The sensitivity of a cell to ripoptocide is determined by the balance between regulatory molecules that enforce and those that disassemble the early checkpoint. As there is evidence suggesting that ripoptocide is inflammatory, individuals may develop inflammation due to ripoptocide brought about by genetic, epigenetic or post-translational alteration of these checkpoint regulators. For these individuals, drugs that reinforce the early checkpoint and inhibit ripoptocide could be useful in ameliorating inflammation.The clinical success of biologics that inhibit TNF (Tumor Necrosis Factor) in inflammatory bowel diseases (IBD), psoriasis and rheumatoid arthritis (RA) has clearly established a pathogenic role for this cytokine in these inflammatory disorders. TNF binding to its receptors activates NFκB and MAPK signaling, inducing the expression of downstream pro-inflammatory genes. This is thought to be the primary mechanism by which TNF elicits inflammation. TNF is also a well-known trigger of caspase-dependent apoptosis or caspase-independent necroptosis. Whether cell death has any role in TNF-mediated inflammation has been less clear. Emerging data from animal models now suggest that cellular demise caused by TNF may indeed provoke inflammation. The default response of most cells to TNF stimulation is survival, rather than death, due to the presence of two sequential cell death checkpoints. The early checkpoint is transcription-independent involving the non-degradative ubiquitination of RIPK1 to prevent RIPK1 from becoming a death-signaling molecule. The later checkpoint requires the induction of pro-survival genes by NFκB-mediated transcription. When the early checkpoint is disrupted, RIPK1 initiates cell death and we suggest the term  Tumor necrosis factor (TNF) was first described in 1975 as a serum factor that could lyse tumor cells present in bacillus Calmette-Guerin (BCG)-infected mice that were challenged with endotoxin . It was Another explanation for why TNF failed as an anti-tumor agent is that TNF is a poor inducer of tumor cell death when used as a single agent, contrary to its initial description as a cytotoxic factor. The initial experiments were carried out using Meth A and L929 mouse tumor lines , which aThe early observation that transcription or translation inhibitors sensitized cells to TNF-induced death pointed toward the presence of a transcription-dependent cell death checkpoint. In the mid-1990s, this checkpoint was attributed to NFκB-dependent transcription of pro-survival genes . One criripoptocide to describe cell death that is dependent on RIPK1, be it apoptosis or necroptosis. Another route to flip on the death switch on RIPK1 is to activate CYLD, a deubiquitinase that preferentially dismantles K63-linked ubiquitin chains, including from RIPK1. On the other hand, the late checkpoint can be disrupted by pharmacological or genetic inhibition of NFκB-dependent gene expression. This leads to a failure in replenishing c-FLIP, which normally acts as a brake on CASPASE 8. Without c-FLIP present, CASPASE 8 undergoes auto-processing and initiates apoptosis  also reversed the skin inflammation in the cpdm mice , which can activate innate immune cells to mount an inflammatory response . Since eficiency . SHARPINficiency . LUBAC ificiency . SHARPINficiency . In vivon in Tnf  indicatipdm mice . This obinocytes  or a hets . Prior in vitro studies had indicated that NEMO is an essential component of the early checkpoint  did not lead to colitis  or RBCK1 (coding for HOIL1) developed autoinflammation and immunodeficiency, which overlap with the phenotype observed in the SHARPIN-deficient mice. Females with a single copy defect in the X-linked IKBKG gene (coding for NEMO) develop Incontinentia Pigmenti (IP), which is characterized by skin inflammation during the early stages of life  also developed autoinflammation  versus those that disassemble the checkpoint  determine sensitivity to ripoptocide and subsequent inflammation . There iThe toggling of RIPK1 between its survival-signaling and death-signaling functions, regulated by an early ubiquitin switch, provides a molecular explanation for the long-known capability of TNF to induce either cell survival or death . An elabAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


SSc)–related Raynaud's phenomenon (RP) are badly needed. Laser speckle contrast imaging (LSCI) and thermography are noninvasive measures of perfusion that have shown excellent potential. This multicenter study was undertaken to determine the reliability and validity of a hand cold challenge protocol using LSCI, standard thermography, and low‐cost cell phone/mobile phone thermography (henceforth referred to as mobile thermography) in patients with SSc‐related RP.Reliable and objective outcome measures to facilitate clinical trials of novel treatments for systemic sclerosis  for reperfusion/rewarming and maximum blood flow rate/skin temperature after rewarming (MAX). Test–retest reliability was assessed using intraclass correlation coefficients (ICCs). Estimated latent correlations  were used to assess the convergent validity of LSCI and thermography.Patients with SSc) were recruited . LSCI and standard thermography both had substantial reliability, with ICCs for the reperfusion/rewarming AUC of 0.67  and 0.68 , respectively, and ICCs for the MAX of 0.64  and 0.72 , respectively. Very high latent correlations were present for the AUCs of LSCI and thermography  and for the AUCs of standard and mobile thermography .In total, 159 patients (77% with limited cutaneous LSCI and thermography in patients with SSc‐related RP. LSCI and thermography both demonstrated good potential as outcome measures. LSCI, standard thermography, and mobile thermography had very high convergent validity.This is the first multicenter study to examine the reliability and validity of cold challenge using  Systemic sclerosis (SSc)–related digital vasculopathy is painful and disabling, and has significant impact on quality of life. Raynaud's phenomenon (RP) occurs in most patients with SSc 96%) and is consistently the highest ranked symptom of SSc in terms of frequency and impact on daily function % and is http://www.cochranelibrary.com) as well as other reviews have highlighted the lack of evidence base for the treatment of both primary and SSc‐related RP Treatments are far from ideal, and Cochrane reviews . The study was approved by the Cambridgeshire and Hertfordshire National Research Ethics Service Committee , and all patients gave written consent to participate.The study aimed to recruit 180 patients with SSc  Cold challenge. Patients were requested to wear light clothing and refrain from vigorous exercise, caffeine, and alcohol for 4 hours prior to the assessment. Upon arrival, patients were seated comfortably for 20 minutes and acclimatized, and clinical research forms were completed. Immediately prior to the cold challenge, a baseline image of both hands  was obtained with LSCI and both thermal cameras. As required for LSCI imaging, all images were acquired in low‐lit rooms.The patient's hands were placed on a black, thermally insulated surface . Small sticky dots were used to mark the location of each finger at baseline. Both hands were placed in nitrile gloves and immersed to the metacarpophalangeal joints for 1 minute into cooled water  in 2 standard containers, one on either side of the patient. After the cold challenge, the gloves were removed and the hands were returned to their original position on the insulating surface, secured by double‐sided sticky tape to avoid movement between images.Reperfusion/rewarming after application of the cold challenge was imaged simultaneously by LSCI at 15 frames per minute, and thermography at 4 frames per minute, for 15 minutes . Mobile thermography did not allow for continuous video images to be obtained, and thus single images from which data could be extracted were acquired at set time points: baseline, 0 minutes after cold challenge, and 15 minutes after cold challenge. At the end of the 15 minutes, 1 extra image was obtained for LSCI and standard thermography, to allow the reperfusion/rewarming gradient from the last data point to be calculated; thus, a total of 225 images/scans were obtained for LSCI, 61 for thermography, and 3 for mobile thermography during the 15 minutes of measurement.Analysis of the images was performed using Moor Instruments Laser Perfusion Imager software (version 4.0) for LSCI, and Research IR Max  for standard and mobile thermography. Patients completed the RCS (possible score range 0–10) at each visit (a measure referred to as “RCS on the day”), measuring the severity and impact of their RP for that day The cold challenge was repeated 1 day later (on day 2), as close as possible to the same time of day in order to minimize variation due to circadian rhythms Image analysis for summary measures of response. Image analysis was carried out locally by an internal, nonblinded observer at each center. These were the same observers who had obtained the images. Regions of interest (ROIs)  L, AUCT, and AUCM representing the values based on LSCI, standard thermography, and mobile thermography, respectively) was calculated manually, not by automation  and the gradient of reperfusion/rewarming in the first 2 minutes post–cold challenge  were determined. Data were averaged for all fingers, as was done in previous studies  Figures B and C wn Figure  . Mobile thermography image analysis was carried out only at the central site.Assessment of feasibility. The feasibility of each technique was assessed at the end of the study based on the individual opinion of the clinical scientist or technician. Feasibility was measured as the ease of use and the ease of analysis , and preference of LSCI over thermography (standard or mobile thermography) for acquiring and analyzing images.Measurement of room temperature. A prerequisite of the cold challenge, and thus a criterion for center participation, was a temperature‐controlled room at each center. All measurements were obtained in a temperature‐controlled room  to assess the impact of temperature on measurements, with an interest in examining whether reliability could be improved by achieving greater temperature control.Identification of edge effects from LSCI. It became apparent when the study began that the blood flow appeared to be lower at the edges of the LSCI images than in the center. This implied that the distribution of the laser light across the hands was not equal, with less light incident toward the edges than at the center of the image. If true, then the consequence of this would be an artificially lower value for perfusion in the little fingers (edge of image)  Figure B compareStatistical analysis. Sample size calculation. Analyses of the data were performed using R version 3.2.3 Determination of test–retest reliability. To determine the test–retest reliability of each technique, intraclass correlation coefficients (ICCs) were obtained using linear mixed effects models. Each summary measure was included as a dependent variable, with center included as a fixed effect.Determination of between‐observer reliability. The data over both visits for each patient were averaged, and the resulting average values were compared between the central observer and the center‐specific observers by calculating the difference  in the paired mean values (details available from the corresponding author upon request). It is not possible to calculate a valid interobserver ICC from these data, since it would require at least some of the participants to have traveled to all sites for imaging and a large subset of images to be analyzed by all observers Determination of validity of the techniques. Convergent validity  was assessed using bivariate linear mixed models, which included fixed center terms and separate random patient intercepts for 1) LSCI and standard thermography, and 2) standard and mobile thermography. We estimated the latent correlation coefficients . For clarity, the statistical analysis protocol for this joint model is available from the corresponding author upon request.A post hoc analysis was conducted in which the responses to the RCS corresponding to the study day were assessed for correlation with all measurements, using linear mixed models.Feasibility data. Descriptive statistics were used to summarize the feasibility data.Mixed effects models accounting for room temperature. The mean room temperature at each patient visit was added to the mixed effects models for each summary measure. ICCs were recalculated, and these values were compared to the previously calculated estimates.Analysis of edge effects. Edge effects were investigated in a post hoc analysis by calculating the trend across fingers for LSCI measurements, and then comparing these measurements to those from thermography. Linear mixed models were used to assess any linear trends in the measurements from the index finger to the little finger. Fingers were numbered. Finger‐level summary measures of response were then regressed on finger number for both LSCI and thermography; this linear approximation was crude, but sufficient. Random intercept and slope terms were included to account for the fact that there was variation from patient to patient in these trends, not attributable to the imaging techniques. Measurements were standardized prior to analysis, thereby allowing for comparisons to be made between LSCI and thermography.Characteristics of the patients. In total, 159 patients were recruited . Among the patients, 157 (99%) fulfilled the American College of Rheumatology/European League Against Rheumatism 2013 classification criteria for SSc Test–retest reliability of the techniques. There was at least moderate to substantial reliability in the DDDL, DDDM, and DDDT, the AUCL, AUCT, and AUCM, and the MAXL and MAXT. The GRADL and GRADT had fair to substantial test–retest reliability , we considered that if the measures were perfectly reliable, the subplot for each center would look like 2 identical ladder plots . Our data suggest that there were systematic differences between the central observer and one of the centers  in extracting data from LSCI images. For thermography, agreement between the central and local observer was generally high for all centers, albeit with a large discrepancy in the data from several patients at one of their visits (results available from the corresponding author upon request).Validity of the techniques. The latent correlation between LSCI and standard thermography  was as follows: for the DDD, ρ = 0.65 ; for the reperfusion/rewarming AUC, ρ = 0.94 ; and for the MAX, ρ = 0.87 . In contrast, for the GRAD, the latent correlation between LSCI and standard thermography was only 0.52   for the AUC, and 0.90  for the DDD (Table T of −0.15 ± 0.07 for a 1‐point increase in the RCS), we found no evidence of correlation between the summary measures and the RCS.With the exception of some weak evidence of a decreasing DDD as measured on thermography with increasing RCS score (mean ± SEM change in the DDDFeasibility. Standard thermography was deemed to be more feasible than LSCI (see Discussion). The proportion of raters giving a score of ≥7 for ease of use was 50% for LSCI, 75% for standard thermography, and 38% for mobile thermography. Ease of analysis was rated as ≥7 by 25% of raters for LSCI and by 50% of raters for standard thermography. One center preferred LSCI to thermography for acquiring images, and 1 center preferred LSCI to thermography for analyzing images. Conversely, the number of centers preferring standard thermography over LSCI was 3 for acquiring images, and 4 for analyzing images. The remaining centers showed no preference.Models including room temperature. When included as a covariate, room temperature was not associated with any of the summary measures, as measured by either LSCI or thermography. Additionally, the ICCs were not affected by the inclusion of room temperature in the analysis. This does not mean that a regulated room temperature is not important, but that small changes in temperature are acceptable (see Supplementary Table http://onlinelibrary.wiley.com/doi/10.1002/art.40457/abstract).Edge effects. When moving from the thumb to the little finger on imaging, all of the trends in the AUC, MAX, and GRAD were in the opposite direction for the 2 modalities, with a decrease in these values when assessed by LSCI and an increase in these values when assessed by thermography (see Supplementary Table http://onlinelibrary.wiley.com/doi/10.1002/art.40457/abstract) Estimates of the DDD were positive with both techniques, but this was attenuated on images obtained by LSCI. This is consistent with the notion of an edge effect artificially producing lower values for the little fingers when LSCI is used for imaging. The cause of the edge effect was attributed to the distribution of light over the imaging area, due to LSCI being used at the upper limit of the suggested imaging distance in order to fit both hands into the imaging area. These data indicate that care must be taken to understand the variations over the field of view, so that these can be accounted for; decreasing the field of view would minimize this effect in future studies.T was slightly superior to the MAXL. Other than this, there were no substantive differences in reliability between the 2 techniques.To date, LSCI techniques and thermography have been insufficiently studied as outcome measures in clinical trials. Those studies in which they have been included show very little consistency in terms of protocol design M and DDDM showed adequate reliability for use as outcome measures. Moreover, there was strong correlation between mobile thermography and standard thermography data. The technique of mobile thermography was added at a late stage in this project (since it had only just come on the market). Our reason for including it was primarily for feasibility assessment. While it is clear that further work is required to validate mobile thermography, the performance in the present study is highly encouraging, because, as a low‐cost tool, it could potentially be readily available for widespread use among rheumatologists.The AUCAlthough it was not our primary objective, we examined differences between observers. Systematic differences between observers at different centers would not be particularly problematic for a multicenter randomized controlled trial (RCT), provided that the randomization would be stratified by center. We note that this should be the default for any multicenter trial, since differences between centers may otherwise bias the estimated treatment effect. This is particularly true in small populations, since simple randomization is less likely to produce balance within centers. Standardized training would reduce measurement variation across centers, and centralized extraction and analysis of LSCI data, conducted in a blinded manner, might also minimize variation by removing multiobserver differences in an RCT setting. Given the small sample size at each center, we are unable to determine whether truly systematic differences were observed. Ideally, a study to assess interobserver reliability would involve participants having images analyzed by all observers.Convergence between the techniques was shown to be very high for the AUC and MAX (particularly for the AUC). This finding provides evidence that the same underlying construct is being measured when using these summaries of response. Convergence appeared to be weaker  for the DDD. Convergence was weakest for the GRAD, which may be a reflection of the lag between reperfusion and rewarming, whereby tissue reperfusion (measured using LSCI) is translated into skin rewarming (measured using thermography), during the 2 minutes immediately following cold challenge.Since there is no gold standard to which we may compare either imaging technique, and we are comparing 2 techniques that measure perfusion by very different methods (skin temperature and a measure of red blood cell concentration and speed by light), it is possible to measure convergence between these techniques for validity The OMERACT review of 2003 Patient‐centered outcome measures are crucial for evaluating the effectiveness (rather than just the efficacy) of treatments. However, patient‐centered outcomes often comprise more “noise” compared to more objective measures of response, and therefore necessitate larger sample sizes to ensure adequate power in clinical trials. For small populations, there is therefore a tension between direct relevance to patients and feasibility of conducting a trial. One solution may be to power studies on the basis of objective measures, such as those considered herein, and to additionally (and consistently) report patient‐centered outcomes to facilitate an eventual meta‐analysis. Another solution might be to seek confirmatory evidence for the vasodilatory potential of candidate interventions, using objective measures, before proceeding to larger, phase III clinical trials.The relationship between 2 measures is limited by the reliability of each With regard to feasibility of the techniques, it has been noted that LSCI is sensitive to movement, vibrations, and lighting, indicating the importance of environmental conditions during the imaging. For mobile thermography, present limitations in feasibility include the battery life (LSCI is mains operated but standard thermography is powered by long‐life batteries), a fixed focusing distance, and lack of analysis for video images, as well as mounting difficulties; however, if the correlation between mobile and standard thermography can be replicated in future studies, these limitations may be acceptable in light of the lower cost and ambulatory (convenient) nature of the technique. When comparing the feasibility of LSCI to the feasibility of thermography, it should be noted that most centers were familiar with thermography but not with LSCI, and therefore this may have influenced the assessment of feasibility.One limitation of the study was that we did not recruit the planned number of participants, due to a seventh center not participating as planned. However, the study was designed to be robust to under‐recruitment. Although the 95% CIs for our estimates were wider than they would have been had the target been met, we were still able to demonstrate sufficient reliability and convergent validity of the AUC and MAX to observe differences that would indicate that the performance of DDD was weaker, and to show that the performance of GRAD was relatively poor.In conclusion, our design was relatively pragmatic, with the aim of establishing the performance of the different techniques as they would be employed in a multicenter clinical trial. Our study successfully established a working group of tertiary care centers for SSc, and together, the group developed a consensus calibration and cold challenge protocol. The summary measures of AUC and MAX both displayed good reliability and strong convergent validity. There was a possible advantage of thermography in relation to the reliability of MAX, although this was not definitive. We found evidence of edge effects when using LSCI, although our summary measures appeared to be quite robust to these in relation to reliability, perhaps suggesting that these effects were fairly consistent (details available upon request from the corresponding author).The results of this study also confirm that small variations in room temperature are acceptable during the imaging, and that, subject to further validation, mobile phone cameras may be a suitable, affordable, and highly portable alternative to more expensive standard imaging equipment . The mobile phone data obtained in this study will facilitate the design of future validation studies assessing mobile thermography–derived outcome measures. Although the design precluded formal assessment of interobserver reliability, there was a suggestion of systematic differences between the central observer and observers at some of the centers, highlighting the importance of image analysis training and potentially a role for centralized or automated image analysis. For multicenter RCTs, we would also recommend that, where possible or appropriate, randomization be stratified by center to balance any center‐specific effects and prevent bias.In summary, LSCI and thermography should now be incorporated as secondary outcome measures in upcoming treatment efficacy trials. This will allow an assessment of responsiveness to treatment as well as longitudinal validity. The present study leads us to recommend the summary measures of AUC and MAX, measured using either thermography or LSCI , as suitable outcome measures for RCTs in patients with SSc‐related RP.All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Murray had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Wilkinson, Leggett, Marjanovic, Moore, Allen, Anderson, Britton, Buch, Del Galdo, Denton, Dinsdale, Griffiths, Hall, Howell, MacDonald, McHugh, Manning, Pauling, Roberts, Shipley, Herrick, Murray.Wilkinson, Leggett, Marjanovic, Moore, Allen, Anderson, Britton, Buch, Del Galdo, Denton, Dinsdale, Griffiths, Hall, Howell, MacDonald, McHugh, Manning, Pauling, Roberts, Shipley, Herrick, Murray.Wilkinson, Moore, Allen, Anderson, Britton, Buch, Del Galdo, Denton, Dinsdale, Griffiths, Hall, Howell, MacDonald, McHugh, Manning, Pauling, Roberts, Shipley, Herrick, Murray. Click here for additional data file. Click here for additional data file.


We found low TNF-α could reduce the levels of ALT and AST in the plasma of TNF-α−/− rats and promote the proliferation of hepatocyte cells. However, the levels of ALT and AST increased gradually with increasing TNF-α concentration after reaching the lowest value. Moreover, we showed that TNF-α affects the cell proliferation and cell death of hepatocytes by regulating Yap activity. Low TNF-α promoted Yap1 nuclear translocation, triggering the proliferation of hepatocytes. However, high TNF-α triggered the phosphorylation and inactivation of Yap1, preventing its nuclear import and consequently promoting cell death. Collectively, our findings provide novel evidence that the concentration of TNF-α is an important factor affecting its function in liver injury, which may provide a reference for the clinical treatment of liver injury.Previous studies have shown that tumor necrosis factor (TNF)-α is a mediator of hepatotoxicity in liver injury. Moreover, TNF-α has also been reported to have a protective effect in liver regeneration, yet the function of TNF-α during liver injury remains controversial. Here, we report that the concentration of TNF-α determines its functions. High concentrations of TNF-α could aggravate LPS-induced liver injury. However, the TNF-α level was unchanged during APAP-induced liver injury, which exerted a protective effect. We expected that the concentration of TNF-α may affect its function. To test this hypothesis, TNF-α Many studies have focused on the role of TNF-α in the occurrence and development of liver injury. It has been also reported that TNF-α plays a significant role by inducing hepatocyte apoptosis, which mediates hepatotoxicity in lipopolysaccharide (LPS)- or concanavalin A-induced liver injury6. Moreover, TNF-α is important for liver regeneration and tissue repair following acetaminophen (APAP)-induced hepatotoxicity7. Grivennikov et al.8 even showed TNF-α could be either protective, as in host defense, or deleterious, as in autoimmunity or toxic shock. Thus TNF-α has dual function in liver injury, either aggravating or alleviating injury, which presents a challenge for designing treatments to prevent liver injury.Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine in disease pathogenesis such as liver injury, which governs development of the immune system, cell survival signaling pathways, proliferation, and regulates metabolic processes4, etc.) induce hepatic injury, which starts with hepatocytes9. A large number of intermediate metabolites trigger hepatocyte necrosis through a series of metabolic reactions, release intracellular contents, and subsequently induce inflammation10. Therefore, the concentration of TNF-α in the liver differs depending on the cause of liver injury, and whether this affects the function of TNF-α is still unclear.Studies have shown that liver injury induced by different causes, such as viruses, bacteria, etc., has relatively specific inflammatory microenvironment characteristics, which could induce rapid immune response, infiltration of inflammatory cells, and production of inflammatory factors in the liver, thus destroying the immune balance in the liver and inducing a series of liver pathological processes. However, some drugs or chemicals , but exerted a protective effect on APAP-induced liver injury. Further results showed that the concentration of TNF-α determined its protective or damaging effect on liver injury. Moreover, we showed that TNF-α functioned as an important factor in regulating the proliferation and cell death of hepatocytes via Yap1 activity.−/− rats died  rats died within 12 h after challenge with 10 mg/kg LPS, but none of the challenged TNF-αied Fig. . Higher ion Fig. . Histoloats Fig. . Furtherats Fig. . TNF-α iCl3 Fig. . As showoup Fig. . The levoup Fig. . These d−/− rats. The results showed that the survival rate of TNF-α−/− rats was lower than that of WT rats after APAP injection  could reduce the levels of ALT and AST in the plasma. However, the levels of ALT and AST elevated with increasing TNF-α concentrations  and TNFRSF1B (TNFR2). TNFR1ats Fig. , which wats Fig.  and an oats Fig. . Moreoveved Fig. , accompaved Fig. . Morpholats Fig. . Also, Tats Fig. . Immunofats Fig. .Fig. 3TNNext, we used a siRNA against TNFR1 to assess the role of TNFR1 in BRL cells treated with different concentrations of TNF-α. Western blotting was performed to analyze the expression level of TNFR1 protein. The results showed that less TNFR1 protein was detected in the siTNFR1-transfected BRL cells after transfection for 12 h Fig. . In the T183, p-LATS1/2T1079/1041 and p-YapS127 was decreased  was increased, and the expression of p-MST1/2sed Fig. . The expsed Fig. . The flused Fig. . These rsed Fig. . In addised Fig. , Immunofsed Fig. . In LPS sed Fig. . Taken tWe next sought to assess the role of Yap activity in cell fate determination during TNF-α treatment. We used gain- and loss-of-function experiments to evaluate the effect of Yap1. Western blotting assays showed that the expression of Yap1 protein was significantly decreased compared to that in the Yap1 control group Fig. . It was TNF-α is a double-edged sword for liver injury. Deletion of TNF-α or its receptor could alleviate LPS-induced liver injury but aggravate APAP-induced liver injury. In the present study, we showed that the concentration of TNF-α is critical for cell fate determination. High concentrations of TNF-α triggered hepatic cell death, but low concentrations of TNF-α could promote hepatic cell survival. We then discovered that the induction and activation of Yap is the key event in the determination of cell fate induced by TNF-α. These results provide new information for the therapeutic development of acute liver injury.23. We found that TNF-α was significantly increased in the livers of WT rats after LPS injection. Continuous LPS exposure lead to the activation of Kupffer cells, which resulted in the production of TNF-α. However, APAP-induced liver injury occurs in hepatocytes, which are the main metabolic cell of the human body24. APAP could be metabolized by hepatic cytochrome P450 to generate N-acetyl-p-benzoquinone imine (NAPQI). NAPQI is then detoxified by reacting with GSH. However, when hepatic GSH is exhausted, excessive NAPQI binds to cellular proteins covalently, which gives rise to mitochondrial dysfunction, oxidative stress, and ATP depletion26. The oxidative stress in turn leads to the nitration of mitochondrial proteins, the DNA damage of mitochondrial, ultimately resulting in the mitochondrial permeability transition and cell death28, which even lead to death of the rat. We found TNF-α expression did not increase in the early stage of APAP-induced liver injury. Therefore, the characteristics of TNF-α secretion were different between the two models. Our past research found that low doses of etanercept (a widely used inhibitor of TNF-α) alleviated CCl4-induced liver injury, but high doses of etanercept aggravated CCl4-induced liver injury29, which indicated that the function of TNF-α is concentration-dependent. We further elucidated the effect of TNF-α concentration on its functions. We injected different concentrations of TNF-α into TNF-α−/− rats. The results showed that a low concentration of TNF-α could reduce the plasma AST and ALT contents in TNF-α−/− rats and could promote the proliferation of BRL cells. However, a high concentration of TNF-α could increase plasma ALT and AST levels in TNF-α−/− rats and even result in death30. In addition, the cell death rate of BRL cells was increased at a high concentration of TNF-α. LPS was found to stimulate the secretion of TNF-α in a dose-dependent manner in rats  pretreatment could alleviate APAP-induced liver injury. However, LPS pretreatment with too low or too high dose could not alleviate APAP-induced liver injury  NF-κB activation, which translocates to the nucleus and activates the transcription of genes that regulate cell survival and proliferation. In addition, TNFR1 is classified as a death receptor, and classical apoptosis was shown to occur upon TNF binding to TNFR1. Therefore, TNF-α/TNFR1 signal transduction is a constant balancing act between these opposing functions42. We confirmed that the protective or damaging effects of TNF-α were mediated via TNFR1 in liver injury caused by different factors. The molecular mechanisms underlying the roles of the TNF-α/TNFR1 pathway in cell fate determination may contribute to developing pharmacological therapies for liver injury.TNFR1 is expressed on nearly all cells43. It has been shown that Yap translocates to the nucleus and is activated in response to TNF-α in endothelial cells44. Gao et al.45 confirmed that TNF-α triggers IKK-mediated Yap activation in breast cancer cells. Other studies have suggested that TNF-α could phosphorylate Yap by inducing LATS2 expression in oral squamous cell carcinoma46. TNF receptor-associated factor 6 (TRAF6), which is a member of the TNF receptor-associated factor family downstream of the TNF receptor, could regulate Yap signaling by promoting the ubiquitination and degradation of MST1 in pancreatic cancer47. These findings suggest that TNF-α could affect Yap activity. Here, we determined that a low concentration of TNF-α induced Yap1 expression, while knockdown of Yap1 in BRL cells significantly inhibited cell proliferation and enhanced cell death. However, a high concentration of TNF-α induced the phosphorylation and inactivation of Yap1, preventing its nuclear import. Overexpression of Yap1 by a virus vector significantly decreased cell death of BRL cells. Taken together, these results indicate that Yap plays an important role in directing cell fate  rats, TNFRSF1A knockout (TNFR1−/−) rats, and TNFRSF1B knockout (TNFR2−/−) rats were all established by Nanjing Xunqi Biotechnology Co. Ltd. by CRISPR/Cas9-based genome editing48. Exon 1 of the TNF-α gene was targeted to induce sequence deletions with frame-shifts, which were identified by PCR genotyping and sequence analysis. Breeding of TNF-α+/− rats was done to generate TNF-α−/− rats for experiments. DNA sequencing shows a 5-bp deletion in exon1 of TNF-α gene in the TNF-α−/− rat , and the plasma was collected at 12 h after the injection. GdCl3⋅6H2O  given at 24 h before LPS administration was used to inhibit Kupffer cells. In some experiments, rats were treated with different doses of LPS  or saline intraperitoneally 24 h before performing the APAP-induced liver injury model. The rats were randomly assigned to interventions.Male Sprague–Dawley rats  were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences . TNF-α knockout .Alanine aminotranferease (ALT) and aspartate aminotransferase (AST) are enzymes located in liver cells, their levels have been regarded as markers of liver injuryLiver tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections (4 μm) were stained with hematoxylin and eosin (H&E) following standard protocols and subsequently analyzed under a light microscope.Apoptotic cells were detected using an in situ cell detection kit  according to the manufacturer’s instructions. The sections were visualized using standard fluorescence microscopic techniques.The levels of hepatic glutathione (GSH) and the lipid peroxidation product malondialdehyde (MDA) were detected by using commercial kits according to the manufacturer’s protocols .TNF-α in the liver was measured using Rat Inflammation Array Q1  according to the manufacturer’s instructions. The levels of TNF-α in plasma were determined using an ELISA Kit  according to the manufacturer’s instructions.2. BRL cells were planted in 96-well plates or 6-well plates and treated with various concentrations of TNF-α  in DMEM without serum for 6 h.Buffalo rat liver cells (BRL cells) were purchased from Cell Bank at the Chinese Academy of Sciences . Cells were cultured in DMEM containing 10% FBS in a humidified incubator at 37 °C and 5% CO5 cells/well in antibiotic-free normal growth medium supplemented with FBS. BRL cells were transfected with siRNA-TNFR1 or adenovirus plasmid using FuGENE HD according to the manufacturer’s protocol. Western blotting was performed to detect protein levels 12 h post transfection.Small interfering RNA against TNFR1 was synthesized by Biomics Biotechnologies Co. Ltd. . The recombinant adenoviruses carrying short hairpin RNA (shRNA) plasmids against Yap1 and the pAdeno vector containing the wild-type Yap1 insert were provided by OBiO Technology Corp . BRL cells were seeded in 6-well plates at a density of 2 × 10Cell viability was tested 6 h after treatment with different concentrations of TNF-α by a Cell Counting Kit-8 , and the absorbance at 450 nm was measured after 1 h of incubation at 37 °C.Cell death analysis was performed 6 h after treatment with different concentrations of TNF-α using the FITC Annexin V Apoptosis Detection Kit  according to the manufacturer’s protocols. All samples were analyzed on a FACSCalibur flow cytometer .T1079/1041 , anti-LATS1 , anti-p-MST1/2 , anti-MST1 , anti-Yap1 , anti-p-YapS127  antibodies, and developed with the BeyoECL Plus substrate system (Beyotime). The blots were stripped and reprobed with GAPDH antibody  to confirm equal protein loading.Cytoplasmic and nuclear fraction separation was performed using NE-PER nuclear and cytoplasmic extraction reagents . Total cellular proteins were collected using RIPA lysis buffer . Equal amounts of protein were electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A wet transfer method was used to electrophoretically transfer proteins from the native gel to nitrocellulose membranes, which were then probed with anti-p-LATS1/231.Paraffin-embedded liver tissues were cut into 4 μm serial sections. Immunohistochemistry (IHC) was performed using anti-CD68 . The detailed method has been published previouslyCells were cultured on glass coverslips, fixed with 4% paraformaldehyde, permeabilized in PBS containing 0.4% Triton X-100, and blocked with 1% bovine serum albumin (BSA). Cells were then incubated with anti-Yap1  overnight at 4 °C. The glass coverslips were washed with PBS and incubated with a fluorescent secondary antibody for 1 h at 37 °C. Then, the glass coverslips were washed with PBS and stained with DAPI. Images of the sections were obtained using Image-Pro Plus 4.5 software .p-value < 0.05 was considered statistically significant. Quantitative data are presented as the mean ± SD.Survival curves were calculated by the Kaplan-Meier method, and differences in survival rates were compared by using the log-rank test. Statistical differences were assessed using one-way analysis of variance followed by Bonferroni post-hoc test. A Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure Lgends


The combined capacity of Jute Fibers (JF), the reinforcement, and Ground-Granulated Blast-Furnace Slag (GBFS), the binder, was examined as a sustainable solution towards ameliorating the inferior engineering properties of micaceous clays. A total of sixteen JF + GBFS mix designs, i.e., JF  = {0, 0.5, 1.0, 1.5} and GBFS  = {0, 3, 6, 9}, were tested for unconfined compression (UC) strength; for those mix designs containing GBFS, curing was allowed for 7 and 28 days prior to testing. Scanning electron microscopy (SEM) studies were also carried out to observe the evolution of fabric in response to JF, GBFS and JF + GBFS amendments. The greater the JF content the higher the developed strength and stiffness up to 1% JF, beyond of which the effect of JF-reinforcement led to some adverse results. The JF inclusions, however, consistently improved the ductility and toughness of the composite. The addition of GBFS to the JF-reinforced samples improved the soil–fiber connection interface, and thus led to further improvements in the composite’s strength, stiffness and toughness. The mix design “1% JF + 9% GBFS” managed to satisfy ASTM’s strength criterion and hence was deemed as the optimum choice in this investigation. Finally, a non-linear, multivariable regression model was developed and validated to quantify the peak UC strength as a function of the composite’s index properties. The proposed model contained a limited number of fitting parameters, all of which can be calibrated by little experimental effort, and thus implemented for preliminary design assessments. Soils are the most common and readily accessible of all materials encountered in construction operations. Most soils, however, are characterized as problematic, as their intrinsic mechanical features, e.g., strength and bearing capacity, are often less than ideal for common civil engineering applications ,2. MeanwCommon solutions to counteract the adversities associated with problematic soils, and most likely micaceous clays, include soil replacement or attempting to amend the low-graded soil by means of stabilization . The forA sustainable soil stabilization scheme can be characterized as one that maintains a perfect balance between infrastructure performance and the social, economic and ecological processes required to maintain human equity, diversity, and the functionality of natural systems. Traditional stabilization agents including cementitious binders and synthetic reinforcements, although proven effective, are not financially competitive in terms of materials procurement, labor and equipment usage. Furthermore, these solutions often suffer from serious environmental drawbacks attributed to their significant energy and carbon emissions footprints ,60. As sThe present study examines the combined capacity of Jute Fibers (JF), the reinforcement, and Ground-Granulated Blast-Furnace Slag (GBFS), the binder, as a sustainable solution towards ameliorating the inferior engineering characteristics of micaceous clays. A series of unconfined compression (UC) tests were carried out on various mix designs to evaluate the effects of JF-reinforcement and/or GBFS-treatment on the strength, ductility, stiffness and toughness of the micaceous clay. Scanning electron microscopy (SEM) studies were also carried out to observe the evolution of soil fabric in response to JF, GBFS and JF + GBFS amendments. Finally, a non-linear, multivariable regression model was developed and validated to quantify the peak UC strength as a function of the composite’s index properties. A sensitivity analysis was also carried out to quantify the relative impacts of the independent regression variables, namely JF content, GBFS content and curing time, on the composite’s strength.clay with intermediate plasticity (CI) and silt with intermediate plasticity (MI) in accordance with the Unified Soil Classification System (USCS). The standard Proctor compaction test (ASTM D698–12) indicated optimum water contents of wopt = 19.84% and 23.52%, along with maximum dry densities of ρdmax = 1.63 g/cm3 and 1.56 g/cm3, for K and MC, respectively. Such trends can be attributed to the spongy nature  and high water demand of the mica mineral [Commercially-available Kaolin (K) and Ground Mica (GM), sourced from local distributors, were used to artificially prepare a desired Micaceous Clay (MC) blend for further experimental work. The choice of GM content for the MC blend was selected as 20% (by dry mass of K), as it represents an upper boundary prerequisite to simulate adverse mechanical attributes commonly exhibited by natural micaceous clays, i.e., compactability issues and low shear strength/bearing capacity ,11,16. T mineral .2) and aluminum trioxide (Al2O3) with mass fractions of 64.9% and 22.2% for K, and 49.5% and 29.2% for GM, respectively. The pH for slurries of K and GM was, respectively, found to be 7.4 and 7.8, from which both materials were classified as neutral substances. Other material properties included a specific surface area of SSA = 11.2 m2/g and 5.3 m2/g for K and GM, respectively.The chemical compositions of K and GM, as supplied by the manufacturers, are outlined in Corchorus capsularis , was used as the reinforcing agent. Its biochemical composition, as commonly reported in the literature, consists of 56–71% cellulose, 29–35% hemicellulose and 11–14% lignin [FD = 30–40 μm; they were cut into segments of approximately FL = 15 mm, thus resulting in an aspect ratio of FAR = FL/FD = 375–500 , manufactured from % lignin . The raw–500 see a,b. The –500 see . The phy2/g; the latter is approximately two-fold greater than that of ordinary Portland cement [2) with mass fractions of 44.7% and 27.1%, respectively. The former, the calcium oxide, acts as a precursor agent, initiating a series of short- and long-term chemical reactions in the soil–water medium, i.e., cation exchange, flocculation–agglomeration and pozzolanic reactions, thereby amending the soil fabric into a unitary mass of enhanced mechanical performance (see A large quantity of Ground-Granulated Blast-Furnace Slag (GBFS) was sourced from a local manufacturer in South Australia, and was used as the cementitious binder. The physical properties and chemical composition of GBFS, as supplied by the manufacturer, are outlined in d cement . The cheance see .xF = x% JF; yS = y% GBFS; and zT = z days of curing.In this study, a total of sixteen mix designs consisting of one control , three JF-reinforced, three GBFS-treated and nine JF + GBFS blends were examined see . HereaftFc = JF content; Sc = GBFS content; wc = water content; mJF = mass of JF; mGBFS = mass of GBFS; mMC = mass of micaceous clay ; and mW = mass of water.The JF, GBFS and water contents were, respectively, defined as:wc = 23.52%, the standard Proctor optimum water content of the natural soil (ASTM D698–12), was added to each blend and thoroughly mixed by hand for approximately 15 min. Extensive care was taken to pulverize the clumped particles, targeting homogeneity of the mixtures. A special split mold, similar to that described in the literature, was designed and fabricated from stainless steel to accomplish static compaction [ρd = 1.56 g/cm3 . The surface of the first to fourth compacted layers was scarified to ensure adequate bonding between adjacent layers of the mixture. Samples containing GBFS were enclosed in multiple layers of cling wrap and transferred to a humidity chamber, maintained at 70% relative humidity and a temperature of 25 ± 2 °C, where curing was allowed for 7 and 28 days prior to testing.The natural soil, JF and GBFS were blended in dry form as per the selected mix designs outlined in mpaction ,43,49. TF0S0T0 , F1.0S0T0, F0S6T0 and F1.0S6T0 were examined, and the results are provided in To ensure uniformity of fabric and hence consistency in behavior, the variations of dry density and water content should be measured along the height of the compacted samples . In thisF0S0T0 , F1.0S0T0, F0S6T28 and F1.0S6T28. In this regard, the standard deviation (SD) and the coefficient of variation (CV) for the triplicate peak UC strength data were found to range between SD = 3.74 kPa and 11.19 kPa, and CV = 3.23% and 5.15%; these low values corroborate the repeatability of the adopted sample preparation technique, as well as the implemented UC testing procedure.Unconfined compression (UC) tests were carried out in accordance with ASTM D2166–16. The prepared samples see  were axiF0S0T0 , F1.0S0T0, F0S6T28 and F1.0S6T28 were examined. The desired samples, prepared as per 3 in volume, and were further subjected to SEM imaging at various magnification ratios ranging from 250× to 20,000×.The scanning electron microscopy (SEM) technique was implemented to investigate the evolution of fabric in response to JF, GBFS and JF + GBFS amendments. SEM imaging was carried out by means of the Philips XL20  scanning electron microscope. Apparatus specifications included a resolution of 4 μm and a maximum magnification ratio of 50,000×. In this regard, typical mix designs consisting of xSyTzF where x = {0, 0.5, 1.0, 1.5}, y = {0}, and z = {0}—are provided in Stress–strain curves for the natural soil and various JF-reinforced samples—Fc = 1%, beyond of which JF-reinforcement was found to adversely influence strength development in the composite. The natural soil exhibited a peak UC strength of qu = 82.15 kPa, while the samples reinforced with Fc = 0.5% and 1% resulted in higher values of qu = 119.35 kPa and 138.21 kPa, respectively. The higher JF inclusion of 1.5% changed the peak UC strength to 132.24 kPa, which still holds a notable advantage over the natural soil, as well as the sample reinforced with 0.5% JF. The axial strain at failure, denoted as εu, is an indication of the material’s ductility; higher εu values manifest a more ductile (or a less brittle) character. Improvement in ductility is often quantified by means of the deformability index ID [εuS = axial strain at failure for the stabilized soil sample; and εuN = axial strain at failure for the control  sample.As demonstrated in index ID :(5)ID=εuεuN = 4.73%). As a result of JF-reinforcement, the deformability index exhibited a monotonically-increasing trend, and resulted in ID = 1.24, 1.39 and 1.81  for Fc = 0.5%, 1% and 1.5%, respectively.The deformability index exhibited a monotonically-increasing trend with JF content, thus indicating that the greater the JF content the more ductile the sample’s response to compression. By definition, the natural soil corresponds to a deformability index of unity  and various GBFS-treated samples—xSyTzF where x = {0}, y = {3, 9}, and z = {7, 28}—are provided in F0S0T0) and various JF-reinforced samples treated with 6% GBFS—xSyTzF where x = {0, 0.5, 1.0, 1.5}, y = {6}, and z = {7}—are provided in Typical stress–strain curves for the natural soil  as a criterion for characterizing an effective stabilization scheme [F1.0S9T28 promotes a 361.06 kPa improvement in the peak UC strength and hence satisfies the aforementioned criterion.Tc = 7 and 28 days, respectively. Similar to the natural soil reinforced with JF, for any given GBFS content and curing time, the greater the JF content the higher the deformability index, following a monotonically-increasing trend. For any given JF content, however, the greater the GBFS content and/or the longer the curing period, the lower the developed ductility. The deformability index for various JF + GBFS blends was cross-checked with that of the natural soil (or ID = 1) to arrive at the optimum cases. In this regard, nine cases (out of 28) manage to satisfy the ID ≥ 1 criterion, and thus are deemed as optimum with respect to ductility improvement. The nine optimum cases and their corresponding ID values include F0.5S3T7 (ID = 1.10), F1.0S3T7 (ID = 1.34), F1.5S3T7 (ID = 1.68), F1.0S3T28 (ID = 1.09), F1.5S3T28 (ID = 1.34), F1.0S6T7 (ID = 1.16), F1.5S6T7 (ID = 1.32), F1.5S6T28 (ID = 1.10), and F1.5S9T7 (ID = 1.08).The deformability index, a measure of the material’s ductility, was also calculated for various JF + GBFS mix designs, and the results are provided in E50 against JF content for the natural soil and various GBFS-treated samples tested at 7 and 28 days of curing, respectively. The variations of E50 exhibited a trend similar to that observed for the peak UC strength given in F1.0S0T0, F1.0S3T7, F1.0S3T28, F1.0S9T7 and F1.0S9T28 resulted in E50 = 3.70 MPa, 5.39 MPa, 7.81 MPa, 12.30 MPa and 18.92 MPa, respectively. Moreover, for any given GBFS content and curing time, stiffness enhancements were only notable for samples with up to 1% JF inclusions. In this regard, the samples F0S6T28, F0.5S6T28, F1.0S6T28 and F1.5S6T28, for instance, resulted in E50 = 8.25 MPa, 9.47 MPa, 11.21 MPa and 10.23 MPa, respectively.F0S6T28, F0.5S6T28, F1.0S6T28 and F1.5S6T28 resulted in peak strain energies of Eu = 3.99 kJ/m3, 6.30 kJ/m3, 9.71 kJ/m3 and 10.70 kJ/m3, respectively. Similarly, for any given JF content, the greater the GBFS content and/or the longer the curing period, the higher the developed toughness. As typical cases, the sample F1.0S0T0 resulted in Eu = 6.11 kJ/m3, while the aforementioned value increased to 8.02 kJ/m3, 8.22 kJ/m3, 8.78 kJ/m3 and 9.88 kJ/m3 for F1.0S3T7, F1.0S3T28, F1.0S9T7 and F1.0S9T28, respectively.E50 and Eu against qu for various JF + GBFS mix designs, respectively. The variations of E50 were situated within the 0.054qu < E50 < 0.025qu domain . For Eu, however, a broader domain in the form of 0.063qu < Eu < 0.018qu  was noted. The former, the E50, exhibited a rather strong correlation with qu. On the contrary, the peak strain energy was poorly correlated with the peak UC strength. In this regard, simple correlative models in the forms of E50 = 0.038qu (with R2 = 0.836) and Eu = 0.029qu (with R2 = 0.449) can be derived; the former can be implemented for indirect estimations of E50.i) frictional resistance generated at the soil–fiber interface, owing to the fiber’s rough surface texture; and (ii) mechanical interlocking of soil particles and fibers [The JF inclusions are able to amend the soil fabric through improvements achieved in two aspects: , which immediately substitute cations of lower valence  and/or same-valence cations of smaller ionic radius  in the vicinity of the clay particles. These cation exchanges lead to a decrease in the thickness of the Diffused Double Layers (DDLs), owing to the development of strong van der Waals bonds between adjacent clay particles in the matrix, which in turn promote aggregation and flocculation of the clay particles [2+) and hydroxide (OH–) units, released from the water–binder complex, gradually react with silicate (SiO2) and aluminate (Al2O3) units in the soil, thereby leading to the formation of strong cementation products/gels, namely Calcium–Silicate–Hydrates (CSH), Calcium–Aluminate–Hydrates (CAH) and Calcium–Aluminate–Silicate–Hydrates (CASH); these products encourage further solidification and flocculation of the soil particles, which in turn accommodate the development of a dense, uniform matrix coupled with enhanced strength performance [Calcium-based binders, in this case GBFS, initiate a series of short- and long-term chemical reactions in the soil–water medium, which alter the soil fabric into a unitary mass of improved mechanical performance. Short-term chemical reactions consist of cation exchange and flocculation–agglomeration; their amending roles are often negligible when paired with neutrally-charged soil particles such as gravels, sands and silts. For fine-grained soils containing a notable fraction of negatively-charged clay particles, however, short-term reactions lead to significant improvements in the soil’s plasticity/workability, early-age strength, swelling potential and consolidation capacity ,74,75,76+ < Ca2+ . GBFS-trarticles ,79. Longformance ,33,76,79F0S0T0 , F1.0S0T0, F0S6T28 and F1.0S6T28, respectively. The microstructure of the natural soil sample manifested a partly-dense, non-uniform matrix, accompanied by a notable number of large inter- and intra-assemblage pore-spaces, respectively, formed between and within the soil aggregates; such morphological features warrant the existence of an edge-to-edge dispersed fabric , as evident with the experimental results discussed in i) JF content Fc (in %); (ii) GBFS content Sc (in %); and (iii) curing time Tc (in days). Therefore, the peak UC strength problem for various JF + GBFS blends can be expressed as:f = an unknown functional expression which is to be obtained through trial and error.For a given type of soil reinforced with JF and/or treated with GBFS, the independent variables governing the peak UC strength f should involve a simple algebraic structure, constructed by a minimal number of model/fitting parameters (or regression coefficients), capable of arriving at a reliable estimate of the problem at hand [β0 to β9 = model/fitting parameters (or regression coefficients); and β0 = peak UC strength of the natural soil, since setting Fc = 0, Sc = 0 and Tc = 0 leads to qu = β0.A suitable regression model can be characterized as one that maintains a perfect balance between simplicity, i.e., ease of application, and accuracy, i.e., acceptable goodness of fit and low forecast error. As such, any suggested functional expression for  at hand ,80. The  at hand ,55,59,81F–test and Student’s t–test, were then carried out to examine the model’s statistical significance. In addition, statistical fit-measure indices, such as the coefficient of determination R2 (dimensionless), the root-mean-squared error RMSE (in kPa), the normalized root-mean-squared error NRMSE (in %) and the mean-absolute-percentage error MAPE (in %), were adopted to assess the model’s predictive capacity [quA = actual peak UC strength, as presented in quP = predicted peak UC strength; b = index of summation; and N = number of experimental data points used for model development  was fitted to the experimental peak UC strength data RM2 (=0.964) and low RMSE (=17.28 kPa), NRMSE (=4.78%) or MAPE (=6.19%) values warrant a strong agreement between actual and predicted peak UC strength data. The R2 index merely surpassed 0.95, thus indicating that leastwise 95% of the variations in experimental observations are captured and further explained by the proposed regression model. The NRMSE index was found to be slightly less than 5%, thus signifying a maximum offset of 5% associated with the predictions. However, the P-value associated with some of the regression components, namely Sc, Tc, Sc2, Tc2 and FcTc, was found to be greater than 5%, implying that these components are statistically-insignificant and hence make no or little contribution towards the predictions. Statistically-insignificant terms can be eliminated to accommodate the derivation of a simplified model with unanimously-significant regression components [The regression analysis outputs with respect to Equation (7) are summarized in mponents . As such2 = 0.951, RMSE = 20.00 kPa, NRMSE = 5.54% and MAPE = 7.28%, which are on par with that observed for Equation (7). In essence, Equation (11) suggests a more practical path towards predicting the peak UC strength while maintaining a performance similar to that offered by the more complex Equation (7). Moreover, the p-values associated with all of the regression components were unanimously less than 5% , all of which can be calibrated by little experimental effort, as well as simple explicit calculations, and hence implemented for preliminary design assessments, predictive purposes and/or JF + GBFS optimization studies. Assuming that the peak UC strength of the natural soil (or β0) is at hand, the four fitting parameters can be adequately calibrated by a total of four UC tests carried out on four arbitrary JF + GBFS mix designs.The regression analysis outputs with respect to Equation (11) are summarized in Fc, Sc and Tc, on the dependent variable qu. The overall relative impact, both positive and negative, of an independent variable, i.e., ax = Fc, Sc or Tc, on the dependent variable qu, commonly referred to as sensitivity, can be defined as:aD = partial derivative of qu or Equation (11) with respect to ax = Fc, Sc or Tc; σ(ax) = standard deviation of ax data; σ(qu) = standard deviation of predicted qu data; b = index of summation; and N = number of observations , measures the likelihood of qu increasing or decreasing as a result of an increase in ax. As such, the likelihood of increase or decrease in qu as a result of an increase in ax can be, respectively, defined as:MP(ax) = number of observations where aD ≥ 0; and MN(ax) = number of observations where aD < 0.The partial derivative term, ax = Fc, Sc or Tc, on the dependent variable qu can be, respectively, defined as:The positive and negative impacts of an independent variable, i.e., SP(ax) and SN(ax) are, respectively, positive and negative fractions of the sensitivity parameter, S(ax) or Equation (12), meaning that for any given ax, S(ax) = SP(ax) + SN(ax).It should be noted that SP(ax) or Equation (15), is of interest for further analysis. The positive-sensitivity parameter can be expressed in terms of percentage to facilitate a more practical comparison between the independent variables [FP(ax) = positive contribution offered by an increase in ax resulting in an increase in qu (in %); and K = number of independent variables .The principal objective of any introduced soil stabilization scheme is to accommodate an increase in the peak UC strength, and as such, the variations of the positive-sensitivity parameter, ariables :(17)(%) Fc ≤ 1.5%, exhibits favorable improvements only up to a particular/optimum fiber content, beyond of which marginal improvements or adverse effects, owing to fiber-clustering, can be expected  + FP(Tc) = 65% >> FP(Fc) = 35%.The sensitivity analysis results with respect to Equation (11) are summarized in Fc = 1%; beyond 1% JF, the effect of JF-reinforcement adversely influenced the development of strength and stiffness. The composite’s ductility and toughness, however, were consistently in favor of JF-reinforcement, meaning that the greater the JF content the higher the developed ductility and toughness.For any given GBFS content and curing time, the greater the JF content the higher the developed strength and stiffness up to For any given JF content, the greater the GBFS content and/or the longer the curing period, the higher the developed strength, stiffness and toughness, following monotonically-increasing trends. The composite’s ductility, however, was adversely influenced by GBFS-treatment, meaning that the greater the GBFS content and/or the longer the curing period, the lower the developed ductility.F1.0S9T28 managed to satisfy ASTM’s criterion and hence can be taken as the optimum design choice.The addition of GBFS to JF-reinforced samples improved the soil–fiber connection interface or bonding, as the fiber units became fully embedded between and within the soil aggregates; this in turn led to a further improvement in the composite’s strength and stiffness. The ASTM D4609–08 strength criterion was used to assess the efficiency and hence applicability of the proposed JF + GBFS mix designs. In this regard, the sample qu as a function of the composite’s basic index properties, i.e., JF content Fc, GBFS content Sc, and curing time Tc. The predictive capacity of the suggested model was examined and further validated by statistical techniques. A sensitivity analysis was also carried out to quantify the relative impacts of the independent regression variables, namely Fc, Sc and Tc, on the dependent variable qu. The proposed regression model contained a limited number of fitting parameters, all of which can be calibrated by little experimental effort, as well as simple explicit calculations, and hence implemented for preliminary design assessments, predictive purposes and/or JF + GBFS optimization studies.A non-linear, multivariable regression model was developed to quantify the peak UC strength The following conclusions can be drawn from this study:


Escherichia coli (E.coli), and assembled into CPV VLPs which could be affected by NaCl and pH. At 250 mM NaCl pH 8.0, the VLPs co-expressed with Tf16 had similar size (25 nm) and shape with the authentic virus capsid under the transmission electron microscopy (TEM), which is also in accordance with the dynamic light scattering (DLS) data. Immunization with these particles could induce high-titer hemagglutination inhibition (1:12288) and neutralizing antibodies (1:6144) in guinea pigs. Splenic cells of them could secrete IFN-γ and IL-4 after stimulation by CPV. Thus, the VLPs produced by the new approach with high yield and immunogenicity could be a potential candidate for CPV vaccine.Canine parvovirus (CPV) has been considered to be an important pathogen, which can cause acute infectious disease in canids. Although current vaccines are effective in preventing CPV infection, safety problems still remain unsolved. In this study, a subunit vaccine against CPV based on virus-like particles (VLPs) with good safety and immunogenicity is reported. Soluble CPV VP2 protein was produced by co-expression of chaperone trigger factor (Tf16) in  Protoparvovirus within family Parvoviridae and has the ability to agglutinate pig erythrocytes [Canine Parvovirus (CPV) disease is a highly contagious infectious disease caused by canine parvovirus, which is manifested as severe hemorrhagic enteritis in dogs of all ages and myocarditis in puppies . Since ihrocytes . It is ahrocytes .CPV-2 was the original virus strain in dogs that spread worldwide, and soon gave rise to three antigenic variants with mutation sites in VP2 protein . CPV-2a Vaccination plays an important role in the prevention of this disease. Attenuated CPV-2 vaccines are effective and widely used, but large scale production of them is usually expensive and laborious. Although the attenuated CPV-2 vaccine strains are stable in limited passages in the dog, a series of CPV-2-like strains were identified and deduced to evolve from the vaccine strains . InactivEscherichia coli (E.coli), such as bovine carbonic anhydrase II [Prokaryotic expression system has been used extensively for recombinant proteins production in laboratory and industrial scale due to its simplicity, rapid growth rate and relatively low cost. Actually, it is not uncommon that overexpressed recombinant proteins fail to reach a correct conformation and are prone to associate with each other to form insoluble aggregates . To copedrase II .E.coli. Under the optimum condition (250 mM NaCl pH 8.0), the VLPs assembled by the VP2 protein were used to immunize guinea pigs. The immunological tests suggested that this kind of VLPs has high immunogenicity and may be a promising candidate vaccine against CPV.In this study, we obtained high-yield soluble VP2 protein by co-expression with chaperone Tf16 in 2 incubator.Feline kidney F81 cells were cultured in Dulbecco’s modified Eagle’s medium , which was supplemented with 100 U/ml of penicillin, 100 mg/ml of streptomycin and 10% fetal bovine serum  at 37 °C in a 5% COThe virus was isolated from feces of an infected puppy as described previously . DNA wasGGATCC (BamHI) ATGAGCGATGGTGCAGT-3′, and the reverse primer was 5’-GATCTCGAG (XhoI) TTAGTACAGTTTGCGCGG-3′. The VP2 gene was digested by the restriction endonucleases BamHI and XhoI, and sub-cloned into pET30a (pET30a-VP2). Then the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells harboring pTf16, which were prepared according to the manufacturer’s protocol .The sequence of VP2 protein was optimized based on CC8601 strain (GenBank accession number GU569948). The complete VP2 gene was amplified by a pair of primers . The forward primer was 5’-GAThe colony, positive for both VP2 protein and Tf16, were cultured into LB medium containing 2 mg/ml L-Arabinose, 0.1 mM IPTG, 0.1 mg/ml chloramphenicol and kanamycin. After 14 h induction at 25 °C, the cells were harvested and lysed by sonication in buffer A  on ice. The supernatant and debris were collected by centrifugation at 10000 g for 20 min and analyzed by SDS-PAGE.The VP2 protein with an N-terminal His tag was purified by Ni-NTA affinity chromatography . Briefly, the clarified supernatant was loaded on the Ni-NTA column at a flow rate of 1 ml/min for 3 times. The column was washed with buffer B  until no protein was detected by CBB staining . The recombinant VP2 protein was thoroughly eluted with buffer C  and analyzed by SDS-PAGE and Western blot.The eluted VP2 protein was dialyzed against 50 mM Tris with different concentrations of NaCl  and pH  at 4 °C for 16 h. To analyze the assembly condition and appearances of VLPs, the collected protein was detected by dynamic light scattering (DLS) and transmission electron microscopy (TEM).n = 5). Group A, B and C were intramuscularly immunized with 10 μg, 30 μg and 50 μg CPV VLPs mixed with Montanide™ ISA 201 VG , respectively; group D was inoculated with 100 μl commercial live-attenuated vaccine as a positive control; group E was injected with 100 μl PBS as a negative control. Booster immunization with the same doses of VLPs was performed at 21 dpi (days post inoculation). Blood samples were collected from the forelimb veil at 0, 14, 21, 28, 35 and 42 dpi.Thirty female guinea pigs housed under pathogen-free conditions were randomly divided into five groups  was assessed by immunoperoxidase monolayer assay (IPMA). The highest dilution of sera showing complete inhibition of CPE was taken as the neutralization titer.All serum samples from guinea pigs were inactivated at 56 °C for 30 min and diluted in a two-fold series starting at 1:30. After incubation with 100 TCIDHemagglutination inhibition (HI) assay was performed as described previously . Briefly4Cl, pH 7.4) for 6 min at room temperature. Then spleen lymphocytes were seeded in 96-well plates and stimulated by CPV. After 48 h’ incubation at 37 °C, cell culture supernatants were tested using commercial IL-4 and IFN-γ ELISA kits  according to the manufacturer’s instructions.Spleens of guinea pigs from each group were removed under sterile conditions at 42 dpi. Cells were washed twice with RPMI 1640 containing 10% FBS and filtered by Cell Strainers . Red blood cells (RBCs) in the cell suspension were lysed using RBC lysis buffer .All values were tested for significance by one-way analysis of variance (ANOVA) using GraphPad Prism version 5.00 . The significance level was set at 0.05 .p < 0.05) than PBS group after the second immunization.To test whether CPV VLPs could induce specific humoral immune responses, guinea pigs were divided into five groups and immunized with different doses of CPV VLPs , 100 μl commercial CPV vaccine and PBS. As shown in Fig. p < 0.001) and 30 μg (p < 0.05) immunized groups than the control group. No significant difference was found between the commercial CPV vaccine and PBS group. The IL-4 content of VLPs vaccinated groups changed in a dose-dependent manner. The VLPs and commercial vaccine groups showed significantly higher (p < 0.01) IL-4 production compared to the control group  which is robust, stable, with low viscosity and easy to inject, compared to traditional oil emulsions. In addition, the low level of HI antibodies (1:192) and neutralization antibodies (1:60) induced by the commercial CPV vaccine may be related to the absence of a host animal. Here, we just regard the commercial vaccine as a positive control and a challenge test will be performed in dogs to evaluate the immune protection in the future.To evaluate the immunogenicity of CPV VLPs, anti-CPV antibodies were tested in the immunized guinea pigs. In VLPs immunized groups, HI antibody titers increased rapidly after the second immunization may because of the stimulated memory B cells. It is remarkable that the maximum titers of HI antibody and neutralization antibody were 1:12288 and 1:6144 respectively at 35 dpi in 50 μg VLPs group. A powerful response was also observed in 10 μg group, despite the lower immune dose compared to the previous study , 26. It + and CD8+ T cells in mice [+ T cells which related to cellular and humoral adaptive immunity, respectively [It has been well documented by previous studies that CPV VLPs could induce proliferation of CD4 in mice , 26. Furectively . In thisectively –31. Howeectively  and requE.coli could assemble into VLPs similar with natural CPV virions in size and shape. The VLPs could stimulate strong immune responses in guinea pigs against CPV. Thus, the Tf16 co-expression system provides a new approach for producing CPV VLPs with high yield and efficiency. With the following improvements, a CPV VLP-based vaccine may be developed successfully in the future.In summary, the CPV VP2 protein co-expressed with Tf16 in


Marine sponges host complex microbial consortia that vary in their abundance, diversity and stability amongst host species. While our understanding of sponge-microbe interactions has dramatically increased over the past decade, little is known about how sponges and their microbial symbionts interact with viruses, the most abundant entities in the ocean. In this study, we employed three transmission electron microscopy (TEM) preparation methods to provide the first comprehensive morphological assessment of sponge-associated viruses. The combined approaches revealed 50 different morphologies of viral-like particles (VLPs) represented across the different sponge species. VLPs were visualized within sponge cells, within the sponge extracellular mesohyl matrix, on the sponge ectoderm and within sponge-associated microbes. Non-enveloped, non-tailed icosahedral VLPs were the most commonly observed morphotypes, although tailed bacteriophage, brick-shaped, geminate and filamentous VLPs were also detected. Visualization of sponge-associated viruses using TEM has confirmed that sponges harbor not only diverse communities of microorganisms but also diverse communities of viruses. Sponges are abundant and ecologically important members of marine benthic communities . Most sp, living in symbiogenesis  recently provided the first insights into the composition and function of viruses inhabiting reef sponges . Consist species .While molecular approaches have substantially improved our understanding of viral-host interactions , biases Sampling was conducted on coral reefs of Orpheus Island, Great Barrier Reef, Australia  and Al Fahal, Red Sea, Saudi Arabia , between December 2015 and February 2016. Sampling in Australia was conducted under the Great Barrier Reef Marine Park Authority permit G12/35236.1, and sampling in Saudi Arabia was authorized by the Saudi Arabian coastguard as the study did not involve endangered or protected species.Stylissa carteri and Carteriospongia foliascens were found at both locations, and sampling was performed in triplicate at both sites. Sponge specimens were photographed in situ before being individually placed within sterile Falcon® tubes and kept on ice until processing. All sampling materials were sterilized prior to and between each sampling. Morphological characterization of sponge species was performed as described in  ultrathin sectioning of sponge tissue ; (ii) pu3 was fixed in 2.5% glutaraldehyde in 0.2M cacodylate buffer and kept at 4 °C for 2–24 h. After fixation, samples were immersed in 1% osmium tetroxide in 100 mM phosphate buffer for 1–2 h, washed in distilled water and stained in the dark with 2% aqueous uranyl acetate for 2 h at 4 °C. Stained tissue was dehydrated through a series of ethanol and propylene oxide then embedded in epoxy resin. Ectosome-choanosome oriented sections (about 65 nm thick) were prepared using a Leica EM UC7 ultramicrotome and placed on TEM copper grids.Histological sections were prepared from fresh sponge tissue based on standard procedures for TEM . Briefly3) and covered with 15 µL of 0.02 µm filter-sterilized  SM buffer , then homogenized with a Craig’s HS30E homogenizer  for 5 to 10 min (min). Tissue homogenate was filtered through a Falcon® 100 µm Cell Strainer , then centrifuged at 500 g for 15 min at 4 °C to pellet the majority of cell debris. The supernatant was used to purify the VLP via centrifugation in Cesium Chloride solution, with density varying from 1.2 g/mL to 1.6 g/mL  and centrifuged at 4,000 g for 10 min. Mucus supernatant was filtered through 0.45 µm filters  and fixed in 1.5% glutaraldehyde. TEM imaging of mucus preparations was performed as described above for CsCl purified samples.To describe the VLPs associated with sponge mucus and the external ectoderm, the sponge surface was carefully scraped with a sterile scalpel blade followed by rinsing three times with filtered (0.02 µm) seawater. This TEM preparation method was based on a viral purification method described for marine hydras . ExtractCarteriospongia foliascens, Stylissa carteri, Xestospongia sp., Pipestela candelabra, Lamellodysidea herbacea, Cymbastella marshae, Echinochalina isaaci and Cinachyrella schulzei; and seven sponge species from the Red Sea: Carteriospongia foliascens, Stylissa carteri, Xestospongia testudinaria, Hyrtios erectus, Mycale sp., Amphimedon ochracea and Crella cyathophora. VLPs were observed within sponge cells, in the extracellular mesohyl matrix, in the mucus/surface biofilm and within sponge-associated microbes. A diverse range of viral morphologies were observed, including hexagonal (tailed and non-tailed), spherical, filamentous, brick-shaped, beaded and geminate VLPs. While we detected numerous viral morphotypes, most were rare and often obscured by vesicles, cell debris and particulate organic matter.TEM analysis revealed that viral particles are diverse constituents of the sponge holobiont. Fifty VLP morphotypes , ranging from 60–205 nm in diameter – 3, 4A. liascens , Xestospngia sp. , E. isaa. isaaci  and S. c carteri . VLPs chschulzei . VLPs ch. isaaci , 1G, S.  carteri  and A. oochracea .Hyrtios erectus where a group of four virions were observed within a vacuole in the mesohyl matrix . The majority of VLPs did not show an envelope outside the capsid, however an envelope was observed in association with a small proportion of VLPs. –3L, 5H. l matrix  and anotl matrix .Xestospongia sp. . In C. foliascens, a FVLP was frequently observed attached to cyanobacteria and within the sponge mesohyl,  and electron-translucent core with outer membrane structures consistent with a glycoprotein coat surrounding the entire membrane  were observed in the sponge mucus, mesohyl matrix, within sponge cells and associated with sponge-associated microorganisms –4L; 5A. ngia sp. , 4D and  carteri . Althougmesohyl, –4G. Thismembrane . In X. t mesohyl –4L. This mesohyl –4L. Anotthophora . It presC. cyathophora mesohyl matrix . However, it is also possible that multiple viruses infect the same host, as has been observed in some bacterioplankton  and coraMorphology is an important feature for viral classification according the International Committee on Taxonomy of Viruses (ICTV). However, there are also some limitations associated with using TEM to identify viruses. For instance, many viral groups lack morphological structures that characterize them as typical viral particles by TEM. Also, as many viruses are small and simple they can be mistaken for non-viral particles such as cellular vesicles or organelles. Although the assignment of viral-like particles in this study was made by comparison to morphologically characterised viruses, the possibility remains that some VLPs may not represent true viruses.Caudovirales-like morphotypes highlights the potential for these VLPs to target sponge symbionts and ultimately control microbial population dynamics within the sponge holobiont. Amongst them, a Siphoviridae”- VLP detected in the surface biofilm of C. schulzei presented similar morphology, although slightly smaller, to the previously described sponge-associated Phage ΦJL001  were detected in both prokaryotic and eukaryotic cells within the sponge holobiont. In ll lysis . A previll lysis .Spiraviridae were detected in the sponge C. cyathophora, with this viral family known to infect Archaea , with each isomer being wider than they are long, contrasting with the isomer dimensions in the A. ochracea VLP. Beaded VLPs were also detected in sponges and their non-isomeric particles comprising a flexible filament strongly resembled the beaded VLPs previously reported from scleractinian corals  ultrathin sections of sponge tissue, (ii) purification via density gradient ultracentrifugation and (iii) ectoderm scraping and filtration of sponge mucus. While density gradient purification facilitated concentration and recovery of VLPs from different areas of the sponge holobiont, it also co-concentrated cellular debris, potentially masking many VLPs. Tissue sectioning enabled direct visualisation of spatial localisation and host-viral interactions but was labour intensive and some VLP structures were distorted during sectioning. Ectoderm scraping and collection of sponge mucus was most effective at preserving delicate viral structures and minimizing the amount of cellular debris, however, it was restricted to recovering VLPs associated with the sponge mucus or ectoderm.This first morphological characterisation of sponge-associated viruses revealed a wide diversity of VLPs infecting both the sponge cells and symbiont compartments of the holobiont. By confirming that viruses are a significant component of the sponge holobiont, this work paves the way for future metaviromic and cell culturing analyses that can characterise the taxonomy and function of the sponge viral community.10.7717/peerj.5625/supp-1Figure S1Amphimedon ochracea, (B) Xestospongia testudinaria, (C) Crella cyathophora, (D) Hyrtios erectus, (E) Mycale sp. GBR and Red Sea sponge species: (F) Stylissa carteri, (G) Carteriospongia foliascens. GBR sponge species: (H) Echinochalina isaaci, (I) Cymbastella marshae, (J), Cinachyrella schulzei, (K), Lamellodysidea herbacea, (L), Pipestela candelabra, (M) Xestospongia sp.. Scale bar = 10 cm. Photos by Cecília Pascelli.Red Sea sponge species: (A) Click here for additional data file.10.7717/peerj.5625/supp-2Table S1Click here for additional data file.


Inhibition of tumor necrosis factor-α (TNF) signaling is beneficial in the management of ulcerative colitis (UC), but up to one-third of patients do not have a clinical response of relevance to TNF inhibitors during induction therapy (i.e. primary non-responders [PNRs]). Through production of prostaglandins (PGs) and thromboxanes, cyclooxygenase-2 (COX-2) affects inflammation and epithelial regeneration and may in this way be implicated in treatment resistance to TNF inhibitors.2, on intestinal epithelial stem cell self-renewal and differentiation using primary human intestinal organoids (“mini-guts”).In this study, COX-2 expression was analyzed in human intestinal biopsies and patient-derived monocytes, and the downstream consequences of COX-2 activity was evaluated by assessing the influence of the down-stream effector, PGECOX-2 expression in monocytes isolated from responders (Rs), whereas COX-2 expression was constitutively high and non-inducible in monocytes from PNRs. Additionally, PGE2 in combination with proliferative signals transformed human intestinal epithelial cells to a proinflammatory state akin to flaring UC, whereas PGE2 in combination with differentiation signals supported robust mucin induction.We found that TNF stimulation induced 2 signaling could be a novel target for the management of PNRs to TNF inhibitors. We additionally demonstrate that COX-2–PGE2 signaling has dual functions during tissue repair and normal lineage differentiation, explaining in part the lack of response to TNF inhibitors among PNRs.Our work indicates that COX-2-PGEThis work was funded by grants from the Novo Nordisk Foundation, the Lundbeck Foundation, the Vanderbilt Digestive Disease Research Center, NIH Grants, Aase and Ejnar Danielsen's Foundation and the A.P. Møller Foundation. •COX-2.Monocytes derived from primary non-responders to therapy with TNF inhibitors have a higher basal expression level of •2 maintains an inflammatory state of TNF-stimulated intestinal epithelium.The sustained induction of PGE•2 pathway might be a druggable pathway to affect TNF inhibitor responsiveness.The COX-2–PGE The COX-2-PGE2 pathway has been suggested to transiently promote intestinal repair in murine studies. Moreover, the use of COX-2 inhibitors in patients with ulcerative colitis (UC) has been linked to an increased risk of flaring disease, although these observations have recently been questioned. Expression analysis using material from patients suffering from UC stratified based on their response to TNF inhibitors, suggests that COX-2 levels are elevated in the group of non-responders prior to TNF inhibitor therapy.2 pathway could explain the downstream consequences of aberrant COX-2 expression and the associated link to a relevant response to TNF inhibitor therapy using patient derived material. We demonstrate for the first time that monocytes from patients not responding to TNF inhibitors have higher basal levels of COX-2 as compared to monocytes isolated from responders. Additionally, PGE2, which is the major downstream effector of COX-2, exacerbates the expression of proinflammatory cytokines upon TNF stimulation in human intestinal epithelial cells, thereby ameliorating the inflammatory response, and potentially it impairs the reestablishment of a functional epithelial barrier.Our aim was to address whether changes in the COX-2–PGE2 pathway should be explored as a target for primary non-responders to TNF inhibitor therapy as well as a prognostic biomarker for the TNF inhibitor responsiveness.The present work indicates that the COX-2-PGEAlt-text: Unlabelled Box1primary non-responders (PNRs) )  (2 stimulation did not affect the expression of carbonic anhydrase II (CAII) or chromogranin A (CHGA), which are markers for enterocytes and enteroendocrine cells, respectively  . Under I< 0.01]) a, confir< 0.01]) e. PGE2 sectively a. Followppressed . At the cultures b, c. Morntiation d. Using ous PGE2 e.2 on intestinal epithelium depends on the presence of concomitant inflammation. During intestinal homeostasis, PGE2 promotes goblet cell differentiation, whereas it drives the epithelial cells into a state with impaired stem cell population during inflammation  and the Lundbeck Foundation (R105-A9755). D.M.A. was supported by a Pilot and Feasibility grant from the Vanderbilt Digestive Disease Research Center and the National Institutes of Health (NIH) Grants DK058404 and AI121796. Y.L. was supported by Aase and Ejnar Danielsen's Foundation, Denmark (F-19215-00-27) and the A.P. Møller Foundation.K.B.J. was supported by grants from the


Although porcine circovirus-like particles can function as a vector to carry foreign peptides into host cells, displaying foreign peptides on the surface of virus-like particles (VLPs) remains challenging. In this study, a plateau, consisting of the middle portion of Loop CD (MP-Lcd) from two neighboring subunits of PCV2 capsid protein (Cap), was identified as an ideal site to insert various foreign peptides or epitopes and display them on the surface of PCV2 VLPs. One of the goals of this work is to determine if the surface pattern of this plateau can be altered without compromising the neutralizing activity against PCV2 infections. Therefore, biological roles of MP-Lcd regarding VLPs assembly, cell entry, and antigenicity were investigated to determine whether this was a universal site for insertion of foreign functional peptides. Three-dimensional (3D) structure simulations and mutation assays revealed MP-Lcd was dispensable for PCV2 Cap assembly into VLPs and their entry into host cells. Notably, substitution of MP-Lcd with a foreign peptide, caused surface pattern changes around two-fold axes of PCV2 VLPs based on 3D structure simulation, but was not detrimental to VLPs assembly and cell entry. Moreover, this substitution had no adverse effect on eliciting neutralizing antibodies (NAbs) against PCV2 infection in pigs. In conclusion, MP-Lcd of the PCV2 Cap was a promising site to accommodate and display foreign epitopes or functional peptides on the surface of PCV2 VLPs. Furthermore, chimeric VLPs (cVLPs) would have potential as bivalent or multivalent vaccines and carriers to deliver functional peptides to target cells. Circovirus genus in the Circoviridae family, has a small, single-stranded and circular DNA genome. There are two main genotypes, namely porcine circovirus type 1 (PCV1) and PCV2, both of which share high levels of nucleotide homology and a common genomic organization  analysis of PCV2 isolates, PCV2 is divided into four main subtypes  , a member of the in vitro  and virus replication associated proteins  Cheung, . The PCV2- and carboxyl-terminus (NT & CT) of the Cap contain a non-structured stretch. The NT, rich in basic amino acids, has a typical nuclear localization signal (NLS), responsible for distribution of the PCV2 Cap in nucleus , and specific antibodies against these tags or foreign peptides were produced in response to these chimeric PCVs  containing amino acid epitope tags in the C terminus of the capsid gene are infectious and elicit both anti-epitope tag antibodies and anti-PCV type 2 neutralizing antibodies in pigs  structure of the PCV2 Cap was elucidated by two research groups  was optimized and synthesized, as we have described . After confirmation by DNA sequencing, recombinant plasmids were transformed into BL21 (DE3) competent cells  for protein expression.Unless otherwise stated, all chemicals were purchased from Sigma Aldrich . PCV2 in vitro of PCV2 Cap wild type and its mutants were done as described . Subtype and cluster of each PCV2 isolate were confirmed via previous phylogenetic analyses . For each Cap mutant, a 3D structural model was generated via homology modeling using a crystal structure of PCV2 Cap (PDB accession number: 3R0R) as a template, with protein modelings done using Modeller . Then, icosahedral structures (3D) of all capsid mutants were generated, based on the monomeric structure of the PCV2 Cap mutant, by applying a VMD 1.9 matrix transformation using Tcl script .Typical Cap sequences from PCV1, 2, and 3 were downloaded from GenBank (n = 2), 2 mo old, were used to prepare anti-PCV2 Cap serum. Rabbits were initially immunized with 2 ml of PCV2 VLPs  mixed with Alhydrogel adjuvant , and then similarly boosted twice more at 2-week intervals (Weeks 2 and 4). Blood samples were collected 6 wk after final immunization and serum was isolated and stored at −20°C.Female New Zealand rabbits  mixed with MontanideTM Gel 01 ST adjuvant (SEPPIC), respectively, whereas Group 3 was immunized with PBS only (negative control). Each pig was injected only once. Sera were collected 21 and 28 d post-primary immunization (dpi) and tested by indirect enzyme-linked immunosorbent assay (ELISA) and PCV2 neutralization assays. All animal care and use protocols in this study were conducted in accordance with the guidelines of the Animal Care and Use Committee of Hunan Agricultural University and were approved by the Institutional Ethics Committee (IEC) of Hunan Agricultural University.Fifteen commercial 3-week-old pigs, serologically negative for PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV), were used. These pigs were randomly allocated into three groups (five per group). Groups 1 and 2 were immunized with intramuscular injections of 200 μg of PCV2 wild type VLPs or cVLPs-M8 (KP112484) for 1 h at 37°C. Then, serum-virus mixtures were inoculated to confluent PK-15 cells cultured in 96-well plates and incubated at 37°C for 72 h. Cells were fixed with absolute ethyl alcohol for 30 min, 0.1% triton for 10 min and then blocked with 3% BSA for 1 h at room temperature. Thereafter, cells were incubated with rabbit anti-PCV2 serum  as the primary antibody and FITC-conjugated donkey anti-rabbit IgG  as second antibody, each for 1 h. Cells were observed using a fluorescent microscope . The reciprocal of the highest dilution at >50% fluorescent focus reduction was the neutralizing antibody titer.PCV2 VLPs assembled from wild type PCV2 Cap (Cap WT) were coated as an antigen to detect specific anti-PCV2 antibodies by ELISA, as described , based on amino acid sequence alignment Figure . There win vitro dialysis   spaced by a “GS” linker . However, when Cap-M7 was assembled into a capsid, the plateaus composed of MP-Lcd around the two-fold axes had disappeared . Antibody titers increased rapidly (P < 0.01) from 21 to 28 dpi, compared to the control group . However, PCV2 VLPs have been developed to carry foreign peptides, with fusion usually done on either the NT or the CT of the Cap , as reported for other VLPs  containing insertions with distinct foreign peptides were capable of self-assembly into VLPs o Figure , and theo Figure . In parto Figure . Therefoo Figure . Finallyo Figure . Morpholo Figure . TherefoRoles of MP-Lcd in PCV2 VLPs assembly, cell entry, and antigenicity were also investigated in the present study. Apparently, removal of MP-Lcd caused the plateau to disappear from the surface, but had no effects on VLPs assembly and cell entry. Therefore, we inferred that MP-Lcd may not be involved in recognition of virus-host cells for PCV2 internalization. Further, deleting MP-Lcd and replacing it with a foreign epitope (Cap-M8) introduced a new surface pattern around the two-fold axes of the PCV2 VLPs, since part of residues of the foreign epitope were present on the surface (orange in Figure in vitro.In conclusion, the MP-Lcd of Loop CD was identified as a very promising site within the PCV2 Cap to develop a functional carrier capable of displaying a variety of foreign peptides or epitopes on the surface of PCV2 VLPs. Of note, it was dispensable for assembly and cellular uptake of PCV2 VLPs. In addition, MP-Lcd was not required for eliciting NAbs against PCV2 infection. Therefore, development of PCV2 VLPs-based bivalent or multivalent vaccines against concurrent infections of PCV2 and other pathogens should be very feasible. Furthermore, based on structure simulation, MP-Lcd functioned as a plateau to project foreign peptides from the surface of PCV2 VLPs. Finally, although insertion size was tested, maximal size for MP-Lcd insertion still needs to be determined, as conditions may be further optimized for VLPs assembly DW, NW, and YY are responsible for experimental design. DW, SZ, YZo, WY, YJ, YZh, and YD did experiments, collected and analyzed data. DW and YY wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.