Autism Res2018, 11: 1129–1137. © 2018 The Authors Autism Research published by International Society for Autism Research and Wiley Periodicals, Inc.Detection of deception is of fundamental importance for everyday social life and might require “mindreading” . People with diminished mindreading, such as those with autism spectrum disorder (ASD), might be at risk of manipulation because of lie detection difficulties. In Experiment 1, performance among 216 neurotypical adults on a realistic lie detection paradigm was significantly negatively associated with number of ASD traits, but not with mindreading ability. Bayesian analyses complemented null hypothesis significance testing and suggested the data supported the alternative hypothesis in this key respect. Cross validation of results was achieved by randomly splitting the full sample into two subsamples of 108 and rerunning analyses. The association between lie detection and ASD traits held in both subsamples, showing the reliability of findings. In Experiment 2, lie detection was significantly impaired in 27 adults with a diagnosis of ASD relative to 27 matched comparison participants. Results suggest that people with ASD (or ASD traits) may be particularly vulnerable to manipulation and may benefit from lie detection training. Detection of deception is of fundamental importance for everyday social life. People with diminished understanding of other minds, such as those with autism spectrum disorder (ASD), might be at risk of manipulation because of lie detection difficulties. We found that lie detection ability was related to how many ASD traits neurotypical people manifested and also was significantly diminished among adults with a full diagnosis of ASD. The ability to detect when one is being deceived by others is of fundamental importance for everyday social life and difficulties detecting deception increase one's risk of being manipulated, with potentially serious consequences. Nonetheless, neurotypical adults tend to assume others are telling the truth (a default “truth‐bias”) and their ability to distinguish truths from lies in experimental situations is only just above chance, albeit statistically significantly so . A corrected hit rate (CHR) of zero would indicate chance‐level judgments on the task. CHR was also calculated separately for the transparent and nontransparent conditions. Finally, the proportion of truth judgments made by participants, independent of accuracy, was calculated. The higher the proportion, the greater the truth bias .The Reading the mind in the eyes (RMIE) task [Baron‐Cohen, Wheelwright, Hill, Raste, & Plumb, We employed a version of the “Animations” task as a second measure of mindreading [e.g., Abell, Happé, & Frith, Each clip was presented to participants on a computer screen. After the clip was finished, participants described what had happened in the clip. An audio recording of participants’ responses was made for later transcription. Transcriptions were scored on a scale of 0–2 for accuracy , based on the criteria outlined in Abell et al. . Twenty Z score was calculated for each mindreading task. The two Z scores were then averaged to form a composite mindreading score. The composite was used in bivariate and partial correlation analyses in order to reduce the number of statistical comparisons and maximize power. However, following an anonymous reviewer's suggestion, we also report post hoc correlations with RMIE and Animations separately.A The Autism‐spectrum Quotient [AQ; Baron‐Cohen, Wheelwright, Skinner, Martin, & Clubley, P values or effect sizes alone [e.g., Dienes, 10) > 3 provide firm evidence for the alternative hypothesis  and values under 1 provide evidence for the null . BF10 values can be considered to reflect the likelihood that the alternative hypothesis is more likely to be true than the null hypothesis. Hence, a BF10 of 3 suggests the alternative hypothesis is three times more likely to be true than the null hypothesis. Bayesian analyses were conducted using JASP 0.8.1 [JASP Team, Details of power calculations can be found in Supporting Information. An increasingly used supplement to power analyses and null hypothesis significance testing in general is to calculate a Bayes factor for each key analysis. Bayesian analyses provide an estimation of the relative strength of a finding for one hypothesis over another , which allows a more graded interpretation of the data than is possible using SD) for performance on the experimental and background tasks are presented in Table F = 1105.13, P < .001, ηp2= .84. One‐sample t‐tests showed that overall accuracy  and accuracy (CHR) in the transparent condition was significantly above chance, ts > 21.20, ps < .001, BF10s > 100. However, CHR in the nontransparent condition was significantly below chance, t = 7.54, P < .001 BF10 >100. Finally, participants showed a significant truth bias, t = 8.01, P < .001, BF10 > 100.Means  or animations  mindreading tasks when they were analyzed individually.Associations between each of the key dependent variables on the lie detection task and performance on each of the background measures are presented in Table rp = −.17, P = .01. Controlling for RMIE or animations task performance separately produced the same results as when the composite mindreading score was controlled (ps ≤ .01). Likewise, the association between CHR in the transparent condition and AQ remained significant after controlling for mindreading composite score, rp = −.25, P < .001. Controlling for RMIE or animations task performance separately produced the same results as when the composite mindreading score was controlled (ps ≤ .001).To investigate further the significant association between lie detection accuracy and AQ, two partial correlation analyses were conducted. The association between overall CHR and AQ remained significant after controlling for mindreading composite score, Given that a number of authors have suggested that significant correlates of lie detection ability may not be replicable across studies [e.g., Aamodt & Custer, rp = −.23, P = .02, and Subsample 2, rp = −.27, P = .005. The association between overall CHR and AQ after controlling for score on the mindreading composite measure was significant in Subsample 1, rp = −.22, P = .02, but marginally nonsignificant in subsample 2 when reported one‐tailed, rp = −.13, P = .08. Fisher's Z tests revealed that the difference in the size of these associations in the total sample, subsample 1, and subsample 2 were all nonsignificant . Note that all results were substantively identical when analyses controlled for RMIE and Animations task performance separately .The association between CHR in the transparent condition and AQ after controlling for score on the mindreading composite measure was significant in both Subsample 1, Twenty‐seven adults with ASD and 27 neurotypical comparison adults took part. All participants completed the Wechsler Abbreviated Scale for Intelligence‐II [Wechsler, Participants from each group completed the same versions of the lie detection task and AQ as participants completed in Experiment 1. In addition, participants with ASD completed the Autism Diagnostic Observation Schedule [ADOS; Lord et al., F = 174.72, P < .001, ηp2= .77, and Group, F = 3.94, P = .05, ηp2= .07, and a significant interaction between these variables, F = 8.48, P = .005, ηp2= .14. Figure M = .46, SD = .36) than among comparison participants , t = 3.16, P = .003, d = 0.86, BF10 = 27.86, albeit significantly above chance among both groups, all ts > 6.65, all ps < .001, all BF10s > 100. In contrast, CHR in the nontransparent condition was almost identical among ASD participants  and comparison participants , t = 0.42, P = .67, d = 0.11, BF10 = 0.30, and nonsignificantly below chance in both groups, ts > 6.65, all ps < .001, all BF10s < 0.33. In sum, participants with ASD showed a significant diminution of overall lie detection accuracy and this was particularly large in the transparent condition. Finally, both groups of participants showed a truth bias and there was no significant difference between participants with ASD  and comparison participants  in the size of the bias, t = 0.41, P = .69, d = 0.15, BF10 = 0.29.3A mixed ANOVA was conducted on lie detection CHR, with Group (ASD/comparison) as the between‐participants variable and Condition (transparent/nontransparent) as the within‐participants variable. This analysis revealed significant main effects of Condition, F, 52 = 17A series of bivariate correlation analyses were conducted to explore the association between lie detection ability and ASD traits/features. However, these were exploratory and not necessarily reliable given the small sample size in Experiment 2 relative to that in Experiment 1.n = 54), overall CHR was nonsignificantly associated with AQ , although the correlation was almost identical in magnitude to that observed among participants in Experiment 1 (Table ps > .67). In the ASD group, ADOS was nonsignificantly associated with overall CHR, r = −.03, P = .88, BF10 = 0.28.Among participant groups combined . However, neither of these correlations was significant when explored in each diagnostic group separately . In the ASD group, ADOS was nonsignificantly associated with CHR in the transparent condition .Among participant groups combined, CHR in the transparent condition was associated significantly with AQ  was significantly above chance, replicating previous studies. However, when looking at each condition separately, it became clear that only judgments in the transparent condition were significantly above chance. This was expected, given previous findings of low lie detection accuracy when judging nontransparent individuals [Levine et al., Arguably, the association between lie detection and ASD traits should not be surprising, given that such judgments are fundamentally social in nature and that ASD is at its core a disorder of social functioning and cognition. However, contrary to predictions, this association was not mediated by mindreading ability. Although there are some questions over the ecological validity of the animations and RMIE tasks [e.g., Cook, Brewer, Shah, & Bird, guess no” to the question “Did any cheating occur when the experimenter left the room?” We suggest that such behavioral cues would cause most neurotypical individuals to suspect deceit, but yet participants with ASD in the current study found it difficult to make such an inference when clear behavioral cues were available. Clearly, this difficulty renders individuals with a full diagnosis at risk of manipulation even by transparent individuals whose lies would be readily detectable by neurotypical individuals.Importantly, the results from Experiment 2 were striking and complemented those from Experiment 1. Adults with a diagnosis of ASD showed significantly diminished lie detection ability, relative to closely matched neurotypical participants. The impairment when judging videos of transparent individuals was associated with a large effect size, reflecting the fact that judgments by participants with ASD about transparent individuals were almost half as accurate as those made by comparison participants. This shows that even when people provide clear behavioral cues about their honesty or deceit, individuals with ASD nonetheless have significant difficulty making accurate judgments. For example, a clear verbal indicator of dishonesty is apparent in one of the videos, in which an individual claims not to have cheated when the interviewer asks “Did any cheating occur when the experimenter left the room?” but makes a Freudian slip and answers “no” to the follow‐up question, “Are you telling me the truth, right now?” before correcting himself and saying “I mean, yes”. In another of the videos, an individual responds, “I The underlying reasons for the observed lie detection difficulties in people with ASD are yet to be established. As argued above, they may be attributable to insufficient learning opportunities, which are the consequence of social impairments. Alternatively, there may be ASD‐specific cognitive differences that make deception detection more difficult. For example, the fact that lie detection inherently carries a high executive load  may mean that people with ASD—who often have executive difficulties—may be overloaded.lack of awareness of the behavioral cues associated with truth‐telling and lying, but because the cues themselves are not consistently strong indicators [Hartwig & Bond, Irrespective of the underlying explanation for lie detection difficulties in ASD, it is important to consider whether training individuals with ASD to detect the behavioral indicators of lying  would be beneficial. Notably, such lie detection training has produced limited success in increasing discrimination accuracy among neurotypical adults [e.g., Frank & Feeley,


Platelets undergo structural, biochemical and functional alterations when stored, and platelet storage lesions reduce platelet function and half-life after transfusion. The objective of this study was to evaluate stored canine platelet concentrates with platelet aggregation, flow cytometry and biochemistry assays. Twenty-two bags of canine platelet concentrates were obtained by the platelet-rich plasma method and were assessed on days 1, 3 and 5 after collection. Parameters such as platelet counts, residual leukocytes, platelet swirling, glucose, lactate, pH, CD62P expression (platelet activation), JC-1  and annexin V (apoptosis and cell death) were assessed.3, pCO2, ATP, pH, swirling and mitochondrial function, associated with a significant increase in lactate levels and pO2. At the end of storage pH was 5.9 ± 0.6 and lactate levels were 2.8 ± 1.2 mmol/L. Results of the quality parameters evaluated were similar to those reported in human platelets studies. The deleterious effects of storage were more pronounced in bags with higher platelet counts (> 7.49 × 1010/unit), suggesting that canine platelet concentrates should not contain an excessive number of platelets.Over the five days of storage there was a significant decrease in glucose, HCOQuality parameters of canine platelets under standard storage conditions were similar to those observed in human platelets. Our results have potential to be used for the routine evaluation and quality control in veterinary blood banks. Recent advances in intensive care and oncology have increased the demand for blood components in veterinary medicine. However, there is limited availability of platelet concentrate (PC) because it is difficult to obtain and has a very short storage period. Although some studies evaluating the quality of canine PC obtained by the platelet-rich plasma (PRP) method under routine blood bank processing and storage have been reported –4, the qDuring storage platelets undergo structural, biochemical and functional changes, also referred as platelet storage lesions (PSL), which are caused by a multifactorial process that includes energy consumption, pH decrease, platelet activation and apoptosis , 6. PSL 10/unit on day 1, and it remained stable over time. All PC showed adequate platelet counts, pH, swirling and leukoreduction  (data not shown) at the first day of assessment, in compliance with the Brazilian regulations for human PC [p < 0.001). None of the canine PC units presented aerobic or anaerobic microbiological contamination.Data on the different parameters analyzed on days 1, 3, and 5 of storage are showed in Table human PC . Swirlin2 levels were detected on day 5 compared with days 1 and 3, whereas a significant reduction of pCO2 levels was detected as storage time increased. Glucose concentration gradually decreased whereas lactate levels increased. There was no significant difference in platelet aggregation when ADP and collagen were used (data not show). However, platelet aggregation increased between days 1 and 5 when arachidonic acid was used (p = 0.0082). Percentage of platelets positive for CD61 and CD62P remained stable during storage.ATP levels and pH decreased over the storage period. Higher pOp = 0.0016) and 3 (p = 0.0373) .Mitochondrial membrane potential (Δψm – JC-1 aggregates) values remained stable between days 1 and 3 of storage. A reduction of Δψm was identified on day 5 when compared with days 1 (73) Fig. . No diffρ = − 0.927, p < 0.001), pH and lactate , and lactate and Δψm . There was a positive correlation between glucose and pH , glucose and swirling , glucose and Δψm , and pH and swirling .There was a negative correlation between glucose and lactate  and one with a higher than average platelet count . PC with lower platelet counts presented better quality parameters than those with higher counts on the last day of storage  . Similar storage . Another storage . Our dat storage , 16. Sti storage , 9.The correlations among the studied parameters determined on day 5 are consistent with the alterations suffered by platelets during storage. A significant correlation between pH decrease and increase in MPV and PDW indexes was observed. MPV increase was also correlated with swirling loss, indicating that platelets shape changed from discoid to spherical. The higher lactate concentration on day 5 of storage was correlated with the increase in MPV and strongly correlated with the decreases in swirling and pH. These correlations were expected due to the cause-effect relationship between medium acidity and morphological changes in platelets .2 values by the end of storage period indicates that glycolytic anaerobic metabolism superseded aerobic metabolism despite the presence of oxygen (Warburg effect) [+ ions [The high pO effect) , as conf [+ ions . As gluc [+ ions .Platelet aggregation capacity increased in response to arachidonic acid, whereas no differences were observed in response to ADP or collagen. However, light transmission aggregometry studies in the clinical practice require fresh platelets, with less than 4 h after blood sampling . This isThe percentage of CD61 expression remained stable during storage, indicating that there was no glycoprotein internalization by endocytosis . P-selecSome authors suggest that ΔΨm loss is the most sensitive marker of platelet quality during storage, because it signals the apoptosis process, even before caspase pathway activation or phosphatidylserine exposure –23. We d10 per unit should be used in less than five days, as exceeding this period may negatively affect PC quality and possibly transfusion efficacy.In the clinical setting, high platelet concentrations are desirable to achieve higher transfusion efficiency. Nevertheless, considering that PC with higher platelet counts experience more severe PSL during storage, it is suggested that canine PC stored in TOTM-plasticized bags with counts greater than 7.49 × 10Our study has some limitations. Samples were not evaluated daily and caspase should have been assessed for longer than 5 days of storage.In conclusion, quality parameters of canine platelets under standard storage conditions were similar to those observed in human platelets. Moreover, as the results of the present study are specific of canine PC, they could be used for quality control in veterinary blood banks.n = 12), German Shepherd (n = 4), Giant Schnauzer (n = 4) and English Mastiff (n = 2). All included dogs had updated vaccines and deworming and were considered healthy.Twenty-two dogs aged between 2 and 7-years old, weighing among 30 kg and 60 kg, with no history of receiving transfusion and not taking any medication were selected. The animals were privately owned. Dogs were individually submitted to physical examination, complete blood counts (CBC), blood chemistry profile, and infectious disease screening . Included breeds were Golden Retriever  were collected in bags using a triple-bag closed system . Primary collection bags had sodium citrate as anticoagulant; and citrate, phosphate and dextrose (CPD) solution as an additive solution for red blood cells. Dogs were kept under gentle physical restraint and with no anesthesia for blood collection. After trichotomy and 70% alcohol asepsis, WB was collected by puncturing the jugular vein , using eWB bags were allowed to stand at 22 °C for one hour after collection. The PRP method was applied to obtain the PC as follows: each WB bag was subjected to light centrifugation (1600 X g) for 6 min at 22 °C . PRP was removed using a manual plasma extractor  and then subjected to a second centrifugation (3300 X g) for 8 min at 22 °C. Fluid weight and density 1.026 g/mL) were used to calculate the final volume of the bag  g/mL wer. PC werePC bags were left undisturbed for one hour after preparation, and then were stored at 20–24 °C in a platelet environmental chamber  under constant stirring . Aliquots were removed for analyses using a sampling site coupler previously sterilized with 70% alchool  on days 1, 3, and 5 [Immediately before aliquot sampling, platelet swirling was assessed. This technique consists in the visual evaluation of the PC against a light source to observe platelet movement. Swirling is classified according to a 0–3 scale, where 0 indicates no platelet swirling and 3 corresponds to platelets with very nice cloudy movements , 28. Swi3, pO2 and pCO2 values were determined using a portable blood gas analyzer , according to the manufacturer’s instructions. On days 1, 3, and 5, ATP was extracted [Lactate and LDH concentrations were determined by dry chemistry . Glucose, HCOxtracted  and sampPlatelet activation was determined according to CD62P (P-selectin) expression. About 500,000 platelets were incubated with titrated amounts of anti-human CD61 FITC  and anti-human CD62P PE  monoclonal antibodies for 20 min at room temperature. Samples were resuspended in phosphate buffered saline (PBS) prior to acquisition. Percentages of platelets positive for CD61 and CD62P were recorded.Assessment of Δψm was performed as described elsewhere . BrieflyPhosphatidylserine exposure was assessed with Annexin V. About 500,000 platelets were incubated with ApoFlowEx® FITC Kit  for 15 min at room temperature, according to the manufacturer’s instructions. Samples were washed once and resuspended in PBS before acquisition. The percentage of platelets binding to Annexin V was recorded.CaspACE™ FITC-VAD-FMK  was used to determine caspases involvement in apoptosis induction. About 5,000,000 platelets were incubated with 1 mM of CaspACE™ FITC-VAD-FMK for 20 min at 37 °C. Samples were washed once and resuspended in 1 mL of PBS before acquisition. Percentage of platelets presenting active caspase was recorded.All flow cytometry experiments were performed on a BD FACSCalibur flow cytometer  using CellQuest™ Pro software, after daily quality control procedures. At least 30,000 events were acquired for each analysis. All flow cytometry data were analyzed using FCS Express 5 software .Platelet aggregation test was performed with light transmission aggregometry  using ADP (5 μM), collagen (5 μg/mL), and arachidonic acid (1 μM) as inducers.p < 0.05. All data were analyzed with GraphPad Prism 6.0 Software .Data analysis was carried out with ANOVA for repeated measurements, and means were compared with Duncan’s test. Correlations among the evaluated parameters were tested with Pearson’s correlation test. Results were considered significant at This study was approved by the Ethics Committee on the Use of Animals of the Universidade Federal do Rio Grande do Sul (CEUA/UFRGS) under protocol #26102. The owners of all evaluated dogs provided written informed consent before inclusion in the study.


Age‐related increased adiposity is an important contributory factor in the development of insulin resistance (IR) and is associated with metabolic defects. Caloric restriction (CR) is known to induce weight loss and to decrease adiposity while preventing metabolic risk factors. Here, we show that moderate 20% CR delays early deleterious effects of aging on white and brown adipose tissue  function and improves peripheral IR. To elucidate the role of CR in delaying early signs of aging, young (3 months), middle‐aged (12 months), and old (20 months) mice fed al libitum and middle‐aged and old mice subjected to early‐onset CR were used. We show that impaired plasticity of subcutaneous WAT (scWAT) contributes to IR, which is already evident in middle‐aged mice. Moreover, alteration of thyroid axis status with age is an important factor contributing to BAT dysfunction in middle‐aged animals. Both defects in WAT and BAT/beige cells are ameliorated by CR. Accordingly, CR attenuated the age‐related decline in scWAT function and decreased the extent of fibro‐inflammation. Furthermore, CR promoted scWAT browning. In brief, our study identifies the contribution of scWAT impairment to age‐associated metabolic dysfunction and identifies browning in response to food restriction, as a potential therapeutic strategy to prevent the adverse metabolic effects in middle‐aged animals. In parallel with these alterations, a low grade of inflammation has also been described in several tissues associated with aging  is the most efficient intervention to delay the deleterious effects of age‐related metabolic diseases   and/or brown AT (BAT), the contribution of specific WAT depots, and the relevance of differentiation/trans‐differentiation to beige AT  is differentially affected at middle age, and whether moderate CR results in beneficial metabolic effects regulating the functionality of these AT depots. We show that several metabolic alterations of old animals are already being developed in middle‐aged animals. These alterations include development of IR, altered WAT and BAT plasticity, as well as alteration of thyroid axis status, which can be mitigated, at least to some extent, by moderate and long‐term CR.22.1First, we investigated the effects of aging on body weight (BW), glucose homeostasis, and insulin response Table  in male Although no significant differences were found in nonfasting glucose levels among mice from different ages, fasting glucose levels significantly increased in older mice respect to both young and middle‐aged mice. Nonfasting and fasting insulin levels increased from 3 to 12 m and maintained high thereafter  did not present significant differences between young and middle‐aged animals, but 20‐m mice showed a significant glucose intolerance compared to 3‐m and 12‐m animals  showed a decrease of the AUC, but this change did not reach significance compared to 20‐m mice. In contrast, ITT showed that CR significantly decreased the AUC in both 12‐m and 20‐m animals, suggesting that CR ameliorates IR at both ages Figure a. The slta Table b, led usAL‐fed animals progressively increased their BW over the study period of 12 months, animals subjected to CR presented an initial drop in BW, and this was followed by a slow recovery of BW gain that remained significantly lower than that of AL‐fed animals  activity in BAT, which is related to T3 activation, was lower in 12‐m than in young animals, whereas it was higher, but not significantly, in 12mCR mice Figure b. Consis2.5AL, before cold challenge; however, core temperature was lower in 12‐m compared to 3‐m mice after the cold challenge. CR decreased body temperature, which decreased even further after cold challenge  in any experimental group. Core temperature was similar in middle‐aged and young mice fed 18F‐FDG uptake in interscapular BAT as a measurement of BAT metabolic activity  were significantly higher in cold‐exposed animals under CR than in 12‐m mice, suggesting that these animals may have presented a better response of BAT. Of interest, the expression of Cd36  show more difficulties to maintain glucose homeostasis, as deduced by the significant increased fasting glycemia, HOMAIR, and the deterioration of pancreatic islets, confirming the progressiveness of this age‐associated alteration. CR significantly improved insulin global sensitivity in middle‐aged animals. Although CR also seemed to ameliorate IR in older animals, this was achieved to a lesser extent, as deduced by the lack of significance in the recovery of several parameters such as the decrease of fasting insulin, the increase of the slope in the ITT, and the HOMAIR data, in spite of a longer period of CR (7 months in 12‐m mice vs. 17 months in 20‐m mice). These findings are in line with previous data in which older animals showed a poorer recovery of IR after three months of CR  SV129 mice already show peripheral IR, as deduced by HOMAWAT is a critical regulator of systemic energy homeostasis, and its remodeling under pathophysiological and challenging conditions is linked to metabolic complications such as IR , they start using fat faster than AL animals, demonstrating metabolic flexibility in this phase.We also studied the physiological response of BAT to cold exposure analyzing glucose uptake by PET. A lower Cidea, Cox7a1, and Cox8b were also described in scWAT from 12mCR animals in the same article. We speculate that increased thyroid function induced by CR and the stimulated β‐adrenergic pathway during cold‐induced thermogenesis could induce BAT to significantly upregulate genes and secrete molecules to promote browning of scWAT in CR middle‐aged mice. Nevertheless, the scarcity of fuel and energy reservoir may limit some of the ameliorating effects of CR on aging metabolism and, particularly, the thermogenic response of BAT under a long‐term cold exposure. The fact that CR has a strong effect on adiposity while improves several metabolic alterations, points to adiposity as one of the causes of the deleterious effects of aging. This also agrees well with the deleterious effects on metabolic parameters and lifespan that increased adiposity, by means of genetic alterations and/or by high‐fat diets, induces in many animal models. However, in spite of the improvement, CR does not fully reverse metabolic alterations suggesting that metabolic alterations associated with aging cannot be solely explained in terms of adiposity. Further work with adiposity‐matched animals would be necessary to clarify the precise role of adiposity in the aging process.It is worth noting the effect of aging and CR on scWAT browning. In parallel to a possible improved BAT function in CR animals, we observed the emergence of beige adipocytes with increased UCP‐1 and TH expression in scWAT, but not in eWAT. This was more evident in cold‐induced thermogenesis, even without cold acclimation as previously described . Male 129S2/SvPasCrl mice (Charles River Laboratories) were used for all studies and were housed individually in climate‐controlled quarters (21ºC) with a 12‐hr light/dark cycle. Food  and water were available ad libitum unless otherwise stated. In some experiments, mice were randomly assigned to undergo 20% CR from 3 months of age, as described earlier  and insulin tolerance test (ITT) were performed as described .Animals were placed in a comprehensive laboratory animal monitoring system . Energy expenditure, the respiratory exchange rate (RER), and locomotor activity were calculated according to the protocols provided by the manufacturer ). The estimate of IR by HOMAIR score was calculated with the following formula: fasting serum insulin (µU/ml) × fasting plasma glucose (mmol/L)/22.5 as described  following the user's manufacturer. Serum adipokines and cytokines were determined with the Bio‐Plex platform  and 1 mM propylthiouracil for 1 hr at 37º C. Results are expressed in femtomoles per hour per milligram of protein.Triiodothyronine (T3) and thyroxine (T4) were quantified by specific and highly sensitive radioimmunoassay in plasma and BAT, after appropriate extraction and purification of tissue extracts as described . Mice were maintained under isoflurane anesthesia during injection, uptake, and imaging. All animals had a CT scan before the PET scan for attenuation correction and anatomical delineation of PET images. All PET images were calibrated in Bq/cm3 and converted to standard uptake value (SUV) units . Thirty‐minute‐long PET scans were acquired 60 min after injection of 4.6Bouin‐fixed samples were gradually dehydrated and embedded in paraffin, cut to 4 µm, and stained with Harris hematoxylin  or incubated with primary antibody UCP‐1 (Abcam), MCP‐1 , insulin (Thermo Fisher), and TH (Millipore) for immunohistochemistry as previously reported  using ImageJ 1.45  and Adiposoft software (http://fiji.sc/Adiposoft).To measure the area of adipocytes, four nonconsecutive sections per animal were quantified (Protein lysates from mouse tissues were electrophoresed in SDS‐PAGE gels (10%), and Western blotting was performed as described before . Results were analyzed with a Shapiro–Wilk normality test. After that, data were analyzed with a Kruskal–Wallis test to check the statistical differences among experimental groups. Finally, experimental groups were compared by Mann–Whitney's nonparametric test. A p value < 0.05 was considered significant.All data are presented as mean ± None declared.P.C, Y.V., M.J.O., and G.M.G. conceived the study, researched data, and wrote the manuscript, which was then read and approved by all the co‐authors . M.R., M.J.O., and M.L. contributed to discussion, and reviewed and edited the manuscript. A.I.L. and C.M. reviewed the manuscript. P.C., M.R., and G.M.G. are the guarantors of this work. The authors declare that there is no duality of interest associated with this manuscript. Click here for additional data file.


Clinical and biologic phenotypes of sepsis are proposed in human studies, yet it is unknown whether prognostic or drug response phenotypes are present in animal models of sepsis. Using a biotelemetry-enhanced, murine cecal ligation and puncture (CLP) model, we determined phenotypes of polymicrobial sepsis prior to physiologic deterioration, and the association between phenotypes and outcome in a randomized trial of prompt or delayed antibiotics and fluids.n = 118) underwent biotelemetry-enhanced CLP, and we applied latent class mixed models to determine optimal number of phenotypes using clinical data collected between injury and physiologic deterioration. In cohort 2 (N = 73 mice), inflammatory cytokines measured at 24 h after deterioration were explored by phenotype. In a subset of 46 mice enrolled in two trials from cohort 1, we tested the association of phenotypes with the response to immediate (0 h) vs. delayed (2 to 4 h) antibiotics or fluids initiated after physiologic deterioration.We performed a secondary analysis of male C57BL/6J mice in two observational cohorts and two randomized, laboratory animal experimental trials. In cohort 1, mice (N = 97) demonstrated a shorter time to deterioration (mean SD 7.3 (0.9) vs. 9.7 (3.2) h, p < 0.001) and lower heart rate at 7 h after injury (mean (SD) 564 (55) vs. 626 (35) beats per minute, p < 0.001). Overall mortality was similar between phenotypes (p = 0.75). In cohort 2 used for biomarker measurement, class 2 mice had greater plasma concentrations of IL6 and IL10 at 24 h after CLP (p = 0.05). In pilot randomized trials, the effects of sepsis treatment (immediate vs. delayed antibiotics) differed by phenotype (p = 0.03), with immediate treatment associated with greater survival in class 2 mice only. Similar differential treatment effect by class was observed in the trial of immediate vs. delayed fluids (p = 0.02).Latent class mixture modeling derived a two-class model in cohort 1. Class 2 (We identified two sepsis phenotypes in a murine cecal ligation and puncture model, one of which is characterized by faster deterioration and more severe inflammation. Response to treatment in a randomized trial of immediate versus delayed antibiotics and fluids differed on the basis of phenotype. Not all septic patients present the same , and theTo support this agenda in precision treatment, recent adult and pediatric human cohort studies propose biologic and clinical phenotypes of sepsis that are both prognostic of outcome and predictive of treatment response –11. Yet,Latent class mixture modeling is an approach to find the best fitting, optimal number of subclasses in longitudinal data, independent of the relationship with outcome. Latent class-based methods are applied in patients with acute respiratory distress syndrome (ARDS) to identify subphenotypes that are predictive of response to fluid management strategies . We sougAll experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (PRO13021581) in accordance with guidelines established by the National Institutes of Health.The primary objective of this study was to determine the feasibility of identifying distinct murine sepsis phenotypes using clinical data from biotelemetry-enhanced cecal ligation and puncture (CLP) prior to physiologic deterioration. This period was chosen to mimic the clinical patient course, physiology, and treatment responses found during the early phases of sepsis. We described clinical outcomes of phenotypes, biomarkers of the host response, and tested the association of phenotypes with differential treatment effects in randomized trial of immediate versus delayed antibiotics and fluids.Male C57BL/6 mice  aged 8 to 12 weeks (mass 25–30 g) were used for all experiments. Mice were housed in specific pathogen-free rooms under 12-h light/12-h dark conditions with an ambient temperature of 23 °C ± 1 °C. Mice were allowed to acclimate to their new surroundings for 1 week prior to any experimentation. Animals were given ad libitum access to water and LabDiet Prolab Isopro RMH 3000 diet pellets . To account for circadian variation, we initiated experiments in the morning between 0700 and 1000.A 1-cm ligation, 21-gauge double puncture model of cecal ligation and puncture (CLP) under anesthesia was performed as previously described . At the We previously developed and validated criteria that define acute physiologic deterioration after cecal ligation and puncture (CLP): (1) 10% decline of heart rate from its peak value and (2) a scaled 10% drop in core temperature calculated as 10% × (peak core temperature − 25 °C) . After CN = 73), heparinized blood was drawn at 24 h after CLP. Blood was centrifuged at 2000g for 15 min, and the plasma was frozen at − 80 °C for future analysis. Plasma concentrations of IL-6, IL-10, and TNF-α were analyzed by ELISA .In a separate cohort of mice prepared under same conditions for biomarker sampling  dose (25 mg/kg) of imipenem/cilastatin administered in a small volume of sterile 0.9% saline (0.15 mL) to minimize lavage of the peritoneal cavity or any potential contribution to additional fluid resuscitation. Fluid resuscitation consisted of 30 mL/kg 0.9% normal saline, injected subcutaneously.We determined murine phenotypes using clinical data prior to physiologic deterioration with latent class mixture models. We used three class-defining variables: (1) hourly measurements of heart rate (beats per minute), (2) hourly measurement of temperature (degrees Celsius), and (3) the total time (min) from CLP to acute physiologic deterioration. Latent class mixture models address issues that arise when longitudinal data is too complex for a linear mixed model framework. These models extend linear mixed models in two key ways: (i) several longitudinal outcomes may be collected and (ii) non-observed heterogeneity may exist in the population. We used a linear transformation to link the latent process to the scale of the observed longitudinal variables. We optimized the number of classes using three criteria: (i) Bayesian Information Criterion (BIC), (ii) clinical significance, and (iii) class size. We prioritized the lowest BIC, followed by clinical significance and adequate class size.We compared clinical and biomarker characteristics between phenotypes using ANOVA, specifically to test for a difference in mean heart rate and temperature at 5 and 7 h after CLP and inflammatory cytokines at 24 h. We compared the time to death and time to deterioration from CLP (in min). The Bonferroni correction was used to adjust for multiple comparisons.To determine if there was a differential treatment effect between phenotypes, we used Kaplan Meier curves, created separately for mice randomized to treatment at immediate vs. delayed time point. We compared survival using the Wilcoxon test with the Bonferroni correction to adjust for multiple comparisons. We tested for class by treatment time point interaction using Cox proportional hazards models. The proportional hazards assumption was checked with a log-log survival plot. Gray’s model was used as a comparison analysis when the proportional hazards assumption was violated.α = 0.05. Statistical analysis was conducted using Stata 14 , R version 3.2.3 package LCMM and SAS 9.3 .All tests were two-sided with The median time from CLP to physiologic deterioration was a 452 min  in cohort 1 and a median 446 min  in cohort 2 for biomarker measurement. The median time to death was 3124 min  in cohort 1.k = 2) model best balanced optimal fit, clinical significance, and class size. Specifically, the Bayesian information criterion was lowest in k = 3  and k = 4  compared to k = 2 (11372), suggesting more model complexity provided better optimal fit. However, the k = 3 and k = 4 models produced classes with two mice, respectively. Although the decrease in BIC would favor the inclusion of additional classes, we retained a two-class model due to the small number of subjects and lack of clinical significance in more complex models. We refer to final model members as class 1  and class 2 . The latent class probability of class membership in class 1 was > 0.8 in 86% of mice compared to 91% of members in class 2. These probabilities suggest moderate to strong likelihood of class assignment.In cohort 1, latent class mixture models suggested a two-class (p < 0.01). To illustrate the behavior of latent class clinical characteristics, Fig. p < 0.01). We observed no difference in overall time to survival among mice in class 2 vs. class 1 (Table p = 0.75).As shown in Table N = 73), we derived similar phenotypes using latent class mixture models (Table p = 0.055) and IL10 (mean (SD) 10,588  vs. 4418 (7499), p = 0.046) were greater in class 2 than in class 1.In cohort 2 for biomarker measurement N = 7, we derip = 0.04 for overall comparison of arms, Cox p value for interaction = 0.03, Gray’s p value = 0.76, Fig. p = 0.02 across arms). In class 1, mice randomized to receive early antibiotics had no difference in survival compared to those who received delayed antibiotics (Wilcoxon p = 1.0 across arms). A similar analysis among 20 mice randomized to receive immediate vs. delayed fluids reached statistical significance for treatment by class interaction .In trial 1, the 8 mice randomized to receive immediate antibiotics compared to 16 mice who received delayed antibiotics (≥ 2 h) had significantly different survival  compared to k = 3  or k = 4 . Second, to determine the advantage of understanding predictive phenotypes from unsupervised models prior to deterioration compared to a different measure of trajectory, we stratified mice by above or below median time to deterioration (min)  to those below the median time to deterioration (N = 27) had no evidence of statistical interaction for early antibiotics or fluids . In this example, the benefit of immediate antibiotics was similar in both groups.First, we derived optimal class number after excluding the time to deterioration variable. When using only heart rate and temperature as class-defining characteristics, we confirmed statistically that  2 BIC = 1,394 comIn this biotelemetry-enhanced murine model of polymicrobial sepsis, we demonstrate the feasibility of deriving clinical sepsis phenotypes prior to overt physiologic deterioration. The evidence suggests there are two phenotypes with different trajectory of clinical characteristics and time to deterioration. Most importantly, phenotypes were predictive of response to immediate versus delayed antibiotic and fluid treatment in small randomized trials. These data suggest that clinical sepsis phenotypes may be present in a commonly used animal model of sepsis and serve as a platform to explore heterogeneity of treatment effects for current and novel therapeutics.There are several possible explanations for the observed heterogenous response. First, previous studies report differential microbial clearance in CLP models of sepsis . Second,Klebsiella pneumoniae pneumonia: the mouse clinical assessment for sepsis (M-CASS) [Prior work attempted to measure severity among mice with polymicrobial sepsis after CLP, but did not explore data for unsupervised clustering models. The murine sepsis score (MSS) is comprised of seven behavioral and physical characteristics that identify phenotypes representative of progressive stages in the evolution of the septic response . It was (M-CASS) . HoweverInflammatory cytokines may also stratify sepsis in murine models. For example, IL6 measured 6 h post-CLP and dichotomized at a threshold of 2000 pg/mL, effectively classified animals as non-survivors vs. survivors: 90% vs. 30% mortality . Many otA related question is whether or not clinical murine phenotypes have face validity? The class 1 mice were more “fit” insofar that they appeared more tolerant to, or rather, better able to compensate for, the sepsis of CLP. Specifically, these mice exhibited more moderate vital sign changes, delayed clinical deterioration, and less inflammation. In contrast, the predictive class 2 deteriorated quickly, had greater inflammation, and significantly greater perturbation in vital sign parameters. The human correlate to these features are readily identified in multiple human cohort studies, where a subset of septic patients rapidly progress to shock and display unremitting inflammation found on serial cytokine measurements , 24. On Recent studies in human sepsis also identify a hyperinflammatory subset, similar to our murine class 2 . In ARDSWhen sepsis phenotypes were determined prior to enrollment in murine randomized trials, we found heterogeneity of treatment effect for broad-spectrum antibiotic therapy and fluids administered at the time of clinical deterioration. Only mice in class 2, which were the less fit, hyperinflammatory group, was there a significantly different survival with immediate vs. delayed therapy. Prior work in large human cohorts has suggested a similar interaction between illness acuity and prompt antibiotic therapy. For example, Liu et al. report that for there was no significant association between delay of antibiotic therapy for more than 3 h compared to before 3 h among a subset of patients with sepsis but no organ dysfunction . Yet, thThe heterogeneity in response to fluid therapy by class is also not surprising given the conflicting findings in human studies of fluid resuscitation in sepsis. A randomized trial in sub-Saharan Africa found greater mortality among septic patients who were aggressively treated with protocol-guided intravenous fluid vs. usual care , similarThere are several imitations to this analysis. First, the data derive from a small sample of male mice, which may be underpowered in both the tests of interaction in the randomized trials, proportional hazards assumptions in Cox models, and biomarker analyses. Second, latent class mixture models converged using hourly measurements of heart rate and temperature, but more complex repeated measures of entropy or variation could refine phenotype derivation. Notably, class number was similar in sensitivity analyses with reduced model variables. Third, we recognize that the time to antibiotic treatment effects may be subject to the choice of treatment. We utilized single dose imipenem due to its broad antimicrobial activity and standardized use in CLP models, although data suggest alternatives . This drIn conclusion, we identified two phenotypes before clinical deterioration in murine sepsis due to CLP, one of which is characterized by faster deterioration and more severe inflammation. Response to treatment in a randomized trial of immediate versus delayed antibiotics and fluids differed on the basis of phenotype.Additional file 1: Murine sepsis phenotypes and differential treatment effects in a randomized trial of prompt antibiotics and fluids. Supplementary figures  with accompanying legends.


RNA network, circular RNAs (circRNAs) with distinct properties and diverse pathophysiological functions are being increasingly delineated. CircRNAs play roles at the epigenetic, transcriptional and posttranscriptional regulatory levels. Major studies have focused on their functions as efficient microRNA sponges. The validated number of endogenous circRNAs involved in hepatocellular carcinoma (HCC) continues to increase. Altered circRNA expression is associated with HCC occurrence, invasion, and metastasis. Moreover, the aberrant expression of circRNAs is also significantly related to HCC tumor stage, size, differentiation and metastasis. Because they are exceptionally stable, highly conserved and have tissue‐specific expression patterns, some circRNAs, including hsa_circ_0004018, hsa_circ_0003570, and hsa_circ_0005075, may be potential markers for the diagnosis of HCC. We herein summarize the current knowledge of HCC‐associated circRNAs and present their implications for carcinogenesis and their potential value as diagnostic and prognostic biomarkers. Finally, we discuss the future directions of studies on HCC‐associated circRNAs.At present, as hotspot members of the noncoding  When diagnosed at the early stage, HCC patients can achieve marked advances in life expectancy; however, when diagnosed at the advanced stage with metastasis, patients have dismal prognoses, even if they are treated with multikinase inhibitor sorafenib or regorafenib.2CircRNAs are formed through lariat‐circulating or back‐splicing events from all regions of the genome and are derived mostly from exons; however, in rare cases, circRNAs are derived from intergenic, intronic regions, antisense or untranslated region (UTR) regions. The terms circRNA, EIciRNA and ciRNA are used to describe circRNAs originating from the exon, exon and intron, and intron, respectively Figure . CircRNAHow are circRNAs degraded or cleared? Scientists have recently focused on exosomes. Exosomes, which are double‐membrane vehicles containing different functional molecules, such as proteins, microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circRNAs, may mediate cell‐cell communication in a paracrine or endocrine manner33.1cis‐acting manner or a trans‐acting manner  and Polyadenylate‐binding protein‐interacting protein 2 (PAIP2) transcription in a cis‐acting manner.Intron‐containing circRNAs, such as ciRNAs and EIciRNAs, are abundant in the nucleus. How ciRNAs and EIciRNAs are restricted to the nucleus remains vague. These circRNAs may regulate the expression of their host genes in either a r Figure A. Some c3.2CDR1as) is the sponge of miR‐7 in the embryonic zebrafish and has been reported to be related to brain dysfunction and many cancers, such as HCC and gastric cancer.VEGFA), a key member of the VEGFA/VEGFR2/Ras/ERK signaling pathway. Increased circ‐UBAP2 promotes cancer cell growth and inhibits apoptosis both in vitro and in vivo by sequestering miR‐143, thus enhancing its target antiapoptotic Bcl‐2 expression.CDK6) may act as a competing endogenous RNA (ceRNA) by sequestering miR‐106b.HIPK2 gene exon 2 can affect the distinct roles of its target in cell autophagy by sponging miR‐124 (miR124‐2HG), thus regulating sigma nonopioid intracellular receptor 1 (SIGMAR1/OPRS1) expression in astrocyte cells.HIPK3 gene exon 2 can abundantly sponge miR‐558 to suppress the expression of heparanase (HPSE) in bladder cancer and can sponge endogenous miR‐30a‐3p in diabetes mellitus‐associated retinal vascular dysfunction.CircRNAs harbor well‐conserved canonical miRNA response elements (MREs), suggesting that some circRNAs act as miRNA sequesters or sponges.3.3As most circRNAs originate from exons, are located in the cytoplasm, might contain internal ribosome entry site (IRES), and include an open reading frame (ORF), they may be efficiently translated3.4CDR1as, which is associated with Argonaute (AGO) proteins in a miR‐7‐dependent manner.QKI knockdown and fluctuates during the human epithelial‐mesenchymal transition (EMT) process. The circRNA derived from the MBL locus (circ‐MBL) harbors binding sites for the MBL protein itself and prevents MBL protein from binding to other targets when tethered to the circ‐MBL.PABPN1 locus (circ‐PABPN1) harbors binding sites for ELAV‐like protein 1 (HuR) and hence prevents HuR from binding to PABPN1 mRNA and lowers its translation.C‐MYC in the nucleus. Hsa_circ_0004214 enhances C‐MYC binding to the promoters of hypoxia‐inducing factor (HIF)‐1α, cell division cycle 25 homolog A (Cdc25a), ETS domain‐containing protein (ELK1), and JUN, thus promoting cell proliferation, invasion and colony formation.CircRNAs may be used to bind, store, or sequester protein molecules. Most circRNAs may interact with RBPs Figure D. With a3.5MDM2) is an important negative regulator of the p53 tumor suppressor, and MDM2 protein functions as both an E3 ubiquitin ligase that recognizes the N‐terminal transactivation domain (TAD) of the p53 tumor suppressor and as an inhibitor of p53 transcriptional activation. Circ‐Foxo3 can bind to MDM2 and p53, promote MDM2‐induced p53 ubiquitination and subsequent degradation, and thus induce tumor cell apoptosis.Those circRNAs that harbor binding domains for enzymes and their substrates are likely to function as scaffolds to connect two or more proteins Figure E. To dat4In accordance with the pivotal role in epigenetic and genetic regulation mentioned above, the deregulation of circRNA expression has been reported in several diseases, such as cancers, neurological disorders, heart disease, diabetes, and atherosclerosis.4.1SHPRH gene is significantly downregulated in HCC samples and correlated with tumor size and the occurrence of tumor embolus.The early diagnosis of HCC in patients particularly is important. Due to their complex tissue‐ or cell‐type and developmental stage‐specific patterns, resistance to ribonucleases, such as exonucleases and RNase R, and longer half‐lives, circRNAs are presumed to be ideal biomarkers.Single‐nucleotide polymorphisms (SNPs) may affect the susceptibility and clinical outcome of HCC. Guo et alExosomes may serve as good candidates for liquid biopsy, particularly for monitoring and predicting tumor occurrence and metastasis.4.2CDR1as‐overexpressing HepG2 cells. Our team has reported that hsa_circ_0005986 functions as an effective sponge for miR‐129‐5p, thereby regulating Notch1 expression.HIPK3 (hsa_circ_0000284) is significantly upregulated in liver cancer compared with matched normal tissues and directly binds to miR‐124, thereby upregulating Interleukin 6 receptor (IL6R) and Homeobox protein DLX2 expression. Chen et alAs with other tumors, in HCC, an increasing number of studies has shown that circRNAs are good candidates for miRNA sequestration and the subsequent fine‐tuning of target gene expression.Characterized by mitochondrial dysfunction, growth arrest, and apoptosis, fatty liver disease is a high‐risk factor for HCC. Gou et al4.3CDR1as, a risk factor of hepatic microvascular invasion, is inversely correlated with the activities of miR‐7.CDR1as might be involved in the EMT process of HCC.The epithelial‐mesenchymal transition (EMT) is defined as the process although which epithelial cells lose their differentiated properties and obtain mesenchymal characteristics. EMT plays important roles in HCC occurrence and progression.4.4As mentioned above, circRNAs are intimately correlated with HCC carcinogenesis. What is the goal of circRNA application in HCC treatment? Hepatitis C virus (HCV) is an important latent carcinogenic factor for HCC. MiR‐122 is a validated dispensable molecule for the HCV cell‐life cycle. In recent times, Jost et al4.5http://gb.whu.edu.cn/CSCD) is a comprehensive database for exploring cancer‐specific circRNAs.An increasing number of circRNAs is involved in the deregulation of cancer pathophysiological processes, and these circRNAs have shown great potential in cancer diagnosis, prognosis, and therapy.None declared.


Participants received VIT‐2763 5/15/60/120/240 mg or placebo in the SAD phase and VIT‐2763 60/120 mg once daily, VIT‐2763 60/120 mg twice daily, or placebo for 7 days in the MAD phase. Seventy‐two participants completed treatment. VIT‐2763 was well tolerated and demonstrated a similar safety profile to the placebo. There were no serious or severe adverse events, or discontinuations due to adverse events. VIT‐2763 absorption was relatively fast, with detectable levels 15 to 30 minutes post‐dose. Following multiple dosing there was no apparent change in absorption and accumulation was minimal. Mean elimination half‐life was 1.9 to 5.3 hours following single dosing, and 2.1 to 3.8 hours on Day 1 and 2.6 to 5.3 hours on Day 7, following repeated dosing. There was a temporary decrease in mean serum iron levels with VIT‐2763 single doses ≥60 mg and all multiple doses; mean calculated transferrin saturation (only assessed following multiple dosing) also temporarily decreased. A shift in mean serum hepcidin peaks followed administration of all iron‐lowering doses of VIT‐2763. This effect was less pronounced after 7 days of multiple dosing (aside from with 120 mg once daily). These results support the initiation of clinical studies in patients with non‐transfusion‐dependent thalassemia and documented iron overload due to ineffective erythropoiesis.Restriction of iron availability by ferroportin inhibition is a novel approach to treating non‐transfusion‐dependent thalassemia . This first‐in‐human, Phase I study ( Thus, erythropoietic demand for iron is a primary regulator of iron absorption.Phenotypes of thalassemia and their severity can be characterized according to dependence on blood transfusions. Patients are conventionally described as having transfusion‐dependent thalassemia  when they have a life‐long requirement for regular transfusions, which results in iron overload.The clinical management of β‐thalassemia generally involves blood transfusions, iron chelation, and treatments to alleviate symptoms of iron overload.The aim of this first‐in‐human, Phase I study (EudraCT no. 2017‐003395‐31) was to assess the safety and tolerability, and to determine the pharmacokinetics (PK) and pharmacodynamics (PD), of single‐ and multiple‐ascending oral doses of VIT‐2763 in healthy volunteers.22.1Healthy male or female volunteers aged between 18 and 65 years were eligible. Exclusion criteria comprised any history of iron storage diseases; history or clinical findings of iron utilization disorders; known hemoglobinopathy; use of intravenous iron therapy, erythropoietin stimulating agent therapy and/or blood transfusions in the previous 3 months, and/or excessive oral iron or oral iron‐containing products in the previous 4 weeks. A complete list of inclusion and exclusion criteria is provided in 2.2This Phase 1 study was a randomized, double‐blind, placebo‐controlled, parallel‐group, dose‐escalation study, and comprised a single‐ascending dose (SAD) phase and a multiple‐ascending dose (MAD) phase. The study was conducted at a PRA Health Sciences Early Development Services study site in Groningen, Netherlands, according to the principles of the Declaration of Helsinki. The study was also conducted according to the International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Guidelines, following approval from the appropriate Independent Ethics Committee. All participants provided written informed consent.max ≥3805 ng/mL; equivalent to the no observed effect level in dogs) dosing at that level or higher would not continue.Participants were admitted on Day −1 and received VIT‐2763 or placebo the following day (Day 1 in the SAD phase) or for the next 7 days (Day 1 to Day 7 in the MAD phase). Participants remained hospitalized for assessments up to and including Day 4 or Day 10 in the SAD and MAD phases, respectively, returning for a final study assessment 3 days later. Participants were assigned a randomization number prior to dosing, according to a code generated by the Biostatistics Department of PRA Health Sciences. In the SAD phase, sequential, ascending cohorts received single oral doses of VIT‐2763  or matching placebo. Sentinel dosing was used, whereby the first two participants in each SAD cohort were randomized to receive either VIT‐2763 or placebo. Dosing of the remaining participants at the same dose level occurred at least 48 hours later, following a safety evaluation of the sentinel participants. Escalation to the next dose level occurred following data review by the Safety Review Committee. In the MAD phase, sequential, ascending cohorts received multiple oral doses of VIT‐2763  or matching placebo for 7 days. Dosing in the MAD phase followed an assessment of data from the first four cohorts of the SAD phase. Subsequent multiple‐dose cohorts were initiated once data from the preceding SAD cohort had been evaluated, along with cumulative data from the preceding multiple‐dose cohorts. Starting and maximum doses, which were based on preclinical toxicology studies and physiologically based PK/PD modeling and simulation, were in accordance with European Medicines Agency (EMA) guidelines.2.3Primary endpoints were safety related and comprised the incidence of adverse events (AEs) and serious AEs, which were coded according to Medical Dictionary for Regulatory Activities code (version 21.0), changes from baseline in vital signs, clinical laboratory tests , twelve‐lead electrocardiogram (ECG), cardiac telemetry, and physical examinations. The severity of AEs and relationship of AE to study drug were also assessed. Secondary endpoints were VIT‐2763 PK and PD. A complete list of endpoints is included in 2.4As this was a first‐in‐human study of VIT‐2763, no formal sample‐size calculation was performed. The PK and PD analyses were performed on the full analysis set (FAS), comprising all participants randomized to treatment, who received ≥1 complete dose and had ≥1 post‐baseline PK and PD assessment. The PK and PD analyses were repeated using the per‐protocol set, comprising all participants who fulfilled FAS criteria and had no major protocol violations. The safety set comprised all randomized participants who had received ≥1 dose of study medication. Safety, PK and PD statistical analyses were descriptive. Continuous variables were presented as mean, SD, SE or median, range and 95% confidence interval, as appropriate; categorical and ordinal variables were presented as frequencies and percentages. Results are presented for each VIT‐2763 dose cohort; data for participants receiving placebo were pooled across cohorts. All statistical analyses were performed using SAS Version 9.3 or higher and with WinNonlin Version 6.3 or higher. There was no imputation of missing values, except missing start and end times of AEs, and PK concentrations below lower limit of quantification (LLOQ).2.5https://www.clinicaltrialsregister.eu at the end of October 2019. Detailed inclusion criteria, study endpoints, the most common AEs and all reported AEs are included in this publication.The key aspects of the study protocol and fully anonymized data were submitted to the EudraCT clinical trial registry at 33.12 in the SAD cohorts, and from 21.8 to 24.6 kg/m2 in MAD cohorts. Despite differences in the proportions of female to male participants, mean baseline levels of serum iron, calculated transferrin saturation, transferrin and hemoglobin were generally comparable across treatment cohorts in both the SAD and MAD phases; during the MAD phase, median  values for hemoglobin were similar between treatment cohorts.A total of 72 participants were randomized and completed treatment. The number of participants assigned to each dose level in the SAD and MAD cohorts is shown in 3.2In the SAD cohorts, 23 AEs occurred in 15 out of 29 VIT‐2763‐treated participants (51.7%) and nine occurred in five out of nine placebo‐treated participants (55.6%). In the MAD cohorts, 75 AEs occurred in 24 out of 26 VIT‐2763‐treated participants (92.3%), and 21 occurred in five out of eight placebo‐treated participants (62.5%). Headache was the most commonly reported AE overall, occurring in 18.4% and 26.5% of participants in the SAD and MAD cohorts, respectively, and, at a similar frequency in the VIT‐2763 and placebo groups. The most common AEs and a complete list of all AEs are presented in 3.3max ranged from 0.50 to 3.00 hours and the geometric mean elimination T1/2 was 1.88 to 5.33 hours  and the received dose divided by the participant's body weight was assessed using scatter plots with a regression line. The R2 and P value suggest that the linear model relating PK parameters to dose/weight ratio fits well to the data .Unlike VIT‐2763, which is given orally, other agents in development are administered intravenously or subcutaneously. Parenterally administered drugs avoid first‐pass metabolism, can have a faster onset of action than orally administered medications, and may be beneficial if the patient is unlikely to adhere to self‐administered capsules or tablets. However, oral formulations may be more suitable for a pediatric population and do not require administration by a healthcare professional, which is costly as well as burdensome and inconvenient for patients. VIT‐2763 has a shorter half‐life than some of the parenteral formulations in development, such as luspatercept, which has a half‐life of 15 to 16 days.As a life‐long disease, β‐thalassemia requires chronic treatment. Patients with TDT rely on frequent, regular blood transfusions throughout their life for survival, while those with NTDT may require occasional, or sometimes frequent, transfusions for defined periods of time.Strengths of the current study include the wide range of safety, PK and PD endpoints assessed, both following single and multiple dosing which will support and inform the design of future trials. A limitation of the study is that blood samples for PK and PD analysis were not taken between 12 and 24 hours post‐dose, which may have impacted some observations, particularly with regards to serum iron in the twice daily dosing cohorts. Another potential limitation is that plasma VIT‐2763 PK parameters were calculated using non‐compartmental analysis; compartmental analytic techniques may have produced different results.5When VIT‐2763, an oral ferroportin inhibitor, was administered at single oral doses up to 240 mg, or multiple oral doses up to 120 mg twice daily, it had a comparable safety profile to the placebo, and was well tolerated in healthy volunteers. VIT‐2763 achieved rapid and prominent decreases in circulating iron levels, consistent with the iron‐lowering PD effect observed in preclinical models, and also reduced transferrin saturation. Thus, results from the current study suggest that limiting iron availability for erythropoiesis via inhibition of ferroportin activity may be a potential approach to treating NTDT, and other indications that involve ineffective erythropoiesis, excessive iron absorption or excessive erythropoiesis. Supported by these findings, clinical studies of VIT‐2763 in patients with ineffective erythropoiesis are warranted.F.R., B.R., T.H., U.‐M.G. and F.D. are all employees of Vifor. J.J.v.L. is an employee of PRA Health Sciences, Early Development Services, Groningen, Netherlands.All authors contributed to the research, conception and design of the study, data analysis and interpretation. All authors reviewed and revised drafts of the manuscript and approved the final draft for submission.Appendix S1: Supporting InformationClick here for additional data file.


Candida isolates, especially Candida albicans, has increased continuously. To overcome the resistance, research on antifungal agent sensitizers has attracted considerable attention. Omeprazole and lansoprazole were found to inhibit the growth of sensitive C. albicans and hyphae formation in a high dose, respectively. This study aimed to determine the interactions of common clinically proton pump inhibitors (PPIs) and fluconazole both in vitro and in vivo and to further explore the possible mechanisms. In vitro, the tested PPIs all acted synergistically with fluconazole against both resistant C. albicans planktonic cells and biofilms preformed for ≤12 h with the minimum inhibitory concentration of fluconazole decreased from >512 μg/mL to 1–4 μg/mL. In vivo, PPIs plus fluconazole prolonged the survival rate of infected Galleria mellonella larvae by two-fold compared with that for the fluconazole monotherapy group and significantly reduced the tissue damage of infected larvae. Mechanism studies showed that PPIs significantly suppressed efflux pump activity, which is the common resistance mechanism of C. albicans, and significantly inhibited the virulence factors: phospholipase activity and morphology switching. These findings will provide new insights into antifungal agent discovery and potential approaches for the treatment of candidiasis caused by resistant C. albicans.The incidence of resistant  Candida species2. Candida species can cause superficial infection of the skin, mouth, or mucous membranes and can also cause invasive infection, such as candidemia and biofilm-related infection3. In Candida infections, C. albicans is still the most commonly isolated strain. Data from the Prospective Antifungal Therapy Alliance registry showed that among the 7526 fungi isolated from 6807 invasive fungal infections, the isolation rate of Candida species was highest , and C. albicans accounted for 47.8% of its isolation rate4. Owing to its great efficacy and low toxicity, fluconazole (FLC) has been extensively used in clinical practice to prevent and treat candidiasis. However, along with the increased in frequency of infections and extensive use of FLC, drug-resistant strains have frequently emerged6. To overcome fungal resistance, research on antifungal sensitizers has attracted considerable attention.The incidence of invasive fungal infections has increased continuously, especially those caused by +/K+-ATPase in the cell membrane and have become the first choice in the treatment of acid-related diseases8. PPIs with a wide range of clinical applications include omeprazole (OME), lansoprazole (LAN), pantoprazole (PTP), rabeprazole (RAB), esomeprazole (ESO) and ilaprazole (ILA). OME was found to cure acute oesophageal necrosis and candidal oesophageal when it was combined with FLC in the clinic11. Studies on the antifungal activity of PPIs found that LAN and OME at a dose of >600 µg/mL could inhibit the growth of sensitive C. albicans and hyphae formation, respectively13. In addition, although some studies showed that PPIs combined with fluconazole in vitro have no synergistic effects against sensitive C. albicans15, other studies found that BM2, a D-octapeptide inhibitor of the plasma membrane proton pump, enhanced the efficacy of FLC against resistant C. albicans and Candida dubliniensis17. However, no study has reported the interaction of commonly used PPIs and FLC against resistant C. albicans. In this study, we evaluated in vitro and in vivo interactions of PPIs combined with FLC against resistant C. albicans, and the underlying mechanism of the interactions of PPIs and FLC was further explored.Proton pump inhibitors (PPIs) inhibit the Hin vitro antifungal activity of PPIs alone or combined with FLC was determined by the microdilution method, and an XTT assay was conducted to evaluate the antibiofilm effects of the drug combination. In addition, the in vivo interaction of the drug combination was evaluated by the establishment of a G. mellonella larvae infection model. Of note, with OME and RAB as representative PPI drugs, synergistic mechanisms were evaluated by assessing extracellular phospholipase activity, morphology switching and the efflux pump activity.In the present study, the C. albicans are listed in Table C. albicans strains, indicating strong resistance of these C. albicans strains. The MICs of RAB, ILA and the others were 128–512 μg/mL, >256 μg/mL and >512 μg/mL, respectively, showing that RAB possessed a weak intrinsic antifungal activity and a very limited intrinsic efficacy for the other PPIs. However, when used in combination with FLC, PPIs could significantly decrease the MICs of FLC from >512 μg/mL to 0.5–4 μg/mL, indicating a significantly increased sensitivity of resistant C. albicans to FLC caused by PPIs. Of these six PPIs, when the MIC of FLC was decreased to ≤2 μg/mL, the concentrations of PPIs required were 8 µg/mL for ILA, 16–32 µg/mL for RAB, 16–32 µg/mL for LAN, 32 µg/mL for OME, 32 µg/mL for ESO and 32–64 µg/mL for PTP. Moreover, the FICI values obtained from the FICI model were 0.06 for OME and ESO, 0.03–0.06 for LAN and ILA, 0.06–0.13 for PTP and 0.04–0.25 for RAB. The FICI values were all <0.5, showing a strong synergism induced by PPIs plus FLC. Additionally, this synergistic effect was demonstrated by another evaluation model  of PPIs and FLC against resistant C. albicans strain (CA10) are listed in Table 80 of FLC decreased significantly from >1024 μg/mL to 1–4 μg/mL, with the FICI values 0.06–0.13 for OME and RAB and 0.06–0.25 for LAN, PTP, ESO and ILA. The FICI values were all <0.5, showing strong synergistic antibiofilm effects induced by PPIs and FLC. When combined with PPIs against biofilms preformed over 24 h, there was almost no change in the sMIC80 of FLC compared with that of FLC alone, indicating an indifferent interaction between PPIs and FLC.The sessile minimum inhibitory concentrations (sMIC) of PPIs and FLC against a resistant in vivo experiment, 20 randomly chosen larvae in each group were injected with a C. albicans suspension, and after 2 h of infection, the larvae were treated with drugs. Regarding the survival rate of G. mellonella larvae .In an C. albicans to the larvae.Regarding observation of histological sections Fig. , integraC. albicans. The results showed  to evaluate the phospholipase activity of C. albicans, and the higher the PZ value was, the lower the phospholipase activity. In this study, very high phospholipase activity was observed in the control group and drug monotherapy group with PZ values of 0.64–0.66 . These data indicated that PPIs combined with FLC could synergistically decrease the phospholipase activity of resistant C. albicans.The P66 Table . For theC. albicans. Therefore, in this assay, we used Rh6G as a tracer of FLC to detect the intracellular FLC concentration. As shown in Fig. C. albicans.The fluorescent dye rhodamine 6 G (Rh6G) and FLC both are substrates of drug transporters in C. albicans to current antifungal agents has emerged frequently, and to solve this problem, research on sensitizers to existing antifungal agents or new antifungal drugs has received wide attention. Numerous studies on sensitizers to antifungal agents found that antibacterials18, calcium channel blockers19, phytocompounds20, etc., could enhance the sensitivity of resistant C. albicans to antifungals.In recent years, the resistance of Candida oesophagitis21, PPIs represent an opportunity for joint applications with FLC for Candida oesophagitis in the clinic. For example, Hasosah, M. Y. et al. and Liang, M. et al. reported that immunocompetent patients presenting with Candida oesophagitis were successfully cured by OME combined with FLC23. Studies on the antifungal interaction of PPIs and FLC have also been conducted previously, and some studies showed antagonistic effects against susceptible C. albicans isolates14. However, there was no report about whether PPIs could increase the sensitivity of resistant C. albicans to FLC. Based on these studies, we tested the interaction of PPIs combined with FLC against both susceptible and resistant C. albicans strains. In a preliminary experiment, we observed a strange phenomenon for all the drug combinations against the susceptible strain. The combination of FLC and RAB serves as an example to clarify this phenomenon. As shown in Table C. albicans susceptible strain CA4; however, a higher concentration of RAB (64–128 μg/mL) resulted in a weaker inhibitory effect. To explain this phenomenon, many experiments need to be done. Therefore, we focused only on the resistant strain in this study. More importantly, Monk, B. C. et al. and Hayama, K. et al. found that an inhibitor of the plasma membrane proton pump (BM2) could enhance the efficacy of FLC against resistant C. albicans and C. dubliniensis16. Inspired by the synergism of BM2 with FLC, we evaluated the interaction of PPIs combined with FLC against resistant C. albicans and further explored the underlying mechanisms.As the most potent acid-suppressing drugs, PPIs are considered as the standard treatment for acid-related diseases. Although acid suppression therapy has been reported as a risk factor for In vitro, we found that PPIs exerted weak anticandidal effects with MICs ≥ 128 μg/mL, which was in accordance with the studies of Biswas, S. K. et al. and Liu, N et al.13. More importantly, PPIs all acted synergistically with FLC against six tested resistant C. albicans isolates, as interpreted by the FICI and ΔE model. These six PPIs decreased the MIC80 of FLC from > 512 μg/mL to 0.5–4 μg/mL. Among them, OME reduced the MICs of FLC to a maximum extent, while RAB reduced the MICs of FLC to a minimum extent. Additionally, when the MIC of FLC was decreased to the same concentration, the minimum concentration of proton pump inhibitors required is 8 µg/mL for ILA, and the maximum is 32–64 µg/mL for PTP, demonstrating that different PPIs resulted in different enhanced efficacy of FLC.C. albicans24. In clinic, biofilm-related infections are difficult to treat due to their trend to be chronic and easy recurrence. Here, the sMIC80 of FLC for resistant C. albicans biofilms preformed for ≤12 h was decreased from >1024 µg/mL to 1–4 µg/mL by the presence of PPIs, indicating enhancement of the antibiofilm effect of FLC by PPIs. In addition, different PPIs resulted in different enhanced efficacy of FLC. With prolonged preformation time, the biofilm was more mature, and the synergistic effect was weaker. The biofilm preformed for 24 h was much more mature and its structure was more complex. Therefore, no obvious inhibitory action induced by the drug combinations was observed. Studies on the antibiofilm effect of drugs also showed that drugs are less effective in mature C. albicans biofilms than in early-stage biofilms and even not effective in mature biofilms26. Our findings demonstrated a potential use of the drug combination in prevention or early treatment of biofilm-related diseases.Biofilm formation has been shown to be related with drug resistance of G. mellonella larva is a type of infection model that has been used to study the efficacy or toxicity of drugs as well as the virulence of pathogens28. Compared with mammal host models, this infection model can provide a more rapid evaluation of the virulence of pathogens and the in vivo efficacy or toxicity of agents, with significant economic and ethical advantages30. To primarily assess the combined effects of PPIs and FLC in vivo, we used this model to determine survival rates of the larvae infected with CA10. The data obtained showed that the survival rates of the larvae treated with PPIs + FLC were significantly higher than those of the FLC monotherapy group and other groups, demonstrating that the tested PPIs could significantly increase the in vivo efficacy of FLC against resistant C. albicans. Histopathological study of G. mellonella larvae was carried out to further assess the combined effects of PPIs plus FLC in vivo. In this study, the tissue infected with CA10 was stained as black lumps and was much more fragmentary than that of the blank group. This result indicated that the resistant C. albicans CA10 could cause serious damage to the tissues of the larvae. In addition, fewer and smaller black lumps were observed in the drug combination group than in the control group and the drug-monotherapy groups, demonstrating that at the experimental concentration, the drug combinations could suppress the damage caused by resistant C. albicans to the larvae. Taken together, these data show that PPIs could significantly enhance the in vivo efficacy of FLC against resistant C. albicans, which was in accordance with the in vitro results.The C. albicans can switch among different morphological phenotypes, and the morphology switching is a primary virulence factor32. Notably, this switch has been proven to be involved in the pathogenicity and biofilms formation of C. albicans34. Here, we found that RAB possessed a weak inhibitory effect on the morphology switching of both CA10 and a susceptible C. albicans strain (CA4)  Fig. . More imA4) Fig. , and thiC. albicans, is another important virulence factor in C. albicans infections36. Ying et al. found that the phospholipase B1 mRNA and protein expression of resistant strains was higher than that of susceptible strains37. In addition, C. albicans phospholipase D1 has been proven to play a role in promoting the transformation of yeast to mycelium38. These studies demonstrated that the phospholipase activity of C. albicans might be related to the resistance and morphology switching. In the present study, the phospholipase activities of C. albicans in the PPIs plus FLC groups were significantly reduced, with Pz values higher than 0.80. However, drug combinations at the same concentrations could not decrease the phospholipase activity of a susceptible C. albicans (CA4)  Table . InspireC. albicans, overexpression of efflux pumps has become a research hotspot for Candida resistance40. The present study found that compared with the control group, the addition of PPIs significantly suppressed efflux pump activity (P ˂ 0.001). Additionally, Monk, B. C. et al. and Hayama, K. et al. found that the inhibition of efflux pumps may be involved in the enhanced efficacy of FLC to resistant C. albicans and C. dubliniensis caused by a D-octapeptide inhibitor of the plasma membrane proton pump (BM2)17. These findings demonstrated that the synergism between PPIs and FLC may be related to efflux pumps suppression.As one of the most common resistance mechanisms in C. albicans both in vitro and in vivo and that the efflux pump suppression, extracellular phospholipase inhibition and morphology switching suppression may be involved in the synergistic antifungal effects. These findings together with the opportunity for the combined applications of PPIs and FLC in the clinic will provide new insights into antifungal agent discovery and potential approaches for the treatment of candidiasis caused by resistant C. albicans.In conclusion, this paper provides an advance over our recent studies and in the field by first finding that PPIs enhanced the efficacy of FLC against resistant C. albicans strains used in this study are listed in Table C. albicans ATCC10231 was used as a quality control strain to determine the MICs of drugs and was provided by the Institute of Pharmacology, School of Pharmacy, Shandong University . Strains were stored at −80 °C in the Sabouraud dextrose broth and subcultured on the Sabouraud dextrose agar for 24 h at 35 °C before the experiment. Egg yolk agar  was used to test the phospholipase activity.The six resistant PPIs and FLC were all purchased from Dalian Meilun Biotech Co., Ltd. . Stock solutions of OME, PTP, RAB and ESO were prepared in sterile distilled water, while those of FLC, LAN and ILA were prepared in dimethyl sulfoxide. All stock solutions were sterilized using 0.22-μm filters, and that of FLC was stored at 4 °C while stock solutions of PPIs were prepared before each experiment. Rhodamine 6 G was purchase from Acros Trading Company.C. albicans were tested by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The test was conducted in 96-well microtiter plates with yeast (2.5 × 103 CFU/mL) in RPMI-1640 medium (PH 7.0) buffered with MOPS [morpholino (propanesulfonic acid)]. Wells containing only RPMI-1640 medium served as negative controls, and a drug-free well was set as a growth control. After 24 h of incubation at 35 °C, the MICs were determined by both visual reading and measuring the optical density (OD) with a microplate reader at a wavelength of 492 nm. The MIC80 was defined as the lowest concentration of drug with 80% fungal growth inhibition.The antifungal activities of PPIs and FLC against the six resistant C. albicans. Briefly, drugs were serially diluted 2-fold in RPMI-1640 medium, and 0.25–128 μg/mL FLC, 2–128 μg/mL OME, LAN, PTP, RAB and ESO and 0.5–32 μg/mL ILA were added to the wells. Subsequently, yeast at a final concentration of 2.5 × 103 CFU/mL was added to each well. Wells containing only RPMI-1640 medium served as negative controls, and a drug-free well was set as a growth control. After 24 h of incubation at 35 °C, MICs were determined as described above.A checkerboard microdilution assay was carried out to determine the interactions between PPIs and FLC against resistant E model were used to analyse the obtained data. The FICI model is based on the Loewe additivity theory41 and is expressed as FICI = FICA + FICB = E model is based on the Bliss independence theory42 and is expressed as ΔE = EA × EB − Emeasured. In this equation, EA and EB are the experimental fungal growth percentages when each drug acts alone, and Emeasured is the measured growth percentage in presence of the combination of drugs A and B. The mode of interaction is interpreted as synergistic when ΔE and its 95% confidence interval (CI) are positive, antagonistic when ΔE and its 95% CI are negative, and indifferent in other cases. The intensity of interaction is evaluated by calculating the sum percentages of all significant synergistic (ΣSYN) or antagonistic (ΣANT) interactions and is interpreted as strong when interactions are >200%, moderate when interactions are 100–200%, and weak when interactions are <100%.To evaluate the mode and intensity of the drug interactions, the fractional inhibitory concentration index (FICI) model and the ΔC. albicans (CA10) in different growth periods were assessed as previously described, with slight modification43. Briefly, 200-μL aliquots of a yeast suspension (2.5 × 103 CFU/mL) were added to a 96-well plate, and the plates were incubated for four different time intervals  at 35 °C to preform biofilms. Then, the preformed biofilms were washed three times with sterile phosphate-buffered saline (PBS), and drugs were added at final concentrations of 1–1024 μg/mL FLC, 16–1024 µg/mL OME, PTP, RAB and ESO, 8–512 µg/mL LAN and 4–256 µg/mL LAN. Following a further 24 h of incubation at 35 °C, an XTT reduction assay was performed to examine the metabolic activity of the biofilms. Colorimetric changes were measured with a microplate reader at a wavelength of 492 nm. The sessile minimum inhibitory concentration (sMIC) was defined as the lowest concentration of drug with 80% inhibition of biofilm metabolic activity.The interactions between PPIs and FLC against preformed biofilms of Galleria mellonella larvae were used as an in vivo infection model to evaluate the in vivo interactions between PPIs and FLC, and CA10 was used to infect the G. mellonella larvae45. For the survival assay, fourteen groups of 20 randomly chosen larvae with a similar size (ca. 0.25 g) and no grey markings were selected and injected with 10 μL of a yeast suspension (5 × 108 CFU/mL) via the last left proleg. After 2 h of incubation at 35 °C, the larvae were injected with 10 μL of sterile PBS, 160 μg/mL FLC, PPIs, or PPIs + 160 μg/mL FLC via the last right proleg. The concentration of PPIs was 40 μg/mL RAB and ILA, 80 μg/mL OME, LAN and ESO and 160 μg/mL PTP. Then, the larvae were placed in the dark and incubated at 35 °C for 4 days. The survival rate of the larvae was monitored daily, considering death of the larvae when they did not respond to physical pressure. For histological study, OME and RAB were selected to evaluate the interactions between PPIs and FLC on the tissue of infected larvae. Seven groups of larvae were selected and injected with a yeast suspension and drugs as described above. One group of larvae untreated with the yeast and drugs served as a blank control group. After a further 48-h incubation, three larvae from each group were randomly selected and cut into sections (20 μm). Sections stained with periodic acid Schiff (PAS) stain were observed under a fluorescence microscope.C. albicans (CA10) were studied in a microplate-based assay46. In this assay, OME was selected as the representative PPI, and yeast-to-hyphae morphogenesis was induced by RPMI-1640. Yeast cells (105 CFU/mL) were added to a 6-well microplate, and drugs were then added at the final concentration of 8 μg/mL FLC, 128 μg/mL OME and 64 μg/mL RAB. The microplate was incubated at 35 °C for 5 h and then was placed directly under an inverted microscope.Effects of PPIs combined with FLC on the yeast-to-hyphae morphogenesis of resistant C. albicans (CA10) were detected by egg yolk agar plates47, and OME and RAB were selected as representative PPIs. Yeast cells (106 CFU/mL) were incubated with no drug, FLC (1 μg/mL), OME (32 μg/mL), RAB (16 μg/mL), OME (32 μg/mL) plus FLC (1 μg/mL), or RAB (16 μg/mL) plus FLC (1 μg/mL) for 24 h at 35 °C. After the incubation, 10 μL of the cell suspensions were inoculated onto egg yolk agar plates and the plates were then incubated for 72 h at 35 °C. The colony diameter and precipitation zone diameter were measured.Effects of PPIs combined with FLC on the extracellular phospholipase activity of resistant C. albicans was evaluated by the Rh6G efflux assay with some modification48, and a resistant isolate with an efflux pump gene over-expressed (CA10) was used. Briefly, yeast cells were incubated in YPD liquid medium overnight, and the cells were harvested, washed with glucose-free PBS and adjusted to 107 CFU/mL. Subsequently, a Rh6G solution was added at a final concentration of 10 μM to the cell suspension, and the suspension was incubated at 35 °C for 50 min and then exposed to an ice water-bath for 10 min. Cells were harvested and washed with glucose-free PBS. Glucose/PBS (5%) was added to re-suspend the cells, and PPIs were added. The fluorescence intensity was detected every 60 min for 4 h by flow cytometry, with excitation at 488 nm and emission at 530 nm.Whether PPIs interfered with the efflux pump activity of resistant P values < 0.05 were considered statistically significant.Each experiment was performed three times on different days. Graphs were produced with GraphPad Prism 5 and MATLAB 2017a, and statistical analyses were performed with SPSS Statistics v.17.0. The survival curve was analysed by the Kaplan-Meier method and log-rank test. Supplementary Information.