When TG explants were incubated with media containing dexamethasone to stimulate reactivation, significantly fewer β-catenin+ TG neurons were detected. Conversely, TG explants from uninfected mice or mice latently infected with a LAT-/- mutant increased the number of β-catenin+ TG neurons in the presence of DEX relative to samples not treated with DEX. Impairing Wnt signaling with small molecule antagonists reduced virus shedding during explant-induced reactivation. These studies suggested β-catenin was differentially expressed during the latency-reactivation cycle, in part due to LAT expression.When herpes simplex virus 1 (HSV-1) infection is initiated in the ocular, nasal, or oral cavity, sensory neurons within trigeminal ganglia (TG) become infected. Following a burst of viral transcription in TG neurons, lytic cycle viral genes are suppressed and latency is established. The latency-associated transcript (LAT) is the only viral gene abundantly expressed during latency, and LAT expression is important for the latency-reactivation cycle. Reactivation from latency is required for virus transmission and recurrent disease, including encephalitis. The Wnt/β-catenin signaling pathway is differentially expressed in TG during the bovine herpesvirus 1 latency-reactivation cycle. Hence, we hypothesized HSV-1 regulates the Wnt/β-catenin pathway and promotes maintenance of latency because this pathway enhances neuronal survival and axonal repair. New studies revealed β-catenin was expressed in significantly more TG neurons during latency compared to TG from uninfected mice or mice latently infected with a LAT More than 50% of adults in the United States are latently infected with herpes simplex virus 1 (HSV-1), reviewed in . HSV-1 i-/- mutant virus restores reactivation to WT like levels Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: No**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Review of: Herpes simplex virus 1 regulates beta-catenin expression in TG neurons during the latency-reactivation cycleBy Harrison, Zhu, Thunuguntla and Jones,In this manuscript, the authors show that β-catenin is upregulated in TG neurons latently infected with HSV-1 in a LAT dependent manner. The authors further establish that induction of reactivation by dexamethasone treatment results in dramatic downregulation/degradation of β-catenin. This decrease was not observed in TG infected with LAT null virus. Furthermore, expression of β-catenin is beneficial for virus because inhibition of this pathway during reactivation results in reduced viral shedding.The findings reported here are very interesting and valuable for the advancement of the field, and only minor improvements are suggested as described below.1. Line 68: How soon after reactivation does Wnt signaling decrease? In figure 6, no decrease in β-catenin expression is seen at 4hr, 8hr post ex-plantation. Why were these timepoints selected?2. Figures 1 and 2: Was β-catenin detected exclusively in neurons? Did you observe any immune cells/supporting cells expressing β-catenin?3. Line 200: Word “estimated” is a bit unclear- did you count number of β-catenin positive neurons over total number of neurons to determine percentage or “eyeball” percentage of β-catenin positive neurons?4. Figure 4: LAT-/- 4 panel: cells surrounding β-catenin positive cells look different from other panels. Also, not necessary but if you can please replace it with a better image.5. Line 212- What does “GR” stand for?6. Line 235 states that β-catenin staining was detected outside of TG neurons. Can you show this in Fig 4 and elaborate on the significance of this finding?7. Line 251: Authors state that intensity of β-catenin was lower in uninfected TG than in infected TG. This suggests that while higher percentage of cells might stain positive for β-catenin, the overall expression levels may be lower. Could you elaborate on this?8. Figure 6: Dex treatment in uninfected TG results in roughly two-fold higher β-catenin positive neurons when compared to LAT-/-, dex treated TG. These results suggest that viral factors other than LAT may contribute to β-catenin downregulation. Can you please comment on this possibility?9. The finding that the percentage of cells expressing β-catenin is higher than the reported number of infected cells is intriguing, as it suggests that other, surrounding uninfected cells are also producing β-catenin. The authors propose this could be caused by miRNA/sncRNA encoded by LAT that are released in exosomes. Perhaps the authors could speculate the functional significance of causing β-catenin upregulation in surrounding cells .10. It seems the two sncRNAs may play a more important role in latency-reactivation than miRNA based on the authors work and Wechsler’s work. Perhaps the authors could elaborate on this?11. Figure 7 suggests that inhibition of Wnt signaling results in decreased viral replication. This seems counterintuitive considering that Wnt pathway is turned off/ β-catenin expression is decreased during reactivation (as stated on lines 68)? Perhaps the authors could elaborate on this?12. The authors are a bit cautious in their discussion, and could expand the discussion section a bit if they wish.Reviewer #2: Harrison and colleagues have investigated the expression of beta-catenin in the context of herpes simplex virus 1 latency and reactivation using a mouse ocular infection model. The authors determined that beta-catenin expression was enhanced in trigeminal ganglia (TG) of mice latently infected with a wild type (WT) HSV-1 strain compared to uninfected animals, or animals latently infected with an HSV-1 LAT deletion mutant. Reactivation from latency was achieved by explanting latently infected TG in the presence of dexamethasone (DEX). Whereas beta-catenin expression was reduced in TG latently infected with WT virus upon explant in the presence of DEX, beta-catenin expression was significantly enhanced in explanted TG from both LAT infected and uninfected animals that had been treated with DEX. In a final set of experiments the authors show that compounds that inhibit the Wnt/beta-catenin signalling pathway are potent inhibitors of WT HSV-1 reactivation in explanted TG. The authors central conclusions are that the Wnt/beta-catenin signalling pathway is important for HSV-1 reactivation and that beta-catenin is differentially expressed during latency (high expression) and reactivation (low expression) and that LAT somehow regulates beta-catenin expression.While the findings are potentially interesting, the study is incomplete, inadequately controlled, and the data are of variable quality. Thus, the findings are too preliminary to be particularly informative to researchers in this field.Major issues:1) Can the authors confirm that TG neurons are latently infected and quantify this? Are the TG neurons expressing beta-catenin the ones that are latently infected? What proportion of latently infected TG neurons express beta-catenin?2) The uninfected controls in figure 5 do not look like the uninfected control sections in figure 1. Why? The uninfected TG explanted in DEX in figure 5 that are highlighted with arrowheads do not look to be beta-catenin positive. If the primary antibody was excluded in control experiments and the samples double-blinded could beta-catenin positive cells be identified with confidence?3) Figure 6 and sentence on lines 347-349. Authors should include uninfected TG from mice that had not been explanted  to control for the effects of explant on beta-catenin expression.4) Sentence on lines 279-280. What are the numbers of latently infected TG neurons in animals infected with WT virus compared to LAT mutant virus? Need to show this important control.5) Figure 6. What is the degree of reactivation from TG explanted from WT and LAT mutant infected animals? Presumably, the medium associated with the explanted TG analyzed for beta-catenin expression shown in figure 6 was available for virus titration. 6) Figure 7. How do Wnt/beta-catenin inhibitors impact the reactivation of LAT mutants? How would DEX impact the action of the Wnt/beta-catenin inhibitors on virus reactivation?7) Lines 341-343. This is an egregious over-interpretation of the data.Minor issues:1) It is curious that Wnt/beta-catenin agonists are so potent at inhibiting reactivation when the levels of beta-catenin are so low during the reactivation phase. Can the authors comment on this apparent inconsistency?2) Line 27. Why highlight ocular disease here? Line 22 introduces ocular, nasal and oral infection. Consider "recurrent disease".3) Line 35. "increased" compared to what?4) Line 47. Odd phrase. Suggests oral disease upon reactivation after primary infection of the eye? This is confusing. Similar issues in abstract. Consider rephrasing.5) Lines 49-50. This sentence implies LAT is not expressed during lytic infection. I don't think this is the point the authors are trying to make. Consider rephrasing.6) Line 53. Why does the nature of LAT locus products "suggest" functions related to latency? Consider rephrasing.7) Line 76. “Wnt binding” to what?8) Line 170. “rational” should be “rationale”.9) Line 177. “marker rescued” should read “repaired”.10) Sentence on lines 308-309. Alternatively, the data may indicate that the inhibitors were toxic to the explants. Can the authors control for this? Is there a dose response to the inhibitors?**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  2 Mar 2020With respect to the comments of Reviewer #1, the following changes in the revised manuscript were made.Concern #1: Line 68: How soon after reactivation does Wnt signaling decrease? In figure 6, no decrease in β-catenin expression is seen at 4hr, 8hr post explantation. Why were these timepoints selected? Response: �-catenin+ TG neurons were nearly undetectable after TG explants were incubated with MEM, 2% stripped FBS, and DEX. However, �-catenin+ TG neurons were readily detectable when there was no DEX added to TG explants. When TG explants are treated with DEX to stimulate reactivation from latency, there are significantly more caspase 3+ TG neurons at 4, 8 and 16 hours after TG explant relative to TG explants not treated with DEX, Refence #31. Since there was a clear-cut difference in �-catenin expression during early times after TG explant in the presence of DEX, we did not examine �-catenin expression at later times because of increased apoptosis. Concern #2: Figures 1 and 2: Was β-catenin detected exclusively in neurons? Did you observe any immune cells/supporting cells expressing β-catenin? Response: For mice latently infected with wt HSV-1, most β-catenin staining was detected in neurons. In TG explants from mice latently infected with dLAT2903, we saw staining observed in neurons and other cells, which is pointed out in lines 253-258 . Since we did not include any differential stain to establish what types of immune/supporting cells were expressing, it is not clear which cells types are expressing β-catenin.Concern #3: Agreed. Line 200: Word “estimated” is a bit unclear- did you count number of β-catenin positive neurons over total number of neurons to determine percentage or “eyeball” percentage of β-catenin positive neurons? Response: We have replaced the word “estimated” with “counted”; total number of β-catenin positive neurons over total number of neurons to determine percentage because we did in fact count �-catenin+ TG neurons (lines 147-148 and 217-218). Concern #4: Figure 4: LAT-/- 4 panel: cells surrounding β-catenin positive cells look different from other panels. Also, not necessary but if you can please replace it with a better image. Response: A higher quality image has been used for LAT-/- panel in Figure 4.Concern #5: Line 212- What does “GR” stand for? Response: GR is glucocorticoid receptor, and this was defined in the revised manuscript (line 228-230). We have carefully examined the manuscript to ensure all acronyms have been defined.Concern #6: Line 235 states that β-catenin staining was detected outside of TG neurons. Response: Recent published studies demonstrated the Wnt pathway is activated within the CNS after injury, including surrounding non-neuronal cell types such as oligodendrocytes and microglia. Thus, the observation of β-catenin staining outside of neurons may be due to expression in supportive cells. This point is included in the revised manuscript (lines 255-258).Concern #7: Line 251: Authors state that intensity of β-catenin was lower in uninfected TG than in infected TG. This suggests that while higher percentage of cells might stain positive for β-catenin, the overall expression levels may be lower. Could you elaborate on this? Response: This statement was made because the intensity of staining did not appear to be as bright, which is difficult to quantify. However, it was clear staining was brighter, which suggested there is more β-catenin in these samples. These points were clarified in the revised manuscript (lines 273-276). Concern #8: Figure 6: Dex treatment in uninfected TG results in roughly two-fold higher β-catenin positive neurons when compared to LAT-/-, dex treated TG. These results suggest that viral factors other than LAT may contribute to β-catenin downregulation. Can you please comment on this possibility?Response: There have been reports that additional viral gene products can be detected in TG during latency; ICP0, ICP4, and thymidine kinase RNA for example. If these lytic cycle regulatory proteins are expressed (ICP0 and ICP4 in particular) they could influence cellular gene expression. This point is included in the revised manuscript (lines 303-309). Concern #9: The finding that the percentage of cells expressing β-catenin is higher than the reported number of infected cells is intriguing, as it suggests that other, surrounding uninfected cells are also produce β-catenin. The authors propose this could be caused by miRNA/sncRNA encoded by LAT that are released in exosomes. Perhaps the authors could speculate the functional significance of causing β-catenin upregulation in surrounding cells Response: We have expanded on the possibility of LAT-encoded non-coding RNAs being transported to surrounding TG neurons via exosomes and why this may impact surrounding neurons, and perhaps non-neuronal cells during latency (lines 393-410). Concern #10: It seems the two sncRNAs may play a more important role in latency-reactivation than miRNA based on the authors work and Wechsler’s work. Perhaps the authors could elaborate on this?Response: We have expanded this part of the discussion, as suggested (lines 372-391). Concern #11: Figure 7 suggests that inhibition of Wnt signaling results in decreased viral replication. This seems counterintuitive considering that Wnt pathway is turned off/ β-catenin expression is decreased during reactivation (as stated on lines 68)? Perhaps the authors could elaborate on this? Response: This section has been revised to explain how �-catenin may have dual roles during the latency-reactivation cycle. (lines 430-451).Concern #12: The authors are a bit cautious in their discussion and could expand the discussion section a bit if they wish.Response: After addressing Concerns 9-11, I think we have speculated as much as I feel comfortable until we pursue this project more in detail. With respect to the comments of Reviewer #2, the following changes were made.Concern #1: Can the authors confirm that TG neurons are latently infected and quantify this? Are the TG neurons expressing beta-catenin the ones that are latently infected? What proportion of latently infected TG neurons express beta-catenin?  Response: In the discussion, we proposed two mechanisms by which �-catenin may be expressed in latently infected TG neurons as well as a subset of TG neurons that are “bystanders” (lines 393-410). It is not trivial to accurately calculate the % of TG neurons that are latently infected and express ��catenin. To do this, one would have to cut consecutive sections and perform IHC on one section to identify �-catenin+ TG neurons and on the other section perform IHC to identify a viral protein during reactivation. We tried this approach and it was difficult to interpret because during explant-induced reactivation �-catenin+ TG neurons are nearly undetectable. It is possible to identify LAT+ TG neurons using in situ hybridization on one section; on the consecutive section perform IHC to visualize �-catenin+ TG neurons. There are a couple of issues with this approach, which would make it difficult to achieve a clear-cut and accurate result. First, it is well established there are HSV-1 genome+ TG neurons that do not express detectable levels of LAT during latency in mice: however, it is not clear what % of the total latently infected TG neurons do not express LAT. Secondly, TG sections from latently infected mice are very difficult to obtain superior consecutive sections. We routinely obtain excellent consecutive section from calves latently infected with bovine herpesvirus 1, but not mice latently infected with HSV-1. Finally, it is difficult to use two antibodies that are differentially stained on the same TG section, at least in our hands. We have done this with brain tissue, but when the same antibodies were used for TG sections the background was terrible. In summary, I do not believe these studies would yield convincing or accurate results. If necessary, these points can be added to the revised manuscript.Concern #2: The uninfected controls in figure 5 do not look like the uninfected control sections in figure 1. Why? Response: The uninfected sections presented in Figure 1 were dissected as intact TG and then directly placed into formalin post-euthanasia. Uninfected tissue in Figure 5 was minced, explanted in MEM containing 2% stripped FBS, and then incubated for 4 and 8 hours. These small TG pieces were then collected and placed in formalin and paraffin embedded. While the integrity of the tissue is not as good as intact TG immediately placed in formalin, they clearly show the general morphology of TG. This is stated in the revised manuscript (lines 276-278).Concern #3: The uninfected TG explanted in DEX in figure 5 that are highlighted with arrowheads do not look to be beta-catenin positive. If the primary antibody was excluded in control experiments and the samples double-blinded could beta-catenin positive cells be identified with confidence? Response: The TG neurons in Figure 5 are stained  relative to the no primary antibody control. The no primary antibody control was performed using TG sections form mice latently infected with wt HSV-1: samples from explanted TG (no primary antibody control) also do not yield any staining: this is stated in the revised manuscript (lines 271-276). if necessary, we can add the no primary antibody TG sections for uninfected TG that was explanted to Figure 5. All TG sections in this study were analyzed in a blind fashion. This was stated in the materials and methods of the original manuscript (lines 147-148). Concern #4: Figure 6 and sentence on lines 347-349. Authors should include uninfected TG from mice that had not been explanted  to control for the effects of explant on beta-catenin expression. Response: These images were included as the uninfected panels shown in Figure 1 of the original manuscript. Concern #5: Sentence on lines 279-280. What are the numbers of latently infected TG neurons in animals infected with WT virus compared to LAT mutant virus? Need to show this important control. Response: This topic was discussed in the revised manuscript (lines 193-206). Concern #6: Figure 6. What is the degree of reactivation from TG explanted from WT and LAT mutant infected animals? Presumably, the medium associated with the explanted TG analyzed for beta-catenin expression shown in figure 6 was available for virus titration. Response: Studies in Figure 7 and 8 show a comparison between efficiency of wt HSV-1 versus the LAT-/- mutant. The results demonstrated at least 2 logs reduction in the LAT-/- mutant relative to wt. These results are consistent with our recent studies, reference #31 in the revised manuscript.Concern #7: Figure 7. How do Wnt/beta-catenin inhibitors impact the reactivation of LAT mutants? Response: dLAT2903 explant-induced reactivation is significantly reduced compared to WT . Hence iCRT14 was assessed for LAT mutant reactivation at 5 days after explant (the peak effect of iCRT14 observed in WT HSV1 reactivation). dLAT2903 reactivation was decreased in the presence of iCRT14: Figures 7 and 8 (lines 349-369). Concern #8: How would DEX impact the action of the Wnt/beta-catenin inhibitors on virus reactivation? Response: New studies showing this data are presented in Figure 8.Concern #9: Lines 341-343. This is an egregious over-interpretation of the data. Response: Agreed. Our thought on this topic have been revised and are less speculative (lines 393-410). Minor issues:Concern #9: It is curious that Wnt/beta-catenin antagonists are so potent at inhibiting reactivation when the levels of beta-catenin are so low during the reactivation phase. Can the authors comment on this apparent inconsistency? Response: The antagonists are measuring viral shedding in the supernatant 4-9 days post-explant. Under the conditions of our TG explant studies, this is when virus shedding consistently occurred (reference 31). Conversely, IHC analyzing beta-catenin expression was performed 4 and 8 hours post-explant. The integrity of the tissue after 16hrs post-explant is compromised and IHC results would not be a clear-cut. Two previous studies demonstrated �-catenin promotes HSV1 productive infection in cell culture (references 32 and 58). This was discussed in the revised manuscript (lines 430-451).Concern #10: Line 27. Why highlight ocular disease here? Line 22 introduces ocular, nasal and oral infection. Consider "recurrent disease". Response: This was modified (lines 29-30).Concern #11: Line 35. "increased" compared to what? Response: This error was corrected in the manuscript (lines 34-36).Concern #12: Line 47. Odd phrase. Suggests oral disease upon reactivation after primary infection of the eye? This is confusing. Similar issues in abstract. Consider rephrasing. Response: This was modified in the manuscript (lines 48-50).Concern #13: Lines 49-50. This sentence implies LAT is not expressed during lytic infection. I don't think this is the point the authors are trying to make. Consider rephrasing. Response: This has been corrected in the manuscript (lines 52-54).Concern #14: Line 53. Why does the nature of LAT locus products "suggest" functions related to latency? Consider rephrasing. Response: This sentence was modified (lines 55-57).Concern #15: Line 76. “Wnt binding” to what? Response: This has been corrected in the manuscript (lines 79-83).Concern #16: Line 170. “rational” should be “rationale”. Response: This has been corrected in the manuscript (lines 172-174).Concern #17: Line 177. “marker rescued” should read “repaired”. Response: This has been corrected in the manuscript (lines 180-182).Concern #18: Sentence on lines 308-309. Alternatively, the data may indicate that the inhibitors were toxic to the explants. Can the authors control for this? Is there a dose response to the inhibitors? Response: Previous data from our lab has shown iCRT14 does not have any cytotoxicity and no significant dose response in cell culture (31). We repeated this study and performed toxicity studies using KYA1797K in cell culture and found this does not have toxic effects in cell culture up to 50µM. This has been updated in the manuscript (lines 328-330). 11 Mar 2020Herpes simplex virus 1 regulates beta-catenin expression in TG neurons during the latency-reactivation cycle.PONE-D-19-33585R1Dear Dr. Jones,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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DeLuca, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 16 Mar 2020PONE-D-19-33585R1 Herpes simplex virus 1 regulates beta-catenin expression in TG neurons during the latency-reactivation cycle. Dear Dr. Jones:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. 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In order to study the influences of amino silane coupling agents with different grafting densities on the surface of nano silica on the thermomechanical properties of cross-linked epoxy resin, the molecular dynamics method was used to establish an amorphous model and calculate the mechanical properties, glass transition temperature, mean square displacement, hydrogen bond, binding energy, and radial distribution function of the composite models in this paper. The results are as follows: with the increase of the grafting density of an amino silane coupling agent on the surface of nano silica particles, the mechanical properties and glass transition temperature of epoxy resin showed a trend of increasing first and then decreasing. When the grafting ratio was 9%, the mechanical properties and glass transition temperature of the epoxy resin were the largest, and the glass transition temperature was increased by 41 K. At the same time, it was found that the higher the grafting ratio, the lower the chain movement ability, but the higher the binding energy. Besides, the binding energy between the nanoparticles of the grafted silane coupling agent and epoxy resin was negatively correlated with the temperature. By analyzing the hydrogen bond and radial distribution function, the results showed that the improvement of the grafted silane coupling agent on the surface of the nanoparticle to the thermomechanical properties of the epoxy resin was related to the OH···O and NH···O hydrogen bonds. The analysis results indicated that the proper grafting density should be selected based on the established model size, selected nanoparticle diameter, and epoxy resin materials in order to better improve the thermomechanical properties of the epoxy resin.  As the main materials of the basin insulator, epoxy resin is very important for the safe and stable operation of high-voltage and ultra-high voltage equipment ,2,3. How2 nanoparticles. Kung-Chin Chang [Nanoparticles have an excellent surface effect, small size effect, quantum size effect, macro quantum size effect, etc. . Therefoin Chang  et al. fin Chang  comparedThis paper studied the influences of N-(2-aminoethyl)-3-Aminopropyl trimethoxy silane coupling agents with different grafting densities on the surface of the nano silicon dioxide particles on the thermomechanical performance of the cross-linked epoxy resin and calculated thermomechanical parameters such as the dynamics performance, glass transition temperature, mean square displacement, hydrogen bond number, binding energy, and radial distribution function to select the optimal grafting density.3 box in the Forcite module was established, and then DGEBA and BD molecules by a 2:1 ratio were placed into the box. Next, geometric optimization and molecule dynamics running were performed in order to make the established model reach the balance conformation, and finally, the cross-linked reaction was completed by using Perl language. COMPASS was selected as the force field for calculations in all dynamic simulations; Nose and Berendsen were used for temperature control and pressure control, respectively [Bisphenol A epoxy resin (DGEBA) and 1,3 benzenediamine (BD) were selected respectively as the monomer and curing agent molecule of epoxy resin in this paper, and an epoxy resin composite model was established by Materials Studio (MS) software . The C aectively .2 to meet saturated status. The silane coupling agent will hydrolyze to form an Si–O–Si bond on the surface of the nanoparticles [2, and epoxy resin doped with four grafting densities on the surface of the nanoparticles, which are represented as pure, 0, 3%, 6%, 9%, and 12% were established, respectively .According to the literature , the N- is an important characteristic parameter of the thermal performance of polymers, which could be calculated by the specific volume method [Tg values of different models were calculated within 300–650 K by using a specific volume method in this paper, which can be seen in The glass transition temperature  shows the atomic chain movement, mechanical performance, and thermal stability of polymers. The bigger the MSD value, the worse the mechanical performance and thermal stability of the polymers.The MSD of pure, 0, 3%, 6%, 9%, and 12% model were calculated at 300 K in Tg. When the temperature is higher than the Tg, the temperature will intensify the chain movement of the epoxy resin and further make the thermomechanical performance of the epoxy resin reduce quickly.The hydrogen bond is defined as the special intramolecular or intermolecular interaction. Reference  mentioneIn In order to study the interface adhesion energy between the cross-linked epoxy resin and nano silicon dioxide particle grafted with a silane coupling agent, the interaction energies of 3%, 6%, 9%, and 12% models were calculated. The interaction energy between the cross-linked epoxy resin and the nanocomposite could be calculated as follows:totalE indicates all the potential energy of the cross-linked epoxy resin hybrid model, epoxyE indicates the potential energy of the epoxy resin, nanomaterialsE indicates the potential energy of the grafting silane coupling agent on the surface of nano silicon dioxide particles, Eint indicates the interaction energy between the epoxy resin and the nano silicon dioxide particles’ grafted silane coupling agent.The interaction energy between the cross-linked epoxy resin and nanocomposite models in 3%, 6%, 9%, and 12% models at different temperatures is shown in A and B are constant, respectively. The result shows that the high temperature will destruct the interaction energy between the epoxy resin and the nano silicon dioxide particles, which is similar to the change trend before the calculated mechanical performance and the hydrogen bond at a different temperature. Tg of the polymers. It further indicates that the calculated Tg before is reasonable.r for any other atom [i and j are the ith atom and jth atom, respectively, while ABN indicates the number of common atoms between groups A and B.The radial distribution function (RDF) means the possibility of finding another particle at the radius her atom ,32. It iThe RDF values of all the atoms in the 3%, 6%, 9% and 12% models at 300 K temperature are shown in By calculating the RDF of all the atoms of different grafting density models, it can be found that the RDF values of the four models are similar. Meanwhile, the RDF of the adjacent grafting densities model is slightly different, which may relate to the grafting number of the silane coupling agents between the adjacent grafting density models having a slight difference as well. In The RDF values of the C–H atom, N–H atom, and O–H atom for 4 different grafting density models are shown in The above results show that the thermomechanical performance is relatively better when the grafting density is 9%, which may be related to the hydrogen bond between atoms in the nano composite model. By analyzing the RDF values of different grafting density models, the results show that such a hydrogen bond is related to the hydrogen bond type of OH···O or NH···O.2 nanoparticles on the thermomechanical performance of the cross-linked epoxy resin were studied, and the mechanism was analyzed from the micro aspect by molecular dynamics. The conclusions are as follows:(1)2 nanoparticles grafted with silane coupling agents can effectively improve the thermomechanical performance of the epoxy resin. When the grafting density is 9%, it shows a better thermomechanical performance than the other models, and the glass transition temperature increases by 41 K. Duo to nanoparticles grafted with silane coupling agents improving nanoagglomeration, nanoparticles can fully react with the epoxy resin matrix to form a hydrogen bond grid, and such hydrogen bonds are mainly from OH···O and NH···O hydrogen bonds. However, when the grafting density of the silane coupling agents on the surface of the nanoparticles is too high, nanoparticles cannot effectively react with the epoxy resin, which will reduce the thermomechanical performance.The SiO(2)2 nanoparticles, the chain movement ability of epoxy resin decreased, the interaction between SiO2 nanoparticles and epoxy resin was enhanced, and the interaction energy is negatively correlated to the temperature. Based on the different sizes of the established models, different nanoparticle diameters and epoxy resin materials are selected, so a proper grafting density should be selected to better improve the thermomechanical performance of epoxy resin.With the increase of grafting density of SiOThe influences of different grafting densities of SiO


Cyp11b2(AS)Cre mouse line to generate mice with adrenocortical-specific Wnt/β-catenin activation, Trp53 deletion, or the combination of both. Mice with targeted Wnt/β-catenin activation or Trp53 deletion showed no changes associated with tumor formation. In contrast, alterations in both pathways led to ACC with pulmonary metastases. Similar to ACCs in humans, these tumors produced increased levels of corticosterone and aldosterone and showed a high proliferation index. Gene expression analysis revealed that mouse tumors exhibited downregulation of Star and Cyp11b1 and upregulation of Ezh2, similar to ACC patients with a poor prognosis. Altogether, these data show that altering both Wnt/β-catenin and p53/Rb signaling is sufficient to drive ACC in mouse. This autochthonous model of ACC represents a new tool to investigate the biology of ACC and to identify new treatment strategies.Adrenocortical carcinoma (ACC) is a rare and aggressive malignancy with limited therapeutic options. The lack of mouse models that recapitulate the genetics of ACC has hampered progress in the field. We analyzed The Cancer Genome Atlas (TCGA) dataset for ACC and found that patients harboring alterations in both p53/Rb and Wnt/β-catenin signaling pathways show a worse prognosis compared with patients that harbored alterations in only one. To model this, we utilized the  Adrenocortical carcinomas (ACCs) are rare tumors with a highly aggressive clinical phenotype and an annual incidence of 0.5–2 people per million worldwide , 2. ApprStandard treatment for advanced ACC consists of radiotherapy and chemotherapy, including the adrenolytic agent mitotane, which have had limited impact on overall patient survival , 4. The CTNNB1 (the gene encoding β-catenin) and biallelic deletion or loss of function mutation in ZNRF3 (a negative regulator of the Wnt/β-catenin pathway), which lead to constitutive activation of Wnt/β-catenin signaling, are among the most common somatic alterations in ACC [The discovery and successful translation of new therapies for ACC patients will require a more comprehensive understanding of the underlying genomic, transcriptional, and epigenetic programs driving adrenocortical carcinogenesis. Such knowledge will allow for the development of in vitro and in vivo model systems that more closely resemble ACC tumor biology. Recently, genomic analyses of ACC have highlighted several genes, which when mutated appear to function as drivers of sporadic adrenocortical tumorigenesis , 9–11. Gs in ACC .IGF2, a mitogen commonly altered in ACC [Despite the high frequency of mutations in Wnt/β-catenin pathway genes in human ACC, such mutations alone are insufficient to induce malignant transformation in mouse models, although they have been linked to the development of benign adrenocortical tumors –14. Based in ACC , 15. HowTP53, the gene that encodes human p53, have also been associated with ACC in both familial and sporadic forms [TP53 germline mutations [Trp53, the gene encoding murine p53, develop ACC [The p53 protein is a critical tumor suppressor and a potent transcription factor that plays a key role in maintaining genomic stability , 17. Mutic forms , 19. In utations . Consistutations . Despiteelop ACC , 23, argTP53 and CTNNB1 genes are among the most recurrent genetic alterations associated with disruption of these pathways in the ACCs [Recent genomic clustering analyses revealed that p53/Rb and Wnt/β-catenin are the leading pathways altered in the ACC molecular subgroup with the poorest outcome –11, suggthe ACCs , 10. Hern = 43). Analysis of these tumors identified alterations in the following genes linked to these pathways: CTNNB1, ZNFR3, MEN1, and APC (Wnt/β-catenin pathway) and TP53, CDKN2A, MDM2, CDK4, RB1, and CCNE1 (p53/Rb pathway). Specific mutations are described in Table To gain further insight into the genetic changes that arise in human ACC, we reanalyzed genomic data from the ACC TCGA dataset using cBioPortal , 24. PreAS) ASCre/+ mouse strain [Trp53flox/flox [-GOF) allele Ctnnbflox(ex3)/+ [-LOF (ASCre/+:: Trp53flox/flox), PCreAS/+; (2) βcat-GOF (ASCre/+:: Ctnnbflox(ex3)/+), BCreAS/+; and (3) p53-LOF/βcat-GOF (ASCre/+:: Trp53flox/flox:: Ctnnbflox(ex3)/+), BPCreAS/+. ASCre/+ mice served as controls.To investigate the effect of Wnt/β-catenin activation and p53/Rb dysfunction in the adrenal cortex, we crossed the adrenal-specific Aldosterone Synthase /+ . These cAS/+ mice revealed no gross morphological differences at 1 (n = 7) or 3 (n = 6) months of age, compared with controls, with a normal appearing zG and zona fasciculata (zF). Consistent with this, β-catenin IF revealed no difference in zG morphology and was comparable with controls  staining. In addition, we used immunofluorescent (IF) staining for β-catenin (a zG-specific marker)  to assesols Fig. . Analysit Cdkn1a   and 3 (n = 6) months of age, with a corresponding expansion of the β-catenin+ domain  and 3 (n = 7) months of age revealed an even greater expansion of the β-catenin+ domain compared with BCreAS/+, PCreAS/+, and controls, suggesting an enhanced hyperplastic response  at 3 months of age  over time revealed an exponential rate of adrenal growth for BPCreAS/+ mice (~200-fold increase at 12 months) compared with control mice , PCreAS/+ (n = 41), and BCreAS/+ (n = 39) mice  mice revealed the presence of adrenocortical tumors (ACA and ACC) in 38% (16/42) of all mice analyzed  were significantly heavier compared to those with ACA (n = 6) and to age-matched control adrenals (n = 6)  and ACA (n = 5) showed clear evidence of nuclear and cytoplasmic accumulation of β-catenin [Additional analysis of the tumors showed consistent similarities between the BPCre 6) Fig. . Also, i-catenin  and a hi-catenin   of 7–12-month-old mice with ACC showed metastases to the lungs . In agreement with previous results [STAR and CYP11B1 was associated with lower overall survival of ACC patients  leads to ACC in mice [A recent study showed that adrenocortical specific expression of SV40 large T-antigen (AdTAg mice) (driven by a 0.5 kb region of the  in mice . In addi in mice . In addi in mice .In summary, we have demonstrated that Wnt/β-catenin activation cooperates with loss of p53 to promote murine ACC tumorigenesis, which mimics the cardinal features of human ACC. This model will be useful for detailed analysis of tumorigenic mechanisms and clinical manifestations of ACC, contributing to the identification of biomarkers and new therapeutic targets as to preclinical testing of drugs for the treatment of ACC.TCGA data was analyzed using the cBioPortal for Cancer Genomics , 24.Experiments were performed with approval by the IACUC.−ΔΔCT method.Relative expression was assessed using the 2Adrenals were analyzed as previously described , 30, 35.Radioimmunoassays were performed as described previously .t-test or one-way ANOVA followed by Tukey’s analysis was performed using Graphpad Prism 8; data presented as mean ± SEM with significance P < 0.05.Two-tailed Student’s Supplemental MaterialsSupplemental Tables


Hydrocephalus induces mechanical and biochemical changes in neural cells of the brain. Astrogliosis, as the hallmark of cellular changes in white matter, is involved in demyelination process, re-myelination inhibitory effect, and inhibition of axonal elongation and regeneration. The pathophysiology of this process is not well understood. The purpose of the present study is to elucidate the effect of lipid peroxidation product on astrogliosis through WNT/ β-catenin in kaolin-induced hydrocephalic rats.The study used kaolin-induced hydrocephalic rats. Obstructive hydrocephalus was expected to develop within seven days after induction. The hydrocephalus animals were killed at day 7, 14 and 21 after induction. One group of the saline-injected animals was used for sham-treatment.We demonstrated that the hydrocephalic rats exhibited a high expression of 4-hydroxynonenal (4-HNE) in the periventricular area. The expression of β-catenin also increased, following the pattern of 4-HNE. Reactive astrocyte, expressed by positive glial fibrillary acidic protein (GFAP), was upregulated in an incremental fashion as well as the microglia.This work suggests that lipid peroxidation product, 4-HNE, activated the WNT/β-catenin pathway, leading to the development of reactive astrocyte and microglia activation in hydrocephalus. Cell membrane suffers from lipid peroxidation induced by oxidative stress when reactive species level exceeds the antioxidant enzyme level. Some studies revealed the accumulation of lipid peroxidation products, i.e. malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). Other cellular changes documented in many hydrocephalus studies were astrocyte reaction and microglial activation, together known as astrogliosis. Both these reactions are a normal response of the brain to injury in the acute phase. However, it is attributed to its detrimental effect on structural and functional recovery in a chronic setting in the form of periventricular hypertrophy and proliferation of astrocytes and microglia  . MicroglThe relation between lipid peroxidation and activation of WNT/β-catenin in hydrocephalus has not been studied. We hypothesised that lipid peroxidation is the subsequent reaction of oxidative stress-induced activation of WNT/β-catenin. This event led to the development of astrogliosis.n = 24) were used for the experiment. They were housed in standard cages and provided with a normal 12 h dark/light schedule with free access to food and water. The rats in the hydrocephalus group were sacrificed at 7th, 14th and 21st days and the rats in the sham-treated group were sacrificed on the 21st day after injection.Sprague-Dawley rats aged 10 weeks  staining, the paraffin sections were stained with mouse monoclonal antibody  for detecting the expressions of 4-HNE (1:300 dilution), Ki-67 (1:500 dilution), GFAP (1:500 dilution) and Iba-1 (1:500 dilution) according to the manufacturer’s instructions. Images from slides were viewed at 20× and 40× magnifications under Nikon H600L microscope, camera DS Fi2 and analysed using NIS Elements Basic Research imaging software . The results were scored using Immuno Reactive Score (IRS) as suggested by Remmele and Stegner .P-value of 0.05 or less. The test was performed using statistic software SPSS version 24 . Path analysis using Categorical Regression with optimal scaling was performed to determine the correlation coefficient between variables (beta coefficient).Data were presented as mean ± SD. Statistical analysis for comparison among groups was performed using ANOVA. Statistical significance was considered for a P < 0.001) on day 7 (8.97 ± 2.98) and day 21 (11.52 ± 0.43) than sham-treated group (3.61 ± 0.99) (P < 0.001) showed that hydrocephalus was significantly upregulated at 4-HNE level.Immunohistochemical appearance of 4-HNE showed that the lipid peroxidation process was involved in hydrocephalus . The hyd ± 0.99) . ImmunohP < 0.05). Its level followed the fluctuated pattern of 4-HNE. We found β-catenin score increased in day 7 (8.70 ± 0.88), slightly declining in day 14 (8.43 ± 0.42) and increasing further in day 21 (10.41 ± 0.42) (P < 0.001) showed a significant positive correlation between the 4-HNE levels and WNT/ β-catenin. The adjusted R2 revealed that 80% of the WNT/β-catenin activator was through lipid peroxidation products. The correlation of increased 4-HNE with WNT/β-catenin activation was measured in path analysis.We investigated the WNT/β-catenin canonical pathway activation by measuring the cytoplasm β-catenin level . The imm ± 0.42) . There wP < 0.001) than in the normal group (2.96 ± 0.28). In the hydrocephalus group, GFAP expressions increased from day 7 (7.47 ± 0.77) to day 21 (10.21 ± 0.10) (P < 0.001) between WNT/β-catenin and expression of reactive astrocyte.Reactive astrocyte was considered as the pathological hallmark in hydrocephalus. The hydrocephalus group showed a significantly higher GFAP expression ( ± 0.10) . The actP < 0.001) (P < 0.001) showed that hydrocephalus was significantly activated microglia.Increased expression of Iba-1 was found in several areas of the brain, especially at the periventricular area, white matter , and striatum after induction of hydrocephalus . The hyd< 0.001) . The coeHydrocephalus induces injury to the brain through mechanical and biochemical alterations. Hydrocephalic rat model exhibited a hunched-back appearance, less mobility, gait abnormality, and balance impairment. Their hind limbs moved in a short wide-based step. The progressive ventricular enlargement causes mechanical stress to the periventricular white matter and the cerebral blood vessels in that area , 11. TheThe involvement of 4-HNE in modulating downstream signaling has been reported . The assWNTs are secreted cysteine-rich glycoproteins ligands that bind to a complex of frizzled (Fz) receptors and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) and regulate target genes expression through an intracellular signaling pathway . WNT ligActivation of WNT/β-catenin plays a role in astrogliosis reaction in hydrocephalus. Our study showed a similar result that astrogliosis was correlated with the activation of WNT/β-catenin pathway. Currently, there are three theories regarding the origin of reactive astrocytes following brain injury. First, GFAP+ astrocytes come from itself that go through the dedifferentiation process and then divide. Second, it comes from oligodendrocyte progenitors that are found throughout the white matter. Third, it is originated from subventricular zone migrating progenitors. The activation mechanism of reactive astrocyte by WNT/β-catenin involving oligodendrocyte progenitor cell (OPC) is supported by evidence that WNT/β-catenin signaling occurred in GFAP+ cells and progenitor cells. High β-catenin tone inhibited oligodendrocyte progenitor maturation into immature oligodendrocyte. On the other hand, high WNT-3a increased GFAP+ cells –19. As tReactive astrocyte has dual appointments based on its appearance period. Its protective action is expected to appear at the early phase as it can eliminate excessive extracellular glutamate and store and supply energy to neighboring cells. Its protective effect will decline after seven days and the neurotoxic phenotype will replace it . The halMicroglia activation in hydrocephalus involved TLR-4 and RAGE as the receptor of Damage associated molecular patterns (DAMPS) and downstream signaling of the nuclear factor-κB (NF-κB) pathway . ActivatTaken together, the results of our study and the latest theories in hydrocephalic pathology revealed the involvement of hypoxia-induced cascade that eventually activates the microgliaastrocyte crosstalk .The hallmarks of cellular changes in hydrocephalus are development of reactive astrocyte and microglial activation. Hypoxia-induced lipid peroxidation activated WNT/β-catenin signaling pathway through its aldehyde products, 4-HNE, which in turn upregulated GFAP+ reactive astrocytes and microglial activation.


The wound healing that follows myocardial infarction is a complex process involving multiple mechanisms, such as inflammation, angiogenesis and fibrosis. In the last two decades, the involvement of WNT signaling has been extensively studied and effects on virtually all aspects of this wound healing have been reported. However, as often is the case in a newly emerging field, inconsistent and sometimes even contradictory findings have been reported. The aim of this systematic review is to provide a comprehensive overview of studies in which the effect of interventions in WNT signaling were investigated in in vivo models of cardiac injury. To this end, we used different search engines to perform a systematic search of the literature using the key words “WNT and myocardial and infarction”. We categorized the interventions according to their place in the WNT signaling pathway . The most consistent improvements of the wound healing response were observed in studies in which the acylation of WNT proteins was inhibited by administering porcupine inhibitors, by inhibiting of the downstream glycogen synthase kinase-3β (GSK3β) and by intervening in the β-catenin-mediated gene transcription. Interestingly, in several of these studies, evidence was presented for activation of cardiomyocyte proliferation around the infarct area. These findings indicate that inhibition of WNT signaling can play a valuable role in the repair of cardiac injury, thereby improving cardiac function and preventing the development of heart failure. Myocardial infarction (MI) is one of the most frequent cardiovascular events and a major cause of heart failure (HF) development. Obstruction of the blood flow in coronary arteries results in a lack of oxygen and nutrients in the affected regions of the heart, causing the loss of cardiomyocytes (CMs) . DespiteIn the infarcted heart, a wound healing response is initiated, resulting in the replacement of the injured CMs by scar tissue. Over the last decades, this wound healing response has been studied extensively. Following the death of CMs, an inflammatory response takes place first. This is followed by the formation of granulation tissue, rich in newly-formed blood vessels and extracellular matrix-producing cardiac fibroblasts (CFs). This granulation tissue eventually matures into scar tissue, which is characterized by large amounts of matrix with limited numbers of blood vessels and some myofibroblasts (MFs) . NumerouOne of the signaling pathways that has been extensively studied in the context of infarct healing is the WNT signaling pathway. After its initial discovery as a pathway involved in development and cancer, many other diseases and disease processes are now known to be regulated by WNT signaling . Our gro+ progenitor cells in the border zone of the infarct from 7 days post-MI onwards. Similar results were reported in other studies using axin-2 reporter mice [The activation of WNT signaling during cardiac remodeling has been confirmed in many studies. An elegant tool to investigate this is the use of WNT signaling reporter mice. Using an axin-2 promoter-driven LacZ expression model, Oerlemans et al.  were theter mice ,12, TOPGter mice ,14 and ater mice . In manyter mice ,17. It hThe purpose of this systematic review is to provide a comprehensive overview of the studies on interventions in the WNT signaling in infarct healing. Because of the fact that the interplay between the various cell types involved in infarct healing is highly complex, we have decided to focus only on studies in which a direct intervention in WNT signaling was investigated in an in vivo model of MI  or cryoinjury). We have analyzed the effects of the interventions on critical parameters of infarct healing, including infarct size, cardiac function, inflammation, angiogenesis, fibrosis, and regeneration, in order to identify targets in the WNT signaling pathway with the most consistent beneficial effects.WNT signaling was discovered more than 3 decades ago by simultaneous research on genes regulating pattern formation in Drosophila (wingless) and preferential integration sites for mouse mammary tumor virus (int1). A comparison of these studies’ results revealed that homologous genes were involved, resulting in the common name WNT for this gene family . Since tNineteen WNT proteins have been identified , which a2+ pathways [2+ signaling can regulate multiple processes in the body. It has to be noted, however, that only few studies have addressed the role of β-catenin-independent WNT signaling in infarct healing so far.Different signal transduction pathways can be activated by WNT proteins. The most extensively studied is the WNT/β-catenin pathway, where β-catenin serves as the second messenger. This pathway is unusual in the sense that activation of WNT/FZD signaling inhibits the degradation of the second messenger, rather than stimulating its production. Under resting conditions, β-catenin is constantly degraded by a so-called “destruction complex”, consisting of axin, Adenomatous Polyposis Coli (APC) protein, glycogen synthase kinase-3β (GSK3β) and Casein Kinase-1 (CK1). This complex phosphorylates β-catenin, targeting it for degradation by the ubiquitin system. Upon activation of WNT signaling, however, this complex is disrupted and β-catenin is no longer phosphorylated, leading to its intracellular accumulation and migration to the nucleus, where it can interact with transcription factors from the T-cell factor/Lymphoid enhancer-binding factor (TCF/LEF) family to activate the expression of its target genes . On the pathways . The PCPhttps://pubmed.ncbi.nlm.nih.gov/) using the search terms “WNT and myocardial and infarction”. The same combination of search terms was used in the EmBase and Medline search engines, assessed via Ovid at the Maastricht University Library. The final searches took place on 1 July 2020. After removing conference abstracts and reviews, the results were combined in a single database and duplicate publications were removed. Retraction notes, comments, and non-English publications were removed from the database. The results were analyzed according to the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA) [Publications on WNT signaling and infarct healing were identified through the PubMed search engine ((PRISMA) . A flow The abstracts from the publications in the database were assessed for eligibility by scanning for experimental data on interventions in WNT signaling in in vivo models of cardiac injury . When the abstract was insufficiently clear, the full publication was evaluated. Manuscripts containing only in vitro data or biomarker studies in humans, review articles and editorials were excluded. This assessment for eligibility yielded 32 primary research articles. These were supplemented with 6 research articles from our own records that were not identified in the database searches but met the inclusion criteria, yielding a total of 38 research articles in this systematic review.Full texts of the remaining manuscripts were analyzed for experimental data on the following topics: infarct size, cardiac function, CM apoptosis, inflammation, angiogenesis, fibrosis and CM proliferation. Moreover, we classified the interventions for each of the topics according to the level in the signal transduction cascade , as illustrated in Figure 2. The results of this analysis are shown in As already mentioned earlier, myocardial damage is a well-established factor determining the infarct size following an acute ischemic event. The extent of the infarct size is directly associated with mortality  and therIn several studies, the WNT signaling pathway was targeted at the level of WNT production or WNT proteins were directly administered, either via injection or by overexpression. Oikonomopoulos et al. studied the effects of recombinant WNT3a (r-WNT3a) administered via injections in the border zone of the infarct area, immediately after the induction of permanent MI by occlusion of the Left Anterior Descending (LAD) coronary artery. The authors demonstrated that r-WNT3a injections led to a substantial enhancement in infarct size, a finding suggesting that activation of the WNT signaling pathway can be noxious for the infarcted heart, already 1 week post-MI . On the Porcupine inhibition is an attractive strategy to prevent acylation of WNT proteins, thereby suppressing their biological activity . Moon etInterventions aiming to disrupt the interaction between ligand an receptor are a common strategy in the pharmacotherapy of cardiovascular diseases. Along these lines, antagonizing the interaction between the WNT ligand and its receptors would be a logical choice in an attempt to inhibit the WNT signaling pathway. This can be achieved by scavenging WNT proteins via a modulation of the levels of sFRPs or WNT Inhibitory Factor-1 (WIF-1), administering peptide fragments of WNT proteins or applying interventions to reduce the numbers of available receptors, as discussed below.Injection of mice with mouse recombinant FZD-1 protein leads to an immune response against the FZD-1 receptor, attenuating the induction of FZD-1 expression in infarcted mice. Nevertheless, this approach did not confer any effect on infarct size, even though CM hypertrophy was attenuated in immunized mice . Our groThere is a relative abundance of studies focusing on the roles of proteins from the sFRP-family following cardiac ischemia. Genetic enhancement of sFRP1 expression was shown to reduce infarct size following LAD ligation or cryoinjury, either acutely (2 days post-injury) or chronically (15 or 30 days post-injury) . A folloSeveral studies addressed the effect of interventions in the co-receptor LRP5/6 and its endogenous inhibitors from the DKK family. LRP5 KO mice were found to demonstrate an aggravation in infarct size expansion compared to WT counterparts, whether this is expressed as a % of total left ventricular (LV) mass (WT: 10% vs. LRP5 KO: 21%) or as a % of the area-at-risk (WT: 17% vs. LRP5 KO: 30%) . DKK2, aOur search identified seven studies where the effect of targeting the destruction complex on infarct size was investigated. Woulfe et al. utilized an inducible KO model where GSK3β was deleted specifically in CMs  and demoFocusing on non-GSK3β destruction complex targets has also been suggested as an alternative strategy to manipulate the metabolism of β-catenin. Pyrvinium, an agent that was found to prevent the degradation of axin, enhance CK1α and stabilize β-catenin  was used by Saraswati et al. following MI in mice. No effect was demonstrated on infarct size of a single intracardiac injection of pyrvinium compared to controls at 30 days post-MI . In agreThe available literature focusing on the role of the WNT/β-catenin signaling pathway in cardioprotection at the nuclear level  is strongly in favor of suppressing the signaling pathway, with the exception of a study by Hahn et al. and one by Zelarayán et al. In the first study, adenoviral gene transfer of β-catenin in a rat MI model attenuated infarct size by ~25% compared to the control rats. The authors proposed anti-apoptotic and pro-angiogenic mechanisms for this reduction in infarct size . ZelarayIn contrast, several studies  have been identified investigating the role of the inhibition of WNT/β-catenin signaling in infarct size magnitude. In this context, the same group  also useFollowing ischemic injury, the assessment of cardiac function (systolic and diastolic) is an essential step in monitoring the pathophysiologic changes taking place in the LV, the response to any therapeutic treatment and the overall clinical management of the subject. In the majority of cases in animal research, this is routinely performed by echocardiography . Other mActivation of the WNT signaling pathway via injections of r-WNT3a in the border zone had profound negative effects on cardiac function, as demonstrated by robustly reduced fractional shortening (FS) and fractional area change, when compared to vehicle-treated mice. Diastolic interventricular septum (IVS) thickness was also shown to be smaller in the r-WNT3a treatment group, but no difference was observed in the LV internal diameter in diastole (LVIDd) . In a stPorcupine inhibition by administration of WNT-974 for 10 weeks following permanent MI in mice led to a substantial increase in cardiac function  and a suppression of LV dilatation (as attested by a lower ESV in the WNT-974 treated mice) , indicatMacrophage-specific deficiency of Wntless results in substantially improved cardiac function (enhanced EF and FS) and preserved LV architecture (increased LVPWs thickness and suppressed LV end-systolic-area), 30 days post-MI .Fan et al. showed that an immunization strategy against the FZD-1 receptor can have a profound effect on FZD-1 mRNA and protein levels, and a drastic inhibition of active β-catenin levels. This was associated with effects on systolic  and hemodynamic function . Interestingly, the LVIDd was reduced in the immunized mice, while LV anterior wall thickness in diastole was attenuated, signifying cardioprotective effects . The admBarandon et al. showed that the genetic overexpression of sFRP1 can have a beneficial effect on cardiac function 2 weeks post-MI, as attested by improved dP/dt measurements  . In a foThe therapy with the WNT-FZD-LRP interaction inhibitor IGFBP-4/H95P  conferred very impressive effects on cardiac function when injected in mouse infarcts. Mice injected with IGFBP-4/H95P showed improved cardiac function (enhanced EF and FS) from 2 weeks up to 20 weeks post-MI .Knocking-out of LRP5/6 was not shown to improve cardiac function (as demonstrated by EF and FS) or architecture (LVIDd and LVIDs) in a mouse MI model, 2 weeks after infarction. In the same study, a single injection of the DKK1 protein shortly after MI did not result in any observable effects on cardiac function and architecture compared to control mice, 4 weeks after permanent MI . On the Various groups have studied the effects of manipulations at the level of the destruction complex by affecting GSK3β or CK1. Conditional genetic deletion of GSK3β in CMs, was shown to confer cardioprotective effects following permanent MI in mice. Indeed, it was shown that GSK3β KO mice exhibit improved diastolic and systolic function post-MI, with EF improving already at 4 weeks post-MI, preserved LVEDD being evident after 6 weeks and hemodynamic parameters improving at 8 weeks post-MI . InhibitThe CK1α enhancer and axin stabilizer pyrvinium failed to demonstrate a beneficial effect on cardiac function when administered as a one-off intracardiac injection following MI . The groIn our systematic research, only two studies were identified in which the role of the overexpression of β-catenin on cardiac function was investigated. Hahn et al. studied female Sprague-Dawley rats following adenoviral gene transfer of β-catenin (Ad-β-catenin) or control (Ad-GFP) and showed that the upregulation of β-catenin expression led to an improvement of systolic function (enhanced FS) and halting of LV dilatation (reduced LVEDD), 7 days post-MI . AdditioΤhe CM-specific genetic deletion of β-catenin improved systolic dysfunction  compared to control mice, but shows no effect on the halting of dilatation, 2 weeks post-MI . A similA characteristic feature of MI is the loss of cells, both CMs and non-CMs, due to the ischemia in the infarct area. Cells can die either in a non-regulated (necrosis) or regulated  fashion. Extensive research has shown that the cell loss due to permanent ischemia is mainly non-regulated, whereas regulated cell death typically occurs as a result of reperfusion. In the past decades, multiple mechanisms of regulated cell death have been identified . In the +/cTnI+ CMs in mice treated with GNF-6231 [+ CMs was unaltered by the administration of CXG-1321, although a trend towards a reduction could be observed [Two studies have been published in which the effect of administration of a porcupine inhibitor on CM apoptosis was investigated in vivo, using the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique. In both studies, cardiac ischemia was induced by permanent ligation of a coronary artery, which has been reported to induce cell necrosis rather than apoptosis . DespiteGNF-6231 . In contobserved . These oobserved .+ cells in the peri-infarct zone in the knockout and a decrease in the overexpressing mice, upon permanent LAD ligation [+ cells in the infarct area at 7 days after permanent LAD ligation; the apoptotic index was decreased by 50% [+ cells was reported in sFRP5-knockout mice, 24 h after I/R [+ cells in the infarct area, albeit in none of the studies the apoptotic cells’ identity was identified, e.g., by performing additional immunohistochemical characterization.The effect of interventions at the receptor level on CM apoptosis was investigated in six studies. Immunization of mice with recombinant FZD1 protein did not result in a change in relative myocardial apoptosis at 7 days post-MI . Using lligation . Overexpd by 50% . In contd by 50% . Finallyfter I/R . These sApoptosis of cells in/around the infarct area was assessed in two of the seven studies where interventions in the destruction complex were applied. CM-specific deletion of GSK3β was found to substantially reduce the number of apoptotic CMs in the remote myocardium at 8 weeks post-MI . On the + cells in AAV9-β-catenin injected to infarcted rat hearts at 7 days post-MI. Using conditional CM-specific depletion of β-catenin, Zelarayán et al. did not find evidence for an alteration in the rate of CM apoptosis at 4 weeks after permanent LAD ligation [The effect of interventions at the nuclear level on apoptosis were reported in three of the studies. Hahn et al. showed a ~30% reduction in TUNELligation . In contligation . HoweverWithin hours after MI, an acute inflammatory response is initiated, consisting of the infiltration of the infarct area with leukocytes. The first wave of inflammatory cells is formed by polymorphonuclear neutrophils (PMNs), followed by a wave of macrophage infiltration, peaking at ~7 days post-MI . This in+ (leukocyte common antigen) and CD68+ (macrophage antigen) inflammatory cells in the non-infarct region at 1 week post-MI in the AAV9-WNT11 treated group. This was accompanied by a marked reduction in the expression of multiple inflammatory cytokines [+ immune cells and CD11b+/Ly6G+ immune cells at 5 days after cryoinjury [Overexpression of WNT11 via AAV 9-mediated transduction in a mouse model of MI with GFP-labeled bone marrow cells resulted in a more than 2-fold reduction in bone marrow-derived cells in the border zone at 4 and 8 weeks post-MI, and even stronger reductions in the non-infarct area. Additional experiments showed reduced presence of CD45ytokines . CM-specyoinjury ; here ityoinjury .high) to resident (Ly6Clow) monocytes: in WIF-1 knockout mice, a significant increase in inflammatory monocytes was observed, whereas AAV9-mediated overexpression of WIF-1 attenuated the monocyte response at day 4 post-MI [Interventions at the level of WNT receptors and extracellular scavengers were also investigated in the context of inflammation. In the FrzA overexpressing mouse model, the number of myeloperoxidase-positive cells (mainly PMNs) was found to be significantly lower at day 2 and day 7 post-MI, indicating an attenuated first wave of inflammation. Macrophage and lymphocyte counts, on the other hand, were not different between Tg and control mice, suggesting a normal second wave of inflammation . Buildin post-MI -/- mice [-/- mice showed more infiltration of WNT5a-positive macrophages and higher levels of pro-inflammatory cytokines TNFα, IL-1β and MCP-1 compared to WT [In a study where the effects of overexpression and knockdown of DKK3 were compared in a mouse MI model, DKK3 was found to have an anti-inflammatory effect, 1 week post-MI, whereas an increased inflammatory response was observed in the DKK3-/- mice . A similed to WT .The effect of interventions at the level of the destruction complex on inflammation were addressed in a single study. Administration of the allosteric GSK3β inhibitor BIO shifted the macrophages in the infarct area from the M1 to the anti-inflammatory M2 phenotype at 14 days post-MI, suggesting that stimulation of WNT-β-catenin signaling at the level of the destruction complex has an anti-inflammatory effect .Effects on inflammation were not reported in any of the studies in which interventions at the nuclear level of WNT/β-catenin signaling were investigated.The formation of new blood vessels in the area of infarction is essential for successful repair, as the cells that are required in the repair process need an adequate supply of oxygen and nutrients. Early signs of neovascularization can be observed in the first days after MI and these newly-formed vessels are derived from pre-existing blood vessels in the border zone . This pr+ smooth muscle cells at 3 weeks after cryoinjury. Moreover, ~30% more ECs could be detected in the infarct by flow cytometry. The WNT10b overexpression activated β-catenin-mediated WNT signaling in the ECs, as demonstrated by immunohistochemistry [In two studies targeting the pathway at the level of the WNT ligand production and secretion, an increased neovascularization was observed. Mice overexpressing WNT10b in CMs showed a doubling of the vascular density in the infarct area, with larger diameter blood vessels and more α-smooth muscle actinhemistry . A 66% ihemistry . Althoughemistry .In several studies, the effects of interventions at the level of the WNT receptors and extracellular scavengers were addressed. In FrzA Tg mice, capillary density was found to be lower in the scar at 7 days post-MI when compared to WT mice, but higher at day 15 post-MI. Moreover, the percentage of muscularized arteries and the vessel surface in the infarct area were significantly increased in the Tg mice at the later time point . EC-specA threefold increase in blood vessel density was observed in a study from our group, where infarcted mouse hearts were treated with the WNT5a fragment UM206 . AlthougThe β-catenin destruction complex can be modulated by intervening at different components. So far, studies have been published in which the neovascularization was investigated after cardiac injury in the presence of inhibitors of GSK3β (NP12 and SB415286) or CK1 (pyrvinium). Activation of the WNT/β-catenin pathway by the GSK3β inhibitor NP12 resulted in an increased vascular density at 7 and 14 days post-MI. This was paralleled by increased migration of ECs and sprouting of aortic rings . SupportThe effect of direct intervention in nuclear WNT signaling was studied by adenovirus-mediated overexpression of a constitutively active β-catenin mutant in rat MI. At 7 days post-MI, capillary density was increased by ~65% in the Ad-β-catenin group, compared to mock-treated rats. This observation was supported by an increased VEGF-expression in the Ad-β-catenin rats at 7 and 14 days post-MI . In contFibrosis is one of the essential consequences of a MI and a hallmark of the development of HF. CFs and their activated forms, the MFs, are crucial cell types that control the fibrotic progress post-MI and their manipulation–along with strategies aiming to target fibrosis in the infarct and the remote areas–has been repeatedly associated with the WNT signaling pathway . TherefoSystemic overexpression of WNT11 via a rAAV9 vector exhibited an anti-fibrotic effect on mice 8 weeks post-MI. The WNT11-mediated effect led to an attenuation of fibrosis in whole-heart sections, in the remote as well as in the perivascular areas, compared to the AAV9-LacZ group (controls) . CM-spec+ cells (MF) in the peri-infarct area 3 days post-MI (a ~2.2-fold decrease), suggesting that this compound possesses anti-fibrotic properties [We identified three studies targeting Porcupine in order to manipulate fibrosis post-MI. WNT-974 was shown to suppress ECM deposition following MI, as determined by picrosirius red staining, exhibiting a modest–but significant–suppression of collagen-rich scar area in the WNT-974 treated animals . Treatmeoperties . Lastly,operties  did not Our group suggested the administration of peptide fragments of WNT5a as a novel anti-fibrotic strategy following permanent MI. Indeed, in our initial study we showed that the peptide UM206 could robustly attenuate the collagen deposition in the infarct area at 2 and 5 weeks post-MI. This finding was correlated with a strong increase in MF numbers in the infarct, suggesting that UM206 turns WNT/β-catenin signaling off, which modulates the behavior of MFs and thus fibrosis . InteresThe group of Barandon et al. showed a pro-fibrotic effect of sFRP1 following permanent MI. Indeed, 2 weeks post-MI the collagen density in the infarcted area of sFRP1-overexpressing hearts more than doubled compared to hearts of WT mice. Interestingly, the group also showed a striking effect on cardiac rupture, which is the deadly result of an imbalance between ECM deposition and breakdown: overexpression of sFRP1 markedly decreased cardiac rupture incidence, a phenomenon that was associated with effects on MMP-9 levels . This inThree studies were identified on the role of DKK in fibrosis following MI. Injection of DKK1 in mouse hearts led to increased Collagen III mRNA levels in the infarct at 4 weeks post-MI . Local iUsing the model of CM-specific deletion of GSK3β, fibrosis was reported not to be increased in the infarct area compared to WT littermates . On the The anti-fibrotic effects of the CK1 inhibitor pyrvinium were investigated in the study of Murakoshi et al. . The groOne interesting study has been identified, making an association between β-catenin overexpression and MF activation following MI. Although the authors do not report any in vivo data concerning fibrosis deposition or gene expression of pro- and anti-fibrotic genes, they show that following gene transfer, β-catenin is successfully transfected in MFs and leads to an enhanced expression of αSMA (thus increasing transdifferentiation of CFs to MFs), decreased apoptosis and activated cell cycle in activated MFs, all pointing towards a pro-fibrotic phenotype . In contIn the last decade, a growing number of studies has addressed the concept of activating a regeneration process in the injured heart to optimally restore its functionality . TraditiSeveral studies have addressed the concept of cardiac regeneration by interventions at the level of WNT. Injection of the WNT/β-catenin-activating WNT3a in the border zone of the infarct reduced the number of BrdU-positive CM in the border zone at 1 week post-MI. The authors attributed this effect to a limited renewal of the adult cardiac side population cells . In cont+/Ph3+ CMs was also observed in hearts of WNT-974 treated mice, despite of the fact that these hearts were not injured [+ cells in the border zone of the infarct. Because this transcription factor is typically expressed by early differentiation CMs, this suggests the induction of CM proliferation by this treatment, which was confirmed by in vitro studies [Inhibition of the essential WNT lipidation with the porcupine inhibitor CGX1321 increased the number of EdU-positive CMs in tissue sections of the infarct area. This result was confirmed in CMs, isolated from the infarcted hearts, demonstrating that significantly more new, mononucleated CMs were formed in the CGX1321-treated group at 4 weeks post-MI . A 3-fol injured . Treatme studies .+ cells in the infarct area, which was triggered by MI [Only a single report on the effects of extracellular WNT inhibition was found to discuss cardiac regeneration. Injections of sFRP2 in the infarct border zone enhanced and sustained the expression of CM-specific genes in resident c-Kited by MI .+/TnI+ cells almost 3-fold at 4 weeks post-MI [+ CMs in a zebrafish model of CM intoxication with aristolochic acid, treated with the GSK3β inhibitor BIO [+ cells in the peri-infarct and distal myocardium [In several studies, the effects of interventions in the β-catenin destruction complex were studied. CM-specific deletion of GSK3β was reported to increase the number of BrdU post-MI . This obitor BIO . These focardium . Interesocardium . It is uocardium .+ and Sca1+ cells in the subepicardial and subendocardial borders of the infarct area at 4 weeks post-MI, which was less prominent in the WT controls [Interventions at the level of nuclear WNT/β-catenin signaling have also been reported to confer effects on CM proliferation . Using acontrols . Similarcontrols . Using zcontrols . These sThe effect of interventions in the WNT signaling pathway on infarct healing has been subject of study for nearly two decades. It is quite common for such a new research area that the initial studies show variable–and sometimes even conflicting–results. This has motivated us to perform a systematic analysis of studies describing interventions in the pathway in in vivo models of cardiac injury, in order to identify common findings or trends. In total, we identified 32 studies via literature search and added 6 papers from our own archives fitting the inclusion criteria. We analyzed them for data on infarct size, cardiac function, apoptosis, fibrosis and angiogenesis. Moreover, we put specific emphasis on any reported signs of cardiac regeneration, as WNT signaling is increasingly associated with this interesting phenomenon.It is relevant to note that in 29 of these 38 studies, the intervention was aimed at inhibiting WNT signaling, whereas in 4 there was a direct stimulation of the pathway by increasing WNT protein of β-catenin expression. In the remaining studies, an inhibitor of the pathway (mainly GSK3β) was inactivated, leading to an indirect stimulation. The vast majority of the studies (>80%) showed beneficial effects on one or more of the parameters that we included in our analysis. Taken together, this demonstrates that inhibition of WNT signaling at different levels of the pathway is beneficial for infarct healing. The major deviation from this pattern was found in interventions in GSK3β that were reported to be beneficial, despite the fact that GSK3β inhibition activates the signaling pathway.A major problem that we encountered was that the WNT signal transduction pathway is highly complex, with multiple family members at different levels . This has complicated our systematic review, because in many cases only a single report on the targeting of an individual pathway member was found. Fortunately, there are exceptions in which drugs where used that have a more general mode of action. In three studies, inhibitors of porcupine were used which prevent the biological activation of virtually all WNT proteins and all of these studies showed beneficial effects on infarct healing. Along similar lines, in 5 studies the interaction of β-catenin with its transcription complex was targeted and 4 out of these studies showed beneficial effects on infarct healing. On the other hand, in 4 studies it was shown that inhibition of GSK3β with either a drug or a genetic intervention improved infarct healing. Because GSK3β targets β-catenin for degradation, GSK3β inhibition is generally considered to activate WNT signaling. However, it has to be noted that this kinase not exclusively serves in WNT signaling but is actually involved in a plethora of other signaling pathways, questioning the specificity of these interventions for WNT signaling. Along similar lines, WNT/β-catenin signaling does not operate in isolation in the nucleus but may crosstalk with other signaling pathways such as TGFβ and YAP/TAZ signaling . TherefoIn this systematic review, we tried to identify the mechanism(s) that were modulated by the interventions in order to improve the infarct healing. Therefore, we systematically analyzed the studies for results on apoptosis, angiogenesis, inflammation and fibrosis, which all have been described to be regulated by WNT signaling. This analysis turned out to be quite challenging: although in most studies effects on infarct size and cardiac function were reported, there was little uniformity in the evaluation of the underlying responses to injury. Therefore, we cannot draw firm conclusions regarding a single causal mechanism in infarct healing that can be augmented by inhibiting WNT signaling. In this context, it is interesting to mention that, particularly in the more recent publications, increased cardiac regeneration has been mentioned as a driving force behind the beneficial effects of the inhibition of WNT signaling on infarct healing. This fits nicely with the observations in zebrafish and newts that endogenous inhibition of WNT signaling is a prerequisite for the cardiac regeneration that occurs spontaneously in these species upon cardiac injury .a broad targeting of WNT signaling, preferably with a drug/therapeutic agent, appears to be more informative than an intervention aiming at a single member of the extensive familya comprehensive analysis of the wound healing response is recommended, including all the aspects of wound healing that we analyzed in this systematic reviewspecific focus should be on signs of cardiac regeneration and CM proliferation, because this would be of major interest for translation towards the clinic.Based on our systematic analysis of these 38 publications, we provide the following recommendations for future studies:


Staphylococcus aureus was the causative pathogen in 48% of cases in the left-sided group compared to 88% in the right-sided group. Unadjusted and adjusted long-term survival was better in right-sided IE compared to left-sided IE. Independent predictors of long-term mortality were increasing age, end-stage renal disease, nosocomial infection, brain emboli and left-sided IE. Left-sided IE was common in people with IDU but the proportion of females with left-sided IE was low. S. aureus was twice as common in right-sided IE compared to left-sided IE, and the long-term prognosis of right sided IDU-associated IE was better compared to left-sided IE despite the fact that few were operated.The aim of the study was to compare background characteristics, microbiology and outcome of patients with right-sided and left-sided intravenous drug use (IDU) associated infective endocarditis (IE). A nationwide retrospective study using the Swedish Registry on Infective Endocarditis between 2008 and 2019 was conducted. A total of 586 people with IDU-IE were identified and divided into left-sided (n = 204) and right-sided (n = 382) IE. Descriptive statistics, Cox-regression and Kaplan–Meier survival estimates were used. The mean age of patients in the left-sided group was 46 years compared to 35 years in the right-sided group, p < 0.001. Left-sided IE had a higher proportion of females.  Non-sterile injection techniques facilitate the entry of skin bacteria into the blood stream. In addition, particles other than the drugs themselves are introduced into the circulation and are thought to damage the endocardium, particularly at the right-sided heart valves4. People with IDU are a particularly challenging group when it comes to IE treatment due to high rates of continued drug use and recurrent IE6. The risk of death and need for valve replacement increases in cases of reinfection which further complicates the management of IE in people with IDU7.Intravenous drug use (IDU) is strongly associated with infective endocarditis (IE)2. Reinfection, drug abuse relapse, and socioeconomic challenges are thought to play a role in limiting the life span of these persons8. Although the majority of IDU-associated IE episodes affect the right side of the heart, a significant proportion of 20–30% occurs on the left side11.Despite the fact that people with IDU-associated IE are more commonly young and do not have chronic diseases associated with early mortality, the long-term outcome of IDU-associated IE has been reported to be very poor13. Understanding differences in background characteristics and microbiology may provide a better understanding of the difference in pathophysiology of right-sided and left-sided IDU-associated IE.There is limited information about the differences in background data and microbiological etiology between left-sided and right-sided and if there is a difference in outcome between left-sided and right-sided lesions. Chamber pressures differ between the right side and left side of the heart and calcifications occur predominantly on the left side. Indications for surgery are not the same for right-sided and left-sided IEThe main aim of this study was to describe patient characteristics and microbiology in right-sided vs. left-sided IE in people with IDU, to identify determinants of long-term survival in IDU-associated IE patients and to compare long-term survival in patients with right-sided vs. left-sided IDU-associated IE.14. Patients who did not have a Duke’s criteria classification in the database were classified within the present study based on the various clinical variables available in the database that constitute the Duke’s major and minor criteria. IDU-status was based on the physician’s assessment using either direct information from the patients or medical records. As seen in Fig. We conducted a retrospective cohort study with data from the Swedish Registry on Infective Endocarditis (SRIE). The registry includes almost 300 variables including patient background data and comorbidities, microbiology, echocardiography, treatment data, including medical treatment and surgery. The registry variables are entered by treating physicians who are usually infectious disease specialists. The study period was from 2008 to 2019. During this period a total of 5969 episodes of IE were entered into the registry. IE was defined as definitive IE based on the modified Duke’s criteriaThe study protocol was approved by the Swedish Ethical Review Authority (Registration number 2019-03549). As this was a retrospective study, individual consent was waivered. All methods were performed in accordance with the relevant guidelines and regulations.Follow-up on survival was done through the National Population Registry. Follow-up time was defined as the period between hospital admission and the current or latest status of the patient. Follow-up was completed on 8th of October 2019. The median follow-up time was 4.1 years (Interquartile range 1.9–6.8 years) and included a total of 2629 patient years.Categorical variables were presented as number and percentages. Continuous variables were normally presented as mean ± standard deviation if they were normally distributed while non-normally distributed variables were presented as median with interquartile range. Categorical variables were compared using the Chi-square test or the Fisher’s exact test if at least one of the cells had an expected count of less than five. Continuous normally distributed variables were compared with Student’s t-test while non-normally distributed variables were compared with Mann–Whitney U test. Predictors of long-term mortality were analyzed using Cox-regression. Univariable cox-regression was first calculated on variables that are known risk factors and those that were deemed important in this study. Variables that had a p-value < 0.10 were then included in the multivariate Cox-regression. Unadjusted survival was estimated using the Kaplan–Meier method Log-rank test was used to compare survival between groups. Adjusted survival was estimated with multivariable cox regression. A survival plot was performed using the same Cox-regression model as mentioned before except that IE location was a factor variable and not a covariate. The significance level was set at p < 0.05. All of the statistical analyses were completed using the statistical software program SPSS .Patient characteristics for left-sided IE and right-sided IE in IVDUs are listed in Table Staphylococcus aureus compromised 85% of the cases compared to left-sided IE where it compromised 46% of the cases (p < 0.001). Alpha-hemolytic streptococci and Enterococci were more common in left-sided IE than in right-sided IE, 14% and 3.7% vs. 24% and 3.1% respectively (p < 0.001). No difference was found for other bacteria which caused relatively few IE episodes.The causative pathogen for both left-sided and right-sided IDU-IE is summarized in Table The unadjusted survival rates after 1-year and 5-year follow-up were 85%  and 55% (95% CI 47% to 63%) for left-sided IE, respectively. The survival rates for right-sided IE in the same follow-up time were 97% (95% CI 95% to 99%) and 84% (95% CI 80% to 88%), respectively. The long-term survival was thus significantly worse in left-sided IE compared to right-sided IE . One explanation may be that left-sided infections more often is complicated by severe heart failure, systemic embolization and uncontrolled infection whereas invasiveness is uncommon in right-sided involvementThe current study includes patients reported to a nationwide registry. The patients are reported from all hospitals that have an infectious disease department, which decreases the risk of a biased patient selection that is usually seen in reports from tertiary care facilities. However, as reporting is not mandatory, the registry does not contain all IE episodes in Sweden. The retrospective nature of the study has inherent risks of biases. The IE registry does not contain more data on the nature of IDU such as what type of substances were used, and whether the patients continued IDU following surgery. There was no data regarding socioeconomic and demographic factors. People who inject drugs may also get endocarditis from non-IDU related causes such as blood stream contamination following dental surgery, this was not specified in the registry. Thus, our findings are applied to IDU-association.S. aureus occurred twice as much in right-sided IE compared to left-sided IE in IDU-associated IE. Survival in right-sided IE is relatively high and few patients in this group undergo surgery.This study adds important information on IDU-associated IE and the differences seen in right-sided and left-sided IE in this patient group. IDU-associated IE is not limited to the right side of the heart as more than a third of the patients had left-sided IE.