loop and finger 3 loop (F3-loop) in BMPRII make unique interactions. Importantly, we show that BMPRII makes fewer interactions with BMP10 and has lower binding affinity for BMP10 in the ternary complex than in the binary complex, suggesting a mechanism for the transient nature of the BMPRII-mediated signalling. Surprisingly, we found that BMP10 fingertip 3/4 preferentially adopts either an extended or a bent conformation in different protein-protein interaction contexts. Such conformational plasticity is also present in BMP9; which may be another unique feature of BMP9 and 10 contributing to their signalling specificity.Despite two decades of extensive research on BMPRII since the discovery of We solved crystal structures of the BMPRII binary complex with BMP10 in two different crystal forms, at 1.48 Å and 2.4 Å, respectively , which provided a BMP10 conformation in yet another protein interaction context. Two crystal forms  because the expression level is very low, and transfection did not lead to an increase in BMP signalling  by Uniprot32. Once it is made in a recombinant expression system, it can bind to BMP10 with similar affinity to WT BMPRII  to undergo the bent-to-extended conformational change in fingertip 3/4. The insertion in the A-loop in BMPRII comes at a slight cost of complex stability because the loop truncation mutant ΔGDP binds to BMP10 with both a slower on-rate and a slower off-rate.1. Sequence alignment revealed a conserved IAP motif across all BMPRII-binding ligands , mouse 2H-11 endothelial cells (cat. No. CRL-2163) and human embryonic kidney (HEK) EBNA cells (cat. No. CRL-10852) were all purchased from American Type Culture Collection (ATCC). All cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin and were frequently tested for the absence of mycoplasma contamination.31. Briefly, cDNA of full-length open reading frame of human BMP10 (NM_014482) was cloned into pCEP4 and transfected into HEK EBNA in DMEM supplied with 5% FBS. Human full-length FURIN cDNA in pCEP4 was co-transfected to facilitate processing. Serum-free chemically defined (CD) CHO  medium was applied from day 2 and conditioned media were harvested every 3–4 days for 5 harvests. To purify Pro:BMP10, conditioned medium was loaded onto 5 ml Hitrap Q columns, selected fractions containing Pro:BMP10 were concentrated and loaded onto a HiLoad Superdex 200 pg 16/600 gel filtration column. Peak fractions were further purified using a MonoP 5/50 GL column pre-equilibrated with 20 mM Tris, 50 mM NaCl, pH 7.4 and eluted with a NaCl gradient. Target fractions were concentrated and loaded onto an Superdex 200 column pre-equilibrated in 20 mM Tris, 150 mM NaCl, pH 7.4. To facilitate crystallisation, a mutant form of Pro:BMP10 was produced. Using the UCLA MBI-SERp server34, residues K267-E269 and E296-E297 were suggested as residues with high entropy at the protein surface, and mutating which may enhance the protein’s crystallisability via the generation of crystal contacts. Therefore these residues were all mutated to alanine using site-directed mutagenesis. The mutant Pro:BMP10 protein was expressed and purified using the same method as described for the WT Pro:BMP10.Pro:BMP10 was expressed and purified following the published method13. In brief, human ACVRL1  ECD cDNA (amino acids 22-118) was cloned into a pET39b vector. A TEV (Tobacco Etch Virus nuclear inclusion A endopeptidase) cleavage site was introduced at the N-terminus of ALK1 ECD. The plasmid was transformed into Rosetta DE3 bacteria and cultured in 2x YT medium at 37 °C to mid-log phase followed by IPTG  induction at 22 °C overnight. The fusion protein DsbA-(His)6-ALK1 ECD was extracted from the periplasmic compartment according to the pET System Manual (Novagen) followed by purification using a 5 ml HisTrap Excel column . The fusion protein was incubated with a His-tagged TEV protease and dialysed in Tris-buffered saline (TBS) overnight. The cleaved DsbA-(His)6 and TEV were removed from ALK1 ECD using a 5 ml HisTrap Excel column. ALK1 ECD was further purified by a Superdex 75 pg 16/600 column .ALK1 ECD was expressed and purified following the published methodBMPR2 cDNA (NM_001204) encoding amino acids 27-150 was cloned into a pET39b plasmid to create a fusion WT protein DsbA-(His)6-BMPRII ECD. All the mutations and deletions were introduced using a Q5® Site-Directed Mutagenesis Kit (New England Biolabs) following the manufacturer’s protocol and were confirmed by sequencing. The WT and mutant proteins were expressed and purified following the method described above for ALK1 ECD.Human KD was determined by the ratio of binding rate constants kd/ka.Surface plasmon resonance (SPR) experiments were undertaken using the Biacore T200 system . Recombinant human BMP10 growth factor (GF)-domain  was immobilised onto a Series S research grade CM5 sensor chip  by amine-coupling at a density of 1300 resonance units. For kinetic measurements, a series of concentrations of ALK1-Fc , BMPRII-Fc , monomeric BMPRII WT and mutant ECDs were injected in duplicate over the flow cells at a flow rate of 40 ul/min in a buffer containing 10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.5 mg/ml BSA  and 0.005% (v/v) surfactant P20 at 25 °C. For ALK1-Fc and BMPRII-Fc binding experiments, the surface was regenerated with 4 M Guanidine Hydrochloride while for BMPRII WT and mutant ECD experiments, no regeneration was needed. The kinetic rate constants were obtained by fitting the corrected data to a 1:1 interaction model using Biacore T200 Evaluation Software . The equilibrium binding constant P212121 to 1.48 Å using DIALS35 and AIMLESS36 in the CCP4 suite37. The structure was solved by molecular replacement using Phaser38, with BMP10 from PDB entry 6SF3 and BMPRII (PDB entry 2HLQ) as the search models. Model building was performed using Coot39 and refinement using REFMAC540. The final model was validated using MolProbity41. A second crystal form was obtained in 14% PEG 3350, 0.14 M KCl, pH 7.0 in a month and data were collected at I04, DLS at a wavelength of 0.97950 Å. The data were processed in C2 space group to 2.40 Å. The structure was determined by molecular replacement in Phaser using the 1.48 Å BMP10:BMPRII structure as a search model. As above, model building was carried out using Coot, refinements using REFMAC5 and phenix.refine42 and validation using MolProbity. All the data collection, data reduction, structure determination and refinement statistics are summarised in Supplementary Table Pro:BMP10 was denatured in 7 M urea solution overnight and the denatured protein was loaded onto a 5 ml HiTrap S column pre-equilibrated in a binding buffer of 20 mM Tris, 25 mM NaCl, 6 M urea, pH 7.4 followed by elution using a NaCl gradient. Fractions containing the denatured BMP10 GF-domain were concentrated to 1 ml followed by a rapid dilution in 19 ml cold refolding buffer  dimethylammonio)-1-propanesulfonate), 2.5% glycine, 5 mM glutamic acid, pH 4.0). Excess of BMPRII ECD was then added in the refolding buffer and left on a roller at 4 °C for 6 days. The refolded mixture was concentrated and further purified using a Superdex 200 16/600 column pre-equilibrated in 20 mM Tris, 150 mM NaCl, pH 7.4. BMP10:BMPRII ECD was concentrated to 4.8 mg/ml for crystallisation trials using the hanging-drop method with 1 μl protein and 1 μl reservoir solution. Crystals were obtained over 4 days at 21 °C in 14% polyethylene glycol (PEG) 3350, 0.19 M ammonium citrate dibasic, 0.02 M sodium citrate tribasic dihydrate, pH 5.8. Crystals were cryo-protected in 30% glycerol in crystallisation reservoir solution and vitrified in liquid nitrogen. Data collection was undertaken at 100 K at Diamond Light Source  on Beamline I04 at a wavelength of 0.91589 Å, and processed in space group P21 to 3.60 Å using DIALS and AIMLESS in CCP4 suite. The structure was solved by molecular replacement using Phaser. The ALK1:BMP10 complex from 6SF3 was used as the first search model which gave 4 copies of ALK1:BMP10 1:1 complex in the asymmetric unit forming two copies of ALK1:BMP10 2:2 complex. The BMPRII ECD from the 1.48 Å BMP10:BMPRII structure was used as the second search model and only 1 copy of BMPRII was found by Phaser using the default settings. The second copy of BMPRII was found by searching more deeply in the rotation list, using all peaks above 50% of the top peak in the translation search in Phaser. The last 2 copies were found by using the Brute rotation function generating all orientations within 15 degrees of their expected values (found by superposing the BMP10:BMPRII complex structure on BMP10) and using all these orientations for the following translation search. Since the data were highly anisotropic, anisotropic correction of data used for refinement was performed using the STARANISO Server . Model building, refinement and validation were performed as described for the BMP10:BMPRII complexes. All the data collection, data reduction, structure determination and refinement statistics are summarised in Supplementary Table To make the ALK1:BMP10:BMPRII complex, ALK1 was mixed with preformed BMP10:BMPRII complex in a 1.2:1 ratio and the mixture was concentrated to 3.9 mg/ml. Crystallisation was performed by hanging drop method using 1 μl protein and 1 μl reservoir solution. A single crystal cluster was obtained after 15 days at 21 °C in 20% PEG 3350, 0.2 M calcium acetate hydrate, pH 7.5. Crystals were cryo-protected in 30% glycerol in crystallisation reservoir solution and vitrified in liquid nitrogen. Data collection was undertaken at 100 K at Diamond Light Source on Beamline I04-1 at a wavelength of 0.91188 Å and processed in space group C2221 to 2.90 Å using DIALS and AIMLESS in CCP4 suite. The structure was solved by molecular replacement using Phaser, with BMP10 from PDB entry 6SF3 and BMP9 prodomain from PDB entry 4YCI as the search models. Model building, refinement and validation were performed as described above.Pro:BMP10, with mutations to promote crystal contacts, was concentrated to 5 mg/ml and subjected to a crystal screen at 21 °C with 1.5 μl protein and 1 μl reservoir solution. Crystals  were obtained in 19% PEG 3350, 0.15 M ammonium tartrate dibasic, 0.02 M sodium cacodylate trihydrate, pH 6.6 after 15 days. Crystals were cryoprotected in 30% glycerol in crystallisation reservoir solution and vitrified in liquid nitrogen. Data collection was undertaken at 100 K at Diamond Light Source on Beamline I04-1 at a wavelength of 0.91589 Å and processed in space group C2221 to 3.5 Å. The structure was solved by molecular replacement using Phaser, with the crystal form 1 structure as the search model. Model building, refinement and validation were performed as described above for the other structures. All the data collection, data reduction, structure determination and refinement statistics are summarised in Supplementary Table WT Pro:BMP10 was concentrated to 8.4 mg/ml and crystallised in 20% PEG3350, 0.2 M ammonium tartrate dibasic, pH 6.6 by mixing 1 μl protein and 1 μl reservoir solution at 21 °C for about 100 days . Data collection was at 100 K at I04-1, DLS at a wavelength of 0.91188 Å, and processed in space group 43 was used for sequence alignment. Buried interface area were calculated using QtPISA 2.1.0 in CCP4-7.1.Structural analyses were performed using Coot and Pymol , and figures generated using Pymol. Clustal OmegaPro:BMP10 was pre-mixed with BMPRII WT or mutant ECDs (at 1:2 and 1:5 molar ratio) in 20 mM Tris, 150 mM NaCl, pH 7.4 in a final volume of 8 ul for 30 minutes at room temperature before fractionation on a 12% native PAGE. After staining with Coomassie Blue, band intensities were quantified by ImageJ (version 1.51 s). To identify the native gel bands, bands were cut out from the native gel and inserted directly into the wells of a 12% gel for SDS-PAGE using a standard protocol.2, pH 9.8 and absorbance was measured at 405 nm. The experiment was repeated three times, with technical duplicates each time. All values are presented as the ratio of OD405nm of samples to Pro:BMP10 only sample.A high binding 96-well plate was coated with 0.25 μg/well of anti-human BMP10 GF-domain antibody  in phosphate-buffered saline (PBS) and incubated in a humidified chamber at 4 °C overnight. The wells were washed with PBS containing 0.05% Tween 20 (PBST) followed by blocking with 1% BSA in PBS (BSA/PBS) at room temperature for 2 hours. In parallel, 25 ng Pro:BMP10 samples were premixed with BMPRII ECD WT  or mutants (at 1:125 molar ratio) in 1% BSA/PBS at room temperature for 30 minutes. The plate was washed with PBST before samples were added. After incubation for 2 hours, the plate was washed, and antihuman BMP10 propeptide detection antibody  in 1% BSA/PBS was added. After washing, ExtrAvidin®-Alkaline phosphatase  diluted 1:400 in 1% BSA/PBS was added. The assay was then developed with 0.67 mg/ml 4-Nitrophenyl phosphate disodium salt hexahydrate  in 1 M Diethanolamine, 0.5 mM MgCl44, β-gal expression plasmid (transfection control) and mutant or WT pcDEF-BMPR2 plasmids were transfected per well in triplicate. Forty-eight hours post-transfection, the cells were serum-starved overnight before luciferase activity was measured.HepG2 and 2H-11 cells were seeded in 24-well plates and transfected, respectively, with PEI  and Lipofectamine 3000. BRE-luc transcriptional reporter45.Cell lysate was fractionated on 10 or 12% SDS-PAGE and transferred to PVDF membrane. For anti-FLAG blots, after blocking in BSA/PBS, the membrane was incubated with anti-FLAG antibody , followed by wash and horseradish peroxidase (HRP)-conjugated secondary antibody . For phosphor-Smad1 blot, an in-house made phosphor-Smad1 antibody (1:1000 dilution) was used which has been validated previouslyBMPR2 plasmid for 24 hours using Lipofectamine™ LTX with PLUS™ Reagent . Cells were washed twice with ice-cold PBS (containing Ca2+/Mg2+) and incubated at 4 °C for 30 min with 0.33 mg/ml Thermo EZ-Link Sulfo-NHS-SS-Biotin in PBS . Cells were quenched thrice with 50 mM glycine in PBS before lysed on ice for 45 min in 320 μl lysis buffer . Cell lysate was sonicated briefly and centrifuged at 21,000 g for 10 min at 4 °C. Lysate supernatant was loaded (100 μl) in duplicate into 96-well Pierce™ NeutrAvidin™ Coated High Capacity Plates  and incubated for 2 hours at 4 °C. The plate was blocked with 3% BSA in PBS and incubated with anti-FLAG antibody  overnight at 4 °C and then 1 hour at room temperature with anti-mouse HRP secondary antibody . Peroxidase activity was detected by incubating with 100 μl/well SIGMAFAST™ OPD (o-Phenylenediamine dihydrochloride) for 30 minutes at room temperature and absorbance was read at 450 nm. Total FLAG-tagged BMPRII in the cell lysate was assessed by immunoblotting.HEK EBNA cells were seeded in a 24-well plate in duplicate 2 days prior experiment and transfected with 500 ng pcDEF-BMPR2 plasmid. Total RNA was extracted using RNeasy Mini Kit buffers  and Silica Membrane Mini Spin Columns (EconoSpin) following the manufacturer’s instructions. Equal amounts of RNA (~1 μg) were then reverse transcribed into cDNA using a High Capacity Reverse Transcriptase kit (Applied Biosystems). 2 μl cDNA, 1.8 μl associated premixed primer sets , 5 μl 2X SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich), 0.2 μl ROX reference dye (Invitrogen) and 1 μl DEPC-treated water were prepared into one well of a MicroAmp® Optical 384-Well Reaction Plate (Applied Biosystems) which was then put on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). Amplification reactions were initiated with a 2-minute pre-incubation at 95 °C, followed by 50 amplification cycles of 30-second denaturation at 95 °C, 30 seconds annealing at 55 °C and 30-seconds extension at 72 °C. The following primers were used for the qPCR reactions: human ID1: 5′-CTGCTCTACGACATGAACGGC-3′, 5′-TGACGTGCTGGAGAATCTCCA-3′; human β2 microglobulin (B2M): 5′-CTCGCGCTACTCTCTCTTTCT-3′, 5′-CATTCTCTGCTGGATGACGTG-3′. The relative expression levels of ID1 were calculated using the ΔΔCt method by normalizing to B2M. Differences in gene expression are presented as the fold change relative to control.HEK EBNA were seeded at 200,000 cell/well into a 6-well plate followed by 24-hour transfection with 1μg of pcDEF-P < 0.05 were considered significant.Data analyses were performed using GraphPad Prism 6.0 (GraphPad Software). Results are shown as means ± SEMs. Statistical significance was analysed using One-way ANOVA with Dunnett’s post-test comparing with appropriate controls as indicated in figure legends. Values of Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary movie 1Reporting Summary


Octodonta nipae, is a pest of palm trees in Sothern China. To address its increasing damage, environmentally friendly control methods are required. This study aimed to test efficacy of Heterorhabditis bacteriophora and Steinernema carpocapsae on O. nipae and investigated the influence of secondary metabolites, nematodes, and their isolated cuticles on the activation of O. nipae’s prophenoloxidase system using qPCR analysis. Our data revealed that O. nipae were less susceptible to H. bacteriophora than S. carpocapsae and penetrations of infective juveniles were higher with S. carpocapsae treatment than H. bacteriophora. Moreover, expression levels of the serine protease P56, prophenoloxidase activation factor 1, PPO and serine protease inhibitor 28 upon S. carpocapsae and H. bacteriophora infections were generally downregulated at all times. However, upon heating, the cuticles lost their inhibitory effects and resulted in upregulation of the PPO gene. Similarly, the addition of arachidonic acid reversed the process and resulted in the upregulation of the PPO gene compared to the control. Further work is needed to identify toxic substances secreted by these EPNs to evade O. nipae’s immune system.Entomopathogenic nematodes are biocontrol agents of invasive insect pests in soil and cryptic habitats. Nipa palm hispid,  Octodonta nipae (Maulik) is not an exception [Tetrastichus brontispae Ferrière (Hymenoptera: Eulophidae) [Steinernema carpocapsae [Metarrhizium anisopliae [O. nipae are needed.The movement of destructive pest species from one country or region to another is on the rise. Nipa palm hispid xception ,6,7,8,9.xception ,14,15,16xception ,18,19. Oophidae) ,26,27,28pocapsae ,18,19 anisopliae ,30,31,32Diabrotica virgifera LeConte [Phyllotreta spp. [Leptinotarsa decemlineata (Say) found on leaf surfaces [O. nipae larvae was conducted in our laboratory using S. carpocapsae and found to be virulent at different concentrations and time points, as published in Sanda et al. [O. nipae larvae have yet to be conducted.Heterorhabditidae and Steinernematidae are families of entomopathogenic nematodes (EPNs) mainly used for biological control of different economically invasive pests at field and laboratory levels ,35,36,37 LeConte , flea beeta spp. , and Colsurfaces . Similara et al. . HoweverO. nipae, three full-length cDNAs of PPAFs (OnPPAFs) were cloned, namely OnPPAF3, OnPPAF1, and OnPPAF2. They were highly expressed in hemolymph except OnPPAF2, which shows low transcript abundance. However, knockdown of OnPPAF1 and OnPPAF3 showed a reduction in hemolymph phenoloxidase activity and inhibition of hemolymph melanization [Insects employ both humeral and cellular immune reactions in response to attacks by pathogens such as bacteria, fungi, and nematodes. These innate immune mechanisms include nodulation, phagocytosis, encapsulations, production of antimicrobial peptides, and activation of PPO system, which leads to melanization and subsequent pathogen death by septicemia ,47. Insenization ,52,53,54S. carpocapsae use their body surface cuticle to escape host hemocyte encapsulation and at the same time inhibit its proPO system activity [S. carpocapsae and are involved in host immune modulation [Moreover, different insect hosts responded differently to EPNs infections depending on the species and strain. Each nematode species uses different strategies to evade and suppress melanization and escape cellular encapsulation. Our previous study showed that EPNs of the family Steinernematids, activity . These aactivity . These Edulation ,56,57.H. bacteriophora and S. carpocapsae interact with O. nipae humoral immune system remains unknown. The present study, therefore, was conducted to determine the survival rates of O. nipae larvae at different concentrations of H. bacteriophora. Similarly, we evaluated the penetration abilities of both H. bacteriophora and S. carpocapsae against the third instar larvae of O. nipae. Further, we tested the differences in expression levels of four selected genes involved in proPO activation in O. nipae larvae. Finally, the effects of some selected secondary metabolites and isolated cuticles from nematodes on O. nipae proPO gene expression were investigated.Eicosanoids are other insect cellular and humoral immune mediators against several microbial infections ,59. TheyH. bacteriophora H06 (Poinar) and S. carpocapsae All (Weiser) were obtained from the Guangdong Institute of Applied Biological Resources, China [Galleria mellonella [O. nipae adults were collected from Hainan Island, China, and reared with small pieces of fortunes windmill palm, Trachycarpus fortunei (Hook) in the laboratory for many generations, after which healthy third instar larvae were selected and used for this study. The insects were maintained at 25 ± 1 °C, relative humidity of 80 ± 5%, and a photoperiod of 12 light: 12 dark hours, as previously described by Sanda et al. [s, China . Nematodllonella . Infectillonella . O. nipaa et al. .2 inhibitor, dexamethasone -9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3), the eicosanoid precursor, arachidonic acid , serine proteases inhibitor, phenylmethanesulfonyl (PMSF) fluoride, and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich  Trading Co., Ltd. .The phospholipase AO. nipae larvae were explored. In addition, the ability of the IJs to penetrate the O. nipae larvae at 0, 25, 50 and 100 IJs/larva concentrations was also experimented, and complete randomized design (CRD) was used as experimental design. Bioassay was conducted to determine the survival of O. nipae larvae at different concentrations of H. bacteriophora as fully described in our previous experiment using S. carpocapsae [H. bacteriophora and S. carpocapsae. After 72 h of inoculation, ten dead larvae (cadavers) were picked from the well plates, rinsed with distilled water, and dissected under dissecting stereo-microscope -Nikon company, Japan. The number of penetrated IJs were counted and recorded. Distilled water was used as control and each treatment contained 30 larvae, replicated three times to confirm the results.Here, the effects of EPN concentrations on mortality of pocapsae . In the O. nipae larvae infected with the two nematode species. These include Serine Protease P56 (SPP56), prophenoloxidase activation factor 1 (PPAF1), PPO, and serine protease inhibitor 28 (SPI28) genes  genes . Third ia et al. . RibosomO. nipae PPO gene, H. bacteriophora and S. carpocapsae body cuticles were isolated and purified as described in our previous study [O. nipae larvae were injected with 112 nL of purified isolated cuticles from H. bacteriophora and S. carpocapsae and the same amount of phosphate buffered saline (PBS) as control. Another set of the cuticles were subjected to heat treatment at 100 °C for 20 min. The heat-treated cuticles were injected in the same way as mentioned above. Total RNA isolation and qRT-PCR analysis were performed in triplicate for each biological replicate according to Sanda et al. [PPO gene expression levels due to the untreated cuticle, heat-treated cuticle, and control treatments.To determine whether nematode body cuticle have an influence on immune expression of us study . The O. a et al.  to checkO. nipae larvae, as described in our previous study [−1, and at 20 °C. The experiments were run in triplicate to confirm the results.We determined the inhibition effects of Dexamethasone’s presence on the phenoloxidase activity in an in vitro assay with hemolymph of us study . Secondlus study . For conO. nipae larvae with eicosanoid biosynthesis inhibitor, dexamethasone (DEX) and precursor, arachidonic acid (AA) individually and in combination with H. bacteriophora and S. carpocapsae to ascertain their roles on the expression of PPO gene. The inhibitor, DEX, was dissolved in 100% dimethyl sulfoxide (DMSO) at an initial concentration of 1 M and subsequently diluted to 5 mM concentration. Three treatment groups were prepared for injection into the O. nipae larvae at 112 nL using a Nanoliter 2010 injection system . The first group was injected with DEX only to test its inhibitory effects on O. nipae proPO activation system, and PBS was used as control. The second group of the larvae was initially infected with nematodes and then injected with DEX after 8 h post-infection. The last group was infected with nematodes + AA to recover the effects of nematode inhibition. Samples were taken at 24 h after treatment for RNA isolation. Total RNA isolation and qRT-PCR analysis were performed in triplicate for each biological replicate according to Sanda et al. [PPO gene expression levels of each group.We injected the a et al.  to invesp ˂ 0.05), means were compared or separated using least significance differences (LSD). The level of mRNA expression of some selected genes in proPO activation system of O. nipae was transformed by Logarithmic function and analyzed using the Student’s t-test. Differences between mean values were analyzed and considered significant when p < 0.05 or considered extremely significant when p < 0.0001 concerned the control values.All statistical analyses were performed using IBM SPSS Statistics version 22  . Data were corrected for control mortality using Abbott formula  and percS. carpocapsae at different concentrations by plotting the survival curve at different time points [O. nipae larvae at different concentrations were reported and at 24 and 48 h post-treatments [H. bacteriophora on the survival of O. nipae larvae was evaluated. The results showed that the survival ability of O. nipae larvae was significant at different time points and H. bacteriophora IJs concentrations . The O. nipae larvae survived for 140 h longer at lower concentrations of 25 and 50 IJs/larva than at 100 IJs/larvae, as shown in We previously evaluated the pathogenicity of e points . Signifieatments . In thisS. carpocapsae and H. bacteriophora penetrated the hemocoel of the larvae of O. nipae at the concentration of 25, 50, and 100 IJs/larva  than that of H. bacteriophora  in O. nipae larvae, as shown in S. carpocapsae and H. bacteriophora.Similarly, the results for the penetration assay revealed that the IJs of Js/larva . HoweverO. nipae prophenoloxidase enzymes  between H. bacteriophora- and S. carpocapsae-treated larvae at three distinct time intervals. Our qRT-PCR results reveals that SPP56 was significantly upregulated  after H. bacteriophora challenge and insignificantly upregulated  upon S. carpocapsae treatment at 8 h post-infection  of the gene  and H. bacteriophora  treatments  but significantly downregulated at 24 h  after injection of the isolated cuticles from the two nematodes  and S. carpocapsae  heat-treated cuticles injections. At 16 h after injection of S. carpocapsae cuticle, insignificance  upregulation of PPO gene was recorded . However, the addition of AA rescued the phenoloxidase-inhibitory activities caused by DEX treatments .In this study, phenoloxidase-inhibitory activities of eicosanoid biosynthesis inhibitors and DEX treatments compared to controls were observed. There were significant decreases in phenoloxidase enzyme activities in hemolymph of eatments . There wPPO gene in treated larvae samples. Our results revealed that the expression level of PPO gene was downregulated significantly upon treatment of DEX  compared with control  and S. carpocapsae plus DEX  treatments at 24 h. Interestingly, injection of AA (precursor of eicosanoid biosynthesis) to nematode-treated larvae reverses the inhibition effects of both DEX and nematode plus DEX-treated larvae on the PPO gene expression. The mRNA level of PPO gene in H. bacteriophora plus AA  and S. carpocapsae plus AA  treatment were highly significant at 24 h after treatments.Secondly, injections of DEX to nematodes-treated larvae further suppressed the expression level of O. nipae larvae were more susceptible to H. bacteriophora. Compared to our previous results, the virulence of S. carpocapsae [H. bacteriophora can be attributed to their differences in host-searching strategies, as reported by Grewal et al. [G. mellonella hemocytes can recognize H. bacteriophora IJs, but not S. carpocapsae and S. glaseri nematodes [S. carpocapsae efficiently resists being encapsulated and melanized within the O. nipae’s hemolymph and most of the nematodes were observed moving freely in the hemolymph even at 24 h post-incubation, whereas H. bacteriophora was identified, encapsulated and subsequently melanized by O. nipae’s hemolymph [H. bacteriophora was less virulent to carob moth Ectomyelois ceratoniae Zeller (LC50 = 426.9 IJs larva−1) when compared to both S. carpocapsae and S. feltiae (LC50 = 2 IJs/larva) at concentrations similar to this study. Additionally, Sharifi et al. [S. carpocapsae caused higher mortality of Osphranteria coerulescens than H. bacteriophora, as evident by their LC50 of 2.7 and 9.0 IJs larva−1, respectively. This is consistent with quite a number of reports for different insect pest control [Entomopathogenic nematodes modulate their hosts differently, depending on the strategies used to escape the host defense mechanisms. For example, Zang and Maizels  describepocapsae  over H. l et al.  and Koppl et al. . Additioematodes . This isemolymph . Moreoveemolymph  reportedi et al.  have rep control ,78,79,80S. carpocapsae and H. bacteriophora with regards to the mean penetration potential within the host larvae. This is also similar to a report by Susurluk et al. [S. carpocapsae into turf pest, Dorcadion pseudopreissi, and lower in H. bacteriophora treatments. Similarly, higher penetration rates were also reported for Steinernematids in previous works by Phan et al. [In any successful biological control program using EPNs as control agents, the nematodes should have the ability to not only penetrate their hosts but also reproduce inside them. The routes of entry of IJs into the host body cavity differ among nematode species and insect hosts. These require some mechanical processes by the nematodes to bypass the insects’ natural barriers, such as hairs . Accordik et al. , which en et al. , Salari n et al. , Salem en et al. , Koppenhn et al. , and Carn et al. .self’ as in S. carpocapsae. They also suppress the hosts’ immune responses after being recognised as ‘non-self’ [O. nipae infected with S. carpocapsae and H. bacteriophora. From our results, we found that nearly all the genes involved were downregulated in both nematodes and at all time points, except in a few cases. Initially, when the serine protease SPP56 was majorly downregulated, the whole enzymes involved in the activation of proPO cascades are shut down. This is consistent with our previous study where O. nipae’s phenoloxidase activity was found to be slightly suppressed due to infection of both H. bacteriophora and S. carpocapsae at early stages of infection [During host infection, nematodes contend the humoral and cellular host immune responses through passive and active strategies, in which nematodes avoid triggering an immune response by being recognised as ‘on-self’ ,87. In aon-self’ ,89,90. Aon-self’ . Howevernfection .S. carpocapsae isolated cuticles escaped being melanized by O. nipae’s hemolymph in vitro and were found to be completely melanized, however, when isolated cuticles were heat-killed before treatment. This is in line with some previous reports that show success in parasitization of Steinernematids is aided by their body surface cuticles [S. feltiae was shown to prevent the antibacterial immune response in G. mellonella by their body surface [S. carpocapsae or H. bacteriophora cuticles caused suppressions of humoral and cellular immune processes, such as phenoloxidase activity, haemocytes vitality, phagocytosis and antimicrobial activity. In this study, we confirm that the purified, isolated cuticles suppress the expression level of PPO gene in both nematodes’ treatments. However, upon heating, the cuticles lost their inhibitory effects on O. nipae’s immune responses and resulted in the upregulation of the PPO gene at both 16 and 24 h after treatment.Additionally, the cuticles ,91,92,93 surface . Akhurst surface  suggest  surface . Yi et a surface  showed tPPO gene in O. nipae, similar to that caused by treatment with H. bacteriophora and S. carpocapsae. In contrast, the addition of AA reversed the process and resulted in the upregulation of the PPO gene compared to the control treatment. In a study by Yi et al. [G. mellonella. However, injection of larvae with AA reverses the inhibitory effects of these inhibitors. Similarly, benzaldehyde and its derivative treatments resulted in the inhibition of PO activity in Pieris rapae larvae [G. mellonella larvae. Similarly, treatments with BZA (benzylideneacetone) in another study resulted in the immunosuppression of PO activities in Plutella xylostella and S. exigua [Furthermore, eicosanoids mediate basic humoral and cellular immune response mechanisms in insects . These ii et al. , injectie larvae . This ise larvae  in which. exigua ,98,99. WH. bacteriophora and S. carpocapsae for biological control of O. nipae larvae at different concentrations in the laboratory. We further dissected the expression levels of four selected proPO activation enzymes infected with H. bacteriophora and S. carpocapsae and found out that all of the genes were downregulated from both nematodes’ treatments. These were further confirmed by the counter experiments of heat-treated cuticles and AA treatment, which reversed the inhibitory effects caused by the nematodes and the eicosanoid inhibitor. Therefore, we speculated that these nematodes devised some means to evade and suppress the immune system of O. nipae, either by secretion of toxins or by manipulation of the host system. Future work will focus on the identification and functional analysis of the secretions used by S. carpocapsae and H. bacteriophora against O. nipae immune system.Overall, this study provides the first data on the use of


Worldwide, up to 8.8 million excess deaths/year have been attributed to air pollution, mainly due to the exposure to fine particulate matter (PM). Traffic-related noise is an additional contributor to global mortality and morbidity. Both health risk factors substantially contribute to cardiovascular, metabolic and neuropsychiatric sequelae. Studies on the combined exposure are rare and urgently needed because of frequent co-occurrence of both risk factors in urban and industrial settings. To study the synergistic effects of PM and noise, we used an exposure system equipped with aerosol generator and loud-speakers, where C57BL/6 mice were acutely exposed for 3d to either ambient PM (NIST particles) and/or noise (aircraft landing and take-off events). The combination of both stressors caused endothelial dysfunction, increased blood pressure, oxidative stress and inflammation. An additive impairment of endothelial function was observed in isolated aortic rings and even more pronounced in cerebral and retinal arterioles. The increase in oxidative stress and inflammation markers together with RNA sequencing data indicate that noise particularly affects the brain and PM the lungs. The combination of both stressors has additive adverse effects on the cardiovascular system that are based on PM-induced systemic inflammation and noise-triggered stress hormone signaling. We demonstrate an additive upregulation of ACE-2 in the lung, suggesting that there may be an increased vulnerability to COVID-19 infection. The data warrant further mechanistic studies to characterize the propagation of primary target tissue damage  to remote organs such as aorta and heart by combined noise and PM exposure. •Traffic noise and particulate matter (PM) always co-localize in urban environments.•New prototype exposure system for combined exposure of mice to traffic noise and PM.•First mechanistic insights in multi-organ damage by combined exposure to noise and PM.•Characterization of brain damage by noise, lung damage by PM and vascular/systemic damage by combination.•Evaluation of multi-organ gene expression by RNA sequencing and cytokine profiles by array technique. Diseases caused by pollution caused an estimated 9 million premature deaths/year worldwide . The WHO3 and •NO2) markedly contribute to cardiovascular and cerebrovascular disease and excess mortality ) and reactive gases . Forto PM2.5 . In 2020to PM2.5 ,14.In addition to the well-established health risks of air pollution , environAccording to the WHO, at least 1.6 million healthy life years are lost annually from traffic noise in Western Europe . StudiesDespite the urgent need for multi-exposure models, most studies on combined effects of noise and air pollution thus far rely on mathematical rather than experimental models, warranting studies under controlled laboratory conditions. Only a few studies have addressed the mutual effects of traffic noise and air pollution due to their interconnected nature . Here, w1)What is the ideal suspension of PM (NIST) to study the effects of air pollution? Ideally, it should contain a high fraction of PM with a diameter of 2.5 μm and lead to oxidative stress, inflammation and endothelial dysfunction, representing the main adverse effects of PM .2)What are the health effects of individual exposures to noise and air pollution?3)Are there additional health effects from the co-exposure to noise and air pollution related to the cardiovascular and cerebral system and the lungs?4)What is the impact of co-exposure to both environmental stressors on the expression of genes in various affected organs?Specifically, we addressed the following questions:22.1The system for the combined PM and noise exposure is illustrated in suppl. 2.23, as explained in the supplement, which is justified by the short exposure protocol and real world particulate matter concentrations reaching up to 200 μg/m3 in highly polluted cities [All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the U.S. National Institutes of Health and approval was granted by the Ethics Committee of the University Medical Center Mainz and the Landesuntersuchungsamt Rheinland-Pfalz . For the main study, a total of 172 male C57BL/6J mice were exposed to either PM only (n = 47), noise only (n = 40), combination of PM and noise (n = 42) or to fresh air and no noise (n = 43) , a number of highly toxic (nitro) polycyclic aromatic hydrocarbons (PAHs) such as pyrenes and anthracenes as well as several polychlorinated biphenyls (PCBs) and chlorinated pesticides (see SRM1648a datasheet in the supplement). The content of biogenic (e.g. endotoxins) and anthropogenic contaminations  on the particles, partially taken-up in urban space or generated by photochemical reactions during particle atmospheric aging, seems to represent a major determinant of their biological toxicity . The met2.3All other methodological details, e.g., for blood pressure measurements by tail cuff, vasoreactivity studies by isometric tension technique, immunohistochemistry, Western blotting, RT-PCR, envisaging of reactive oxygen species (ROS) formation by fluorescent dihydroethidium microtopography, cytokine array, RNA sequencing and bioinformatical analysis, are provided in the Extended Methods in the online supplement and also in our previous work related to environmental risk factors .33.1We have developed an exposure protocol to study the effects of noise on the cardiovascular system of mice ,42. We f3.2Nox2 and Hmox1 mRNA was mostly driven by PM exposure alone and was highest in the combined exposure group  were only observed in the PM + noise exposure group suppl. . Gene re4Our study yields a novel co-exposure model of noise and ambient PM pollution. NIST2 particles were chosen since the exposure to this PM preparation causes significant endothelial dysfunction, oxidative stress and inflammation comparable to that observed with single exposure to aircraft noise ,42. Our 4.1Nox2 knockout [It is believed that the cognitive perception of noise triggers cortical activation and the release of stress hormones leading to high blood pressure, increased vascular and cerebral inflammation and oxidative stress that in the long turn can enhance cardiovascular risk factors such as diabetes, high cholesterol levels, activation of blood coagulation factors and hypertension that subsequently manifests as cardiovascular disease  ,51. Receknockout  and by hknockout , pointinOxidative stress is also a hall-mark and trigger of the adverse vascular health effects of air pollution ,57. Ultr•NO bioavailability, endothelial cell activation (adhesion molecule expression) and inflammation [Nox2 knockout mice [p47phox knockout mice [Receptors, such as the transient receptor potential cation channel subfamily A member 1 (TRPA1) receptors, in airway sensory neurons can also sense environmental toxicants and aerogenic oxidants, resulting in neurogenic inflammation . Activatammation ,57. Impoout mice  and p47pout mice . These pout mice ,62.In summary, noise and air pollution share a number of pathophysiological aspects, e.g. the dominant role for reactive oxygen species and inflammation in causing endothelial dysfunction and high blood pressure. We exposed our animals in an experimental approach separately as well as simultaneously to these environmental stressors because noise and air pollution share a number of urban/industrial sources.4.2Nox2 and Hmox1 mRNA was mostly driven by PM exposure alone and was highest in the combined exposure group. MCP-1 was also additively upregulated by co-exposure, indicating additive effects of the combination of both stressors on inflammatory processes. Importantly, the protein and mRNA expressions of pulmonary ACE-2, which represents the docking and entry mechanism for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [Exposure to PM is known to increase pulmonary oxidative stress  while thnfection , showed nfection ,66. A renfection . In addinfection . Polymornfection ,68,69. Tnfection .We have previously reported that exposure to noise alone increased systolic and diastolic blood pressure via stimulating the release of neurohormones and by activating the HPA-axis ,42. Sing•NO donor sodium nitroprusside was not modified. Vascular oxidative stress as determined by fluorescence of oxidized dihydroethidium products was increased by the single exposures and showed a trend of additive augmentation in the co-exposure group along with higher nitrotyrosine levels. In addition, the oxidative stress response protein, heme oxygenase-1, was upregulated in an additive manner and the expression levels of the antioxidant enzymes Cu,Zn- and Mn-superoxide dismutase were lower in an additive manner. Thus taken together, a higher degree of oxidative stress may be at least in part responsible for the severe degree of endothelial dysfunction in the co-exposure group.In contrast, endothelial dysfunction of conductance (aorta) and resistance vessels  showed a substantial additive negative effect on vascular function, while vasodilation to the endothelium independent Transportation noise exerts its detrimental health effects to a large extent via the brain, where it initiates stress responses by activating direct and indirect pathways accompanied by increases in circulating catecholamine and cortisol concentrations . AccordiPreviously, we demonstrated significant changes of genes in part responsible for the regulation of vascular function, circadian rhythm, vascular remodeling, and cell death using comparative Illumina sequencing of transcriptomes of aortic tissues from aircraft noise-treated animals . The traThe lung represents one organ for which we identified possible synergistic negative interactions. We detected a robust induction of pathways related to a DNA-damage response upon PM exposure as exemplified by the activation of p53 signaling, mismatch repair, cell cycle changes and apoptosis. In addition, phagocytosis and lysosomal activation are likely to be induced to clear small particles lodged in the lung tissue. PM clearly induces oxidative stress and inflammation , but also appears to induce metabolic responses. We detected altered signaling in the AMPK pathway, one of the major regulators of cellular energy metabolism. Accordingly, we observed that metabolic pathways downstream of AMPK signaling were also regulated by PM. It included both PPAR signaling, which is a crucial regulator of lipid metabolism, as well as the induction of proteins which are involved in oxidative phosphorylation, or the TCA cycle. In addition, we have identified that glutathione synthesis is induced as a response that is likely due to heightened levels of oxidative stress. Intriguingly, the induction of these PM-induced stress pathways is further exaggerated by noise stress, despite the fact that noise exposure alone causes a nearly completely independent transcriptional response. Transcriptomic analysis in the brain, on the other hand, indicated that the combination of noise and PM exposure elicited a transcriptional response that was comparable to the noise-stress response only, with many of the PM-only dependent transcriptional responses being absent in the combination treatment.trans-synaptic signaling and “behavior”, all of which are intimately linked to neuropathological pathways. In the aorta, noise-only exposure triggered the expression of a hypoxic signature. We detected that numerous genes reported to be induced by the hypoxia inducible factor (HIF)-driven transcription were upregulated. This included the induction of genes in the glycolytic pathway, and the downregulation of genes involved in the oxygen-dependent production of ATP, the TCA cycle, lipolysis and the mitochondrial electron transport chain. Suggesting that noise stress may affect the metabolic state of the cardiovascular system, in a similar way to hypoxia. Remarkably, the combination of PM + noise reduced this response, for which we currently have no explanation.Overall, stressors induced multiple signaling pathways that are of relevance for cardiovascular and neurological diseases. In the brain, the most prominent GO cluster induced by noise or co-exposure included ion transport, 5Previous animal studies have shown additive/synergistic adverse cardiovascular health effects via exaggerated oxidative stress by noise and pre-existing arterial hypertension  as well 10.13039/501100008454Boehringer Ingelheim Foundation for the collaborative research group “Novel and neglected cardiovascular risk factors: molecular mechanisms and therapeutics” and through continuous research support from Foundation Heart of Mainz (mostly dedicated to the purchase of the exposure system). M.K., I.K., M.T.B.J., K.V.-M. and S.K. hold or held TransMed PhD stipends funded by the 10.13039/501100008454Boehringer Ingelheim Foundation. L.S. and H.U. hold TransMed PhD stipends funded by the Else-Kröner-Fresenius Foundation. S.S. holds an excellence stipend of the Else-Kröner-Fresenius Foundation (2021_EKES.04). T.J. holds a MD stipend funded by the Heart Foundation Mainz. T.M. is PI and A.D. and P.S. and S.D. are (Young) Scientists of the 10.13039/100010447DZHK (German Center for Cardiovascular Research), Partner Site Rhine-Main, Mainz, Germany. Also financial support by 10.13039/501100000289Cancer Research UK (CRUK Edinburgh Centre C157/A255140) and 10.13039/100010269Wellcome Trust  is gratefully acknowledged. The collaboration of the authors was supported by European 10.13039/501100000921COST Action CA20121: Bench to bedside transition for pharmacological regulation of NRF2 in noncommunicable diseases (BenBedPhar). Webpage: https://benbedphar.org/about-benbedphar/.A.D. and T.M. were supported by vascular biology research grants from the The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.