To model data from multi-item scales, many researchers default to a confirmatory factor analysis (CFA) approach that restricts cross-loadings and residual correlations to zero. This often leads to problems of measurement-model misfit while also ignoring theoretically relevant alternatives. Existing research mostly offers solutions by relaxing assumptions about cross-loadings and allowing residual correlations. However, such approaches are critiqued as being weak on theory and/or indicative of problematic measurement scales. We offer a theoretically-grounded alternative to modeling survey data called an autoregressive confirmatory factor analysis (AR-CFA), which is motivated by recognizing that responding to survey items is a sequential process that may create temporal dependencies among scale items. We compare an AR-CFA to other common approaches using a sample of 8,569 people measured along five common personality factors, showing how the AR-CFA can improve model fit and offer evidence of increased construct validity. We then introduce methods for testing AR-CFA hypotheses, including cross-level moderation effects using latent interactions among stable factors and time-varying residuals. We recommend considering the AR-CFA as a useful complement to other existing approaches and treat AR-CFA limitations. When people respond to multi-item scales, item responses may depend on one another due to the order in which items are presented  and Bayesian approaches , which is stricter than EFA because it omits unhypothesized cross-loadings see , 2004. TThe question of how to address this misfit has generated substantial debate, leading to many alternatives to the IC-CFA including exploratory structural equation modeling (ESEM) and Bayesian approaches , this approach is still often interpreted as a sign of a poorly functioning measurement scale because such specifications deviate from the traditional IC-CFA wherein items are “pure” indicators of one factor. Indeed, in a model with cross-loadings and residual covariances, some researchers may question the substantive meaning of structural effects and the nature of the items and scales themselves . By answering items sequentially, respondents must: (1) interpret the meaning of an item; (2) retrieve relevant beliefs, feelings, or memories; (3) apply these to the item; and then (4) select a suitable response  to answer a question. Memory retrieval works through the spreading activation of nodes  that are linked through an associative network. Once activated, these nodes are primed for further activation, thereby making them more likely to be used for subsequent retrieval. For example, items assessing the same construct deal with similar beliefs and behaviors, and thus retrieving information to respond to similar questions will sequentially rely on access to the same areas in memory Knowles, . TherefoHowever, once a node is activated, the process of spreading activation continues automatically, potentially activating nodes that are irrelevant to the task at hand Posner, . For insAm interested in other people's problems.” Again, such effects are distinct from the intended measurement characteristics of a scale, which include factor loadings that potentially increase over time but also latent covariances among factors due to any true-score covariance .In such cases, the irrelevant  memories that are activated would likely carry over to subsequent questions, but degrade as new memories are retrieved with each subsequent item. As such, previous items would serve as context that inadvertently affect future items due to spreading memory activation, but such AR effects may be short-lived because each new item causes new activations that then subsequently spread. Such AR effects are theorized as being due to carry-over in node activation—a cognitive effect. These effects should be positive when activated nodes are consistent, such as the “Don't talk a lot” and “Am not interested in other people's problems” items. On the other hand, negative effects should arise when nodes are countervailing, such as if the latter item read “Alternatively, mood states are highly susceptible to context and can be primed and then used directly as information by respondents . Furthermore, as with memory activation effects, the AR effects induced by affective priming should fade with time as more items are responded to and previous items recede further into the past. In conclusion, the effects of affective priming typically fade over time, such that the time-varying AR effects of past responses should fade as respondents move from one item to another.Thus, serial AR effects may exist when a previous item causes an affective reaction and responding to a future item relies on the affect generated. In such cases, the affect is likely to carry over to the following item(s) systematically, such that the affect generated by a previous item and used for a future response will cause positive AR effects if the affective implications of the items are positively linked , but negative AR effects may exist when items are negatively linked  as well as greater emotional reactivity to stimuli . Indeed, literature on affective inertia shows people high in neuroticism tend to have larger AR effects that reflect the persistence of emotions over time  allows us to theorize that: (1) AR effects may exist between items measuring the same construct (what we will term “within-construct” effects) even if dispersed in a survey; (2) AR effects may exist between items of different constructs (what we will term “between-construct” effects) when these items are adjacent; and (3) neuroticism will moderate AR effects, such that higher neuroticism will be associated with larger AR effects. Certainly, we could draw on additional theory to justify potential sources of context effects, such as anchoring  referenced in a set of items. This assumption can be formalized as an IC-CFA as follows see :* distinguishing these terms from those in subsequent models; p × 1 vector of p observed items for person i; ν* is p × 1 vector of item intercepts; Λ* is a p × m matrix of factor loadings that uniquely link each observed item to a single latent variable m = 1, 2, … m ; m × 1 vector of standings on m latent variables for person i, distributed as MVN, where α* is an m × 1 vector of latent variable means ; Ψ* is a m × m symmetric matrix of unrestricted latent variable variances and covariances; and, p × 1 vector of latent disturbances for each person i, distributed as MVN, wherein Θ* is an m × m diagonal matrix of residual variances .with a superscript p observed variables can be thought of as entirely caused by (or arising from) the m latent variables (including their latent covariances), wherein m is typically much smaller than p .As with any measurement model, observed scores are specified as being determined by latent factors and disturbances  or for similar wordings or contexts to be referenced across items  can be extended by the logic of an AR model in Equation (3), justified by the idea that item responses can reflect the effects Λ* of latent variables K• that link past and future items in yi In turn, by estimating an IC-CFA without accounting for AR effects, estimates of latent variable (co)variances and measurement model fit may be biased on a regular basis.As we note later, the AR terms in Indeed, the existence of stable latent variables alongside AR effects has received substantial treatment in the longitudinal/panel data modeling literature, and many techniques have been developed to account for both variance in observed variables . Theoretically, this separation is akin to proposing that the context effects due to AR terms cause variance in observed data that is conceptualized as error which is unrelated to the substantive latent variables of interest in a CFA. Theoretically speaking, this is akin to the memory activation and affective priming effects  Bollen, . This is Bollen, , whereasWe formalize this logic with an AR-CFA model that combines the logic of Equations (1) and (3) see , allowinandΛη (as in an IC-CFA), which is conceptually separate from a p × p matrix Λε with on-diagonal elements fixed to unity and off-diagonal elements fixed to zero to capture latent residuals in εi, and we then show a matrix of AR effects among residuals as K, with ultimate residuals now captured by a p-length vector ui, where ui ~ MVN. Also, we remove all superscripts from Equations (4) and (5) to signify that any bias induced by AR effects (omitted in Equation 1) and latent variables (omitted in Equation 3) is no longer present, with the AR-CFA allowing a traditional IC-CFA (or a different) structure for factor loadings Λη and latent covariances Ψ, combined with the AR effects in κ. Also, latent interactions among factors in ηi and residuals in εi can be included in the model by stacking them in the latent vector ε, with any resulting regression coefficients (on the interaction terms) being stacked in Λε. For brevity, we omit a technical description of this point and instead show how to construct and use the latent interactions in Mplus in our Online Appendix A and .zip file  can be estimated with typical methods such as maximum likelihood (ML) or a Bayesian Markov Chain Monte Carlo (MCMC) approach—although latent interactions are only possible under ML with numerical integration. Also, by rearranging (Equation 6) as a difference equation , it becomes clearer how the IC-CFA factor structure is separated from the residual AR terms, which can be shown either as:with all terms as before, but orΣ are essentially adjusted by either the latent factors in η or the residuals in ε to estimate the parameters associated with the other.such that, in both cases, the covariances in K have two desirable properties that are consistent with our theorizing about context effects. First, they exist in residuals and therefore are unrelated to the systematic responding associated with the substantive latent factors of interest in a typical CFA. For example, personality scale items may be ordered as Extraversion (e), Agreeableness (a), Conscientiousness (c), Neuroticism (n), and Openness (o), so that sequential AR effects would be e1 → a1 → c1 → n1 → o1 → e2 → a2 → c2 → n2 → o2. In this case, it may seem that factor loadings and latent covariances would account for the AR effects due to their repeating nature, but this is not the case because of the serial nature of the effects. For example, the similar e1 → a1 and e2 → a2 effects may appear to induce true-score covariance among extraversion and agreeableness. However, because e1 is very distant from a2, a typical CFA factor structure and latent covariances could not account for the AR effects, which would appropriately occur in the residual structure implied in As df : a CFA that reparameterizes AR terms as residual correlations. If the context effects associated with an AR-CFA fade with time , then the AR-CFA should provide a superior fit to the data because correlated residuals cannot capture fading correlations over time as freely-estimated indirect AR effects can. Free correlated residuals will cause model under-identification issues, and the constraints required to identify them in a way that is consistent with a freely-estimated AR structure cannot be known a priori.Second, because of their serial nature, AR effects allow past context effects to fade with time, being multiplied along AR paths as indirect effects. This longitudinal relationship results from Weiner or Markov processes that induce a “simplex” pattern in correlation matrices –(8) can be shown as infinite-order “moving average” models Hamaker, . Howeverm > 1 being measured by a scale, there are multiple ways to specify the AR structure in K. Consistent with a classic interest in determining an AR “lag order”  the next extraversion item due context effects rather than the extraversion factor itself. The AR specification in and they link adjacent items when they reference the same construct. Second, by linking items that reference the same construct, the AR structure in There are three points to make about this second AR structure in p – 2, p – 3, …, p – h past items will quickly fall, but the AR terms still allow for data-driven lagged effects beyond the p – 1 items, particularly in Third, the simpler AR structure in Λ and one with all possible residual covariances in Θ , which was originally presented in Donnellan et al. . This scN = 8,569  survey  by online study announcements, offering course credit for participation or entering a prize draw. Data collection was between September 2008 and October 2009, and the mini-IPIP survey took 1–2 h to complete. Corker et al.  study mahttp://ipip.ori.org/MiniIPIPKey.htm).The mini-IPIP assesses the following five personality traits in this order: Extraversion (e); Agreeableness (a); Conscientiousness (c); Neuroticism (n); and Openness (o). These traits are typical personality constructs used in psychology and elsewhere , as well as a deviance information criterion , we used small-variance priors of 0.01, and for the Bayes CFA with all residual covariances (RC-CFA), we took analysis steps recommended in Asparouhov et al. .p-values should not be understood as reflecting typical null hypothesis tests.In what follows, we first compare model fit statistics and measurement characteristics for all models. Given the AR-CFA's favorable results, we then interpret AR effects and test hypotheses related to them, including their moderation by neuroticism and by using parameter constraints that help address concerns about overfitting. For chi-square difference tests of nested models, we used Satorra-Bentler chi-square correction factors Satorra, . For the2 = 6052.87, df = 160, p < 0.001; CFI = 0.80; TLI = 0.76; RMSEA = 0.07; SRMR = 0.05; AIC = 488895.76; BIC = 489389.69; DIC = 488895.96; PPP <0.00. We then estimated an AR-CFA for comparison with the IC-CFA as well as other models. The df difference is 34, because with 20 items, there are 19 sequential “between-construct” AR terms, and with four items per factor, there are three AR terms per factor, for a total of 15 “within-construct” AR terms . With the addition of the AR terms, model fit improved: χ2 = 3279.08, df = 126, p < 0.001; CFI = 0.89; TLI = 0.84; RMSEA = 0.05; SRMR = 0.04; AIC = 485524.31; BIC = 486258.15; DIC = 485515.23; PPP <0.001. The differences between the nested IC-CFA and the AR-CFA show improved fit in all indices : Δχ2 = 2363.90, Δdf = 34, p < 0.001; ΔCFI = 0.09; ΔTLI = 0.08; ΔRMSEA = 0.02; ΔSRMR = 0.01; ΔAIC = 3371.45; ΔBIC = 3131.54; ΔDIC = 3380.73; ΔPPP = 0. These results point to the benefits of a AR parameters for model fit, but other model comparisons are also useful for interpreting the AR-CFA.We first estimated a typical IC-CFA as a baseline for model comparison. As expected, this model demonstrated problems with model fit: χdf, where all AR terms are reparameterized as autocorrelated residuals, which resulted in worse fit compared to the AR-CFA : Δχ2 = 137.96, Δdf = 0, p < 0.001; ΔCFI = 0; ΔTLI = 0.01; ΔRMSEA = 0.01; ΔSRMR = 0; ΔAIC = 382.53; ΔBIC = 382.52; ΔDIC = 358.86; ΔPPP = 0. These results suggest that residuals may have indirect effects via AR terms that cannot be accounted for by residual correlations, partially supporting an AR-CFA wherein responses along past items affects future responses both directly and indirectly via AR paths.For example, the AR-CFA can be compared to a non-nested alternative model with equivalent 2 = 853.46, Δdf = 60, p < 0.001; ΔCFI = 0.02; ΔTLI = −0.1; ΔRMSEA = −0.01; ΔSRMR = 0.02; ΔAIC = 2578.27; ΔBIC = 2154.89; ΔDIC = 2578; ΔPPP = 0), Bayes CL-CFA , Bayes RC-CFA , as well as Bayes AR-CFA with small-variance priors of 0.01 and M = 0 on AR terms . These results could be used to infer that indicators with common wordings or context impact model fit , or the AR-CFA specification can capture serial dependence even when researchers do not a priori expect AR effects.Beyond this, the IC-CFA shows worse fit across most models and their indices , including EFA2 = 1920.33, Δdf = 26, p < 0.001; ΔCFI = 0.07; ΔTLI = 0.18; ΔRMSEA = 0.03; ΔSRMR = −0.01; ΔAIC = 793.18; ΔBIC = 976.65; ΔDIC = 802.73; ΔPPP = 0), Bayes CL-CFA , Bayes AR-CFA with small-variance priors and M = 0 on AR terms , but worse fit compared to Bayes RC-CFA . Indeed, the Bayes RC-CFA is superior to the AR-CFA in terms of BIC, DIC and the χ2 CI, showing good fit by including zero (95% CI for RC-CFA: −46 to 78.93). Further, AR-CFA Bayes model's 95% CI values are narrower and/or does not contain zero when compared to other Bayes CFAs .Yet, because some researchers are wary of models that include cross-loadings and residual specifications , the AR-CFA does show improved fit across most indices when compared to models such as the IC-CFA and EFA, both of which are very common. Specifically, in relation to model fit, we infer that underlying AR effects among items may be a reasonable conclusion in the observed data, thereby lending some support for the proposition that items in a scale have an AR effect on subsequent items  as 0.15 when compared to other CFA models: CFA = 0.18, CL-CFA = 0.17, and RC-CFA = 0.19. The lower average factor correlations with AR-CFA indicate better discriminant validity and can strengthen the effectiveness of diagnostic feedback . In sum, our results indicate that when researchers use CFA to evaluate measurement model fit . Thus, only the e2 → a2, e3 → a3, and e4 → a4 effects are equivalent, and therefore we focus on such effects for hypothesis testing and model constraints , which implies that responses to the extraversion item that deviate from one's true score along extraversion  persist, leading to slightly higher scores on the agreeableness item that cannot be accounted for by the true score along agreeableness . To understand this, it is reasonable to presume that respondents may reflect on a time that they did not talk a lot in an interpersonal encounter, which they may then infer implies that they are not interested in others' problems and/or may more easily lead to a memory of being disinterested in others' problems. The affect generated by thinking about the experience combined with any similarly activated memory could create the small e2 → a2 effect observed here, such that endorsing the previous item could lead to endorsing the latter in a manner that is not accounted for by latent standings along the factors themselves.Examining the between-construct AR effects, most of them appear reasonable from a substantive perspective. Consider our previous example of the e2 → a2 relationship among the “Don't talk a lot” and “Am not interested in other people's problems” items. This relationship is positive . This runs counter to the way we hypothesized within-construct effects—we presumed that cognitive and affective priming would create positive AR effects—but the pattern of effects in Turning to the within-construct effects, again most  of the effects were statistically significant. More interesting was the fact that many of these effects were negative. For example, the e2 → e3 effect of the “Don't talk a lot” item and the subsequent extraversion items “Talk to a lot of different people at parties” was negative and statistically significant . Although only five of the AR effects in SE = 0.012, t = 10.422, p < 0.001; a → c = 0.114, SE = 0.009, t = 13.236, p < 0.001; c → n = 0.13, SE = 0.011, t = 12.008, p < 0.001; n → o = −0.027, SE = 0.008, t = −3.535, p < 0.001; o → e = −0.012, SE = 0.13, t = −0.937, p = 0.349. For the within-construct effects we find: e → e = −0.192, SE = 0.101, t = −1.901, p = 0.057; aa = −0.153, SE = 0.014, t = −10.988, p < 0.001; cc = −0.173, SE = 0.04, t = −4.341, p < 0.001; n → n = −0.113, SE = 0.027, t = −4.215, p < 0.001; o → o = 0.088, SE = 0.013, t = 6.583, p < 0.001. Taken together, these appear to show, on average, AR effects that are predictably small but systematic. However, computing an average of AR effects does not imply that similar AR effects are actually the same.Next, it is possible to examine the AR effects in an AR-CFA in two additional ways. First, overall averages of AR effects can be computed to test omnibus hypotheses about similar AR effects in a model variance, with item-specific (co)variance remaining.To test for equivalence in similar AR effects, we conducted a series of nested model comparisons by constraining common AR effects to equality. The first set of constraints took the average AR effects as a starting point, so that instead of computing averages each set of effects were constrained to equality, resulting in a single effect for each e → a, a → c, c → n, n → o, o → e, e → e, a → a, c → c, n → n, and o → o effect : no = −0.156, SE = 0.014, t = −11.155, p < 0.001; o → e = −0.044, SE = 0.011, t = −3.81, p < 0.001; e → e = −0.13, SE = 0.011, t = −11.63, p < 0.001; aa = −0.118, SE = 0.015, t = −8.064, p < 0.001; c → c = −0.075, SE = 0.013, t = −5.607, p < 0.001; n → n = −0.102, SE = 0.015, t = −6.893, p < 0.001; o → o = −0.335, SE = 0.049, t = −6.801, p < 0.001. Although this pattern may be due to the fact that many of the similarly scored items are between-construct AR effects whereas the differently-scored items are within-construct AR effects, it does appear that the scoring of items may systematically impact item AR effects.Specifically, the effects for similarly scored items are : e → a = 0.132, A simulation study with AR-CFA and IC-CFA models was conducted with varying sample sizes of 250, 500, and 1,000 as well as AR effects of 0, 0.1, 0.2, and 0.3 . The factor loadings, residual variances and other population parameters were set by relying on typical standards of standardized loadings of 0.8 and factor correlations of 0.2  should include both latent variable and AR effects, the latter of which allow past survey items to affect later item responses in ways that are unrelated to the latent factors of interest in a CFA. This sequential response process and its effects have been investigated by researchers Schwarz, , 2011 anBy grounding AR effects in literature on item context effects, we theorize two key mechanisms that produce AR processes in a survey setting: memory activation and affective priming. Our conceptualization of these mechanisms and the potential moderating effects of stable traits such as neuroticism offer a sound theoretical rationale for expecting AR effects in survey data. By modeling AR effects among item residuals, our method permits operationalizing various kind of AR effects, as well as evaluating such effects by computing their averages or through model constraints—as we demonstrate here. In sum, the AR-CFA model offers improved fit and a more theoretically rigorous approach to model specification when compared to the IC-CFA, while also offering ways to balance the difference between highly a constrained IC-CFA vs. other less-constrained approaches such as an EFA.Our substantive findings help to justify the AR-CFA by showing improved discriminant validity in the form of larger factor loadings and latent factor variances as well as smaller latent factor covariances and lower average factor correlations among personality traits. This is done while accounting for what could otherwise be problematic item-level covariances in form of lagged AR effects. Although many alternative measurement models are possible and our goal here is not to criticize these approaches, our comparisons against competing models , in conjunction with the theoretical justification for an AR-CFA give us some confidence in recommending it as a viable alternative to other approaches.When considering the use of the AR-CFA by researchers or practitioners, two issues arise. The first relates to how scales should be scored if, at the item level, it is reasonable to expect relationships that deviate from an IC-CFA in ways that could bias, for example, scale means. On this point, it is important to point out that, like the RC-CFA, the AR-CFA specifies item-level relationships in a way that can be conceptually and mathematically separated from the latent factors that are typically of interest in a CFA. In turn, it becomes possible to use scoring methods that rely only on the latent factors themselves. These would include specifying an AR-CFA and then computing factor scores for latent factors of interest Grice, , but stiThe next issue to consider is the increasingly popular method of randomizing items when surveys are administered online , the AR-CFA we propose can only account for temporal AR effects when items are ordered in a fixed and shared way across items and respondents. This, the AR-CFA is useful for the many surveys that use a fixed ordering of questions, mailed survey databases, national surveys such as the Household Income and Labor dynamics in Australia (HILDA) dataset, or research that relies on historical datasets that were collected with fixed item orderings via questionnaires.population, with a finite sample size this is not possible, and therefore even fully randomized item orderings should produce some bias, and in all cases will reduce efficiency and therefore lead to inflated standard errors. In addition to addressing this problem, future research may consider how item response times and other time-varying covariates can be incorporated into measurement models to examine the residual covariance induced by item orderings . Certainly, in the case of such randomization, it is interesting to consider how this is an attempt to overcome the kind of order effects that our AR terms capture, but such randomization only does this by introducing unmodeled order effects that vary across participants in the form of unsystematic (and unmodeled) residual covariance. Instead of this, the AR-CFA may be a viable alternative to addressing the concerns that random item ordering across respondents is meant to achieve—a topic for future research by modeling specifying person-specific AR terms. Furthermore, it is worth noting that even though randomized-item surveys may “average out” AR effects at the level of a Furthermore, our AR-CFA in its current form offers only a single AR term for each type of item pairing, effectively implying a kind of single-order AR process linking indicator-item residuals. However, more complex, higher-order AR or moving-average (MA) processes allowing for more complex residual covariance structures could be implemented (for general insight, see Hamaker, In conclusion, since the origination of EFA for modeling survey responses, there have been a vast array of methodological developments, culminating in confirmatory techniques that, today, take a wide variety of forms—most notably a highly restricted IC-CFA. Our AR-CFA is meant to extend and complement these existing approaches for those who seek to add a temporal element into their measurement models while improving model fit. We hope that the AR-CFA is useful for this purpose, and we look forward to future work on how it can be further developed and applied across a wide cross-section of settings, samples, and purposes.This study was exempt from ethics oversight, as per the University of Melbourne human subjects protocol, because the data are anonymized and exist in the public domain.OO and MZ contributed equally to this project to formulate the idea, procure data, analyze it, and draft early versions of the manuscript. AB drafted the theory section and otherwise he, along with MT, MD, and FO, contributed equally to manuscript drafts and the general ideas associated with the project.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.


Due to its central role in the cellular biology of prostate cancer (PC), androgen receptor (AR) still remains an important therapeutic target for fighting this tumor. Several drugs targeting AR have been reported so far, and many new molecules are expected for the future. In spite of their antitumor efficacy, these drugs are not selective for malignant cells and are subjected to AR-mediated activation of drug resistance mechanisms that are responsible for several drawbacks, including systemic toxicity and disease recurrence and metastasis. Among the several strategies considered to overcome these drawbacks, very appealing appears the design of hybrid small-molecule conjugates targeting AR to drive drug action on receptor-positive PC cells. These compounds are designed around on an AR binder, which selectively engages AR with high potency, coupled with a moiety endowed with different pharmacological properties. In this review we focus on two classes of compounds: a) small-molecules and AR-ligand based conjugates that reduce AR expression, which allow down-regulation of AR levels by activating its proteasome-mediated degradation, and b) AR-ligand-based conjugates for targeting small-molecules, in which the AR binder tethers small-molecules, including conventional antitumor drugs  and selectively directs drug action toward receptor-positive PC cells. Prostate cancer (PC) is the leading cause of tumor death in men of Western countries and the malignancy is increasing in developing nations  and PROteolysis Targeting Chimeras (PROTAC) are two classes of compounds endowed with antitumor activity on hormone-refractory PC that knock-down AR (wt and mutant) content via proteasome-mediated degradation.1a, 1b), in vitro and in vivo  have been completed in metastatic CRPC patients. In spite of the important side effects observed (nausea and vomiting), patients treated with AZD3514 showed significant Prostate-Specific-Antigen (PSA) reduction and disease stabilization , Another SARD is ASC-J9 (1d) . Results of the phase I study recently concluded indicate that, due to the toxicity observed, niclosamide is not a suitable drug for repurposing as a CRPC treatment  , a drug 1d) . By redu1d) . The sig1d) , 2017. T1e)  coupled with an AR ligand. The hydrophobic moiety mimics a partially denatured protein state (hydrophobic tagging) that recruits chaperons and in turn induces proteasome-mediated degradation of the receptor   (Toure a1e) . In spit1e) .2a)   (Schneek2a) . The pro2a) .2b) , a compound containing the von Hippel-Landau E3-ligase moiety coupled with enzalutamide for AR targeting  proteins, e.g., BRD 2, 3, and 4. These proteins are AR co-activators that activate receptor-related functions. Compounds that disrupt AR-BRD interaction by inducing BRD degradation impair PC growth   (Raina e2b) . The com2d, 2e. The compound, ineffective on AR-independent PC3 cells, markedly reduced the cell growth of AR-dependent (VCaP and LNCaP) cell lines. Effective and specific proteasome-mediated knockdown of AR and apoptotic cell death (PARP and caspase 3 cleavages) have been reported in VCaP cells treated with 2e. These findings paralleled with the reduced expression of receptor-associated genes , and this behavior reverted when cells were pretreated with the proteasome inhibitor MG132.Specific and Non-genetic Inhibitor-of-Apoptosis proteins (IAPs)-dependent Protein Eraser (SNIPER) is a peculiar type of PROTAC which contains a IAP ligand functioning as an E3-ubiquitin ligase binder  preferentially accumulated in AR-positive cell lines and this finding paralleled with the improved cytotoxicity observed in these cells in comparison with AR-negative cells  against PC cells, the drug has been conjugated with the testosterone-homolog ethisterone, 3a  (Huxley 3a . By usin3b)  . The con3c, 2-modified AR binder) coupled with a pheophorbide α moiety, which allows the production of reactive oxygen species, after light exposure. The compound is totally safe in the dark and following exposure to white light both pheophorbide α and NO donor moieties are activated. The molecule showed nuclear localization in CRPC and, differently to pheophorbide α alone, it by-passes the ABCG2-mediated efflux, thus making this compound interesting for patients resistant to photodynamic therapy. Gaining further insight into compound's action mechanism, the same research group identified the doubtful role played by the NF-kB/YY1/RKIP pathway in mediating NO activity of the conjugate. Specifically, NO mediates pro- or anti-survival activity depending on concentration levels achieved following light activation. Low NO levels activate a pro-survival/anti-apoptotic NF-kB/YY1 pathway; conversely, high NO release inhibits NF-kB/YY1 and, via Snail, activates the anti-survival/pro-apoptotic RKIP leading to antitumor activity. Thus, the two photosensitive moieties synergize and induce cell death following conditions that produce high NO levels.A very intriguing way to pursue for anticancer therapy is the development of compounds that, after light activation, selectively release nitric oxide (NO) into cancer cells , The status of the chromatin is also controlled by histone acetyltransferase (HAT) enzymes. These enzymes catalyze acetylation of histones and produce chromatin decondensation allowing changes in gene expression and protein levels that in turn impact on cellular response to antitumor drugs. By increasing HAT levels, genistein increases p21 and p16 expression and favors cell-cycle arrest and apoptosis. Aimed at directing genistein action against AR-expressing PC cells, bi-functional agent containing a non-steroidal moiety linked to genistein (hydantoin-derived antiandrogen-genistein) has been recently designed  have been reported . This compound consists of two ethisterone moieties (AR binder) linked each other via a peptoid oligomer (in vitro of AR-expressing PC cells (LNCaP), including those resistant to bicalutamide (LNCaP-C4-2) and enzalutamide . Noteworthy, MPC6 showed favorable pharmacologic profile and antitumor potency in vivo against enzaltamide-resistant LNCaP-abl tumor xenografts.The interaction of AR with co-activators critically regulates receptor functions and compounds that interfere with this binding impact on tumor growth. In this regard, two hybrid derivatives containing a AR binder and endowed with AR/co-activator interfering action  the proneness of the two pharmacophores to tolerate chemical modifications without affecting the interaction with the targets; (ii) the physical-chemical properties (size and solubility) of the two moieties, which are fundamental for compound formulation; and (iii) linker length and flexibility, which provide optimal distance between the two pharmacophores and allow them to adapt on the targets. In this regard, ligand-based and/or structure-based computational approaches are very useful for the design and optimization of hybrid molecules.1b, 1c, 1d, 1e, 2b, 2c, 3e, and 3g) have been tested on CRPC and proved efficacy in overcoming drug resistance developed against AR-targeted drugs. Compounds that function by reducing the levels of the receptor are in principle very interesting for patients resistant to ADT due to AR mutations. Indeed, these molecules activate proteasome-mediated degradation of both wt and AR mutant variants. In this context, SARD  and PROTAC (2b and 2c) proved efficacy on CRPC.Some of the conjugates reported  progressed beyond the biochemical/cellular characterization and the clinical setting. Unfortunately, the results of clinical trials conducted so far are not encouraging because of important side effects experienced by patients.It is important to underline that the synthesis of hybrid molecules may result in high molecular weight conjugates . According to Lipinski's rule of five (Lipinski et al., In conclusion, although the compounds here reported may be considered a sort of next-generation AR-targeted drugs, most of them represent proof of concept supporting the feasibility of a pharmacological strategy rapidly evolving and arousing great interest. The benefits for patients suffering from PC have yet to be proven.All 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.


To explore, through face to face interviews with a selection of General Dental Practitioners (GDPs), their views and experiences of having used the International Caries Detection and Assessment System (ICDAS) within primary care research studies for recording caries.This qualitative study involved one on one interviews with eight GDP’s who had previously used ICDAS on patients in their dental practices as part of a research study. The participants were selected from among those who had taken part in two clinical studies in the UK using convenient, but purposive sampling. The interviews were tape-recorded and transcribed; the data analysis was conducted by thematic analysis.GDP’s indicated their beliefs that ICDAS had an important role in caries prevention but reported four main barriers while using the full (6 caries stages) ICDAS coding system in their practices: lack of simplicity of coding, financial implications and time consumption (in both training and use of ICDAS) and inadequate undergraduate training. An overarching theme identified from the GDPS was the willingness to offer potential solutions to their barriers which might improve the utilisation of the system in primary care.The GDPs experienced common obstacles in using ICDAS in the primary care setting, many of which have relatively straight-forward solutions which they put forward themselves such as: incentivisation, undergraduate-level training in ICDAS for both dentists and nurses and computerized data entry. Further qualitative and quantitative research is needed on how to facilitate the utilisation of the system in dental practice. It is also recommended to explore the influences of wider agencies on influencing primary dental care professionals’ caries management, including appropriate recording of diagnosis and risk assessment. Dental caries is a multifaceted disease . In 2010The ICDAS system was developed by dental clinicians and academics from various dental institutes across Europe and America . This deThe ICDAS system has been used in research for about a decade, which is a promising indicator of the adoption of the method in the field of research . The ICDCurrent literature suggests that GDP’s and undergraduate dental students till date are being taught to use the conventional DMF index for assessment of dental caries which was developed by Klein, Palmer and Knuston in 1938 . One of Although some reports suggests that there are a number of dental undergraduate establishments using the ICDAS system for teaching purposes , there iResearch to date has focussed on quantitative methodologies to establish the reliability, feasibility, validity, reproducibility and practicality of using ICDAS. However, to our knowledge none of the studies have looked at the GDP’s perceptions about barriers and facilitating factors while using the ICDAS index in research or clinical settings. There is a gap in the literature regarding what factors exist that may hinder or help practitioners to use the ICDAS index in clinical practice. To address this gap, a qualitative one on one interview based study was conducted in the United Kingdom to explore the views and experiences of GDP’s and to determine the factors responsible for increasing the uptake of the ICDAS system in the future.Twelve GDP’s who had previously taken part in research projects using ICDAS were approached via email and telephone to participate in this research using convenience but purposive sampling methods. Ethical approval was given by the University of Leeds School of Dentistry Research Ethics Committee (DREC Reference: 050416/MK/196).The GDPs were contacted by a member of the research team that they originally worked with (KV) and given sufficient information regarding the aim and rationale to allow them to consider if they would like to take part in the proposed study. This way the details of GDP’s who had taken part in previous studies were not shared with the interviewer (MIK), protecting their personal details.The research experiences of the GDPs varied slightly depending on which previous research study they had participated in. One was a trial involving mixed dentition children while the other was of adults. The training that each received was very similar and from the same ICDAS-experienced clinician. This comprised of face to face didactic photograph-based training after self-directed preparatory training using a 90-minute e-learning package , face toThose wanting further information about the proposed study were put in touch with the interviewer via a brief telephone call, and the interested participants received an email copy of the study information leaflet.In order to explore in depth the insight and experiences of the GDP’s on the ICDAS system, a one on one interview method was chosen for collecting data. One to one interviews were conducted using a topic guide that was developed based on the literature review and was piloted with first interviewee by the researcher (MIK) along with an experienced qualitative researcher (KV), to ensure the questions were clear and facilitated discussion, the data from the pilot was utilised within the study. A total of 8 GDP’s willing to participate were recruited and consented at a personal meeting prior to undertaking the interview on the same day. Those 8 general dental practitioners comprised of four males and four females. The interview participants consisted of 4 general dentists, 2 former general dentists who were waiting to begin their speciality training  and 2 general dentists that had recently started working in the community dental setting.To minimize the burden of participation, the length of the interviews were restricted to around 30 minutes. The interviews took place in participant’s dental practices with only the researcher and participant present.The risk of exposing identifiable information about the participants was countered by giving every participant a penname of their choice before beginning the interview and they were assured that there was no data from which their identity could be revealed. Before the interview took place participants were asked for audio-recording permission, to which all agreed.The participants were given a copy of the ICDAS coding sheet before the interview started, in order to remind them of the coding in case they had not used the ICDAS system recently. They were asked exploratory questions with the purpose of putting them at ease, so that they would be able to express their views and beliefs about the ICDAS system; what it meant to them in the previous research study and in clinical settings.The audio-recorded sessions were transcribed verbatim with anonymization. The participants were given an opportunity to verify that their views and opinions had been represented in a fair manner, none requested any change or correction.Interview transcripts and analysis were organized through NVivo 10 software. Phenomenology was selected as a foundation methodology for this study. It is the approach that helps in explaining and identifying the importance of people’s experiences . A pilotOverall, 12 GDP’s were initially approached to take part in the research out of which 8 GDP’s consented to take part. To achieve in-depth information about the research topic participants were chosen with varying years in practice as GDPs, gender, age and current working role, their main characteristics are shown in .The analysis used an inductive approach to identify themes as highlighted in the thematic map Four main themes for this category were recognised from the data: lack of simplicity, financial implications, time consumption, and training.Lack of simplicity was one of the main themes identified in the findings. In the eyes of most users of the ICDAS system, they felt it was not easy to use, maintain using it and understand. Lack of simplicity was found in two contexts: difficulty deciding between different codes and difficulty while charting. One of the participants mentioned the word ‘complexity’ while incorporating the system in research:Probably, because of the complexity of charting and then trying to analyse that information. So, all these numbers written down for the mesial surface, occlusal surface, for 4a, for 4, 7; it’s probably a little bit too much to sort of analyse it really. That’s probably the hardest way”“When participants had been trained in the use of ICDAS for their respective clinical studies, the trainer returned to participants’ practices on request until participants demonstrated competence. However, all participants interviewed for this study revealed concerns about not completely understanding the ICDAS codes. There were varying levels of understanding; of how to detect and assess caries. Two participants felt that they were not confident about distinguishing between early codes:…it was between the 1’s and 2’s [both non-cavitated enamel caries] that were difficult. Once, there is a great big stonking hole it’s pretty obvious and anybody can see that…”“I think zero, 1, 2 how to differentiate between and sometimes weren’t sure which one is 1 or 2…”“..The ease of use of a new system can vary from one dentist to another and the GDPs differed in their reported level of understanding of the technique. The ability to learn the ICDAS system because of relative complications was remarked by a participant:“I think it was a bit too complicated as a first off to be using it on patients for research and having so many different codes that you have not been used to using.”Financial disadvantage to the GDP’s was one of the most common themes among the participants’ discussions and was one of the important reasons cited for not adopting the ICDAS system, as it has personal cost implications to them as independent contractors. One participant stated:Obviously the incentive financially isn’t there to use it really [outside of research]. So, I think that does have a big, big factor…”“… Three participants revealed that the impetus for using the ICDAS system in their clinical practice to detect and then act on early caries lesions in particular was absent and this was based on the simple rule of cost-benefit analyses:I would say, we as a dentist are not paid for prevention and by the end of the day clinical decision-making are influenced by other factors as well.”“Aside from the implied costs of the ICDAS system taking additional time to complete in clinics, other issues with respect to ‘time’ were raised, such as the amount of time it took the GDP’s to examine the teeth systematically and then chart them using the ICDAS system. This issue was a fundamental problem for them. A participant specified:It does take longer, and sometimes that can be a problem…”“…Two participants indicated that the challenge with time was not only crucial for them as dentists but also other practice staff were not comfortable with designating additional time for thorough examination and charting:So, to start with it was a bit time consuming. Especially, for myself and the nurses as well”“It took a lot longer because we were making so many mistakes. Because and plus you are looking at every single surfaces as well, so it used to get a bit you know, you could shout one code and your five codes ahead and your nurses like three codes behind, so it did get a bit confusing.”“However, another participant expressed how patients becoming frustrated and external pressures were pivotal factors for dentists to base their decisions on:I had to discontinue ICDAS because my patients were taking longer to deal with and because of the works strain, the rest of the waiting list was just getting impatient. So, I had to discontinue it.”“Furthermore, many themes were found to be interrelated, but the complex issue that was identified was probably deficiency of training not only of the dentist during undergraduate studies but more especially amongst the dental nurses who had previously trained in a less time consuming method.As some of them [nurses] didn’t come on the initial [research] trial training that as, as the dentist all of us went on and that wasn’t a fault of the trial, it was our trainer and the chap that owned the practice didn’t release everybody and sometimes they’d been put in and they were so used to charting one way and then we were expecting them to do something completely different and also it was trying to get them on board to help with the paperwork and the booklets that were being expected to be completed as well that. That I think they were used to working in a bit faster paced environment and maybe it slower things down a little bit.”“I think, it’s more of a “faff” more than anything because you don’t get paid for prevention and ultimately. Yes, we do want to help patients, we do care about patients, but, at the end of the day it still is a job for us.”“If it’s not going to benefit the patient or myself, in terms of making my job easier or making the treatment easier and it’s gonna take longer. Largely that’s not really gonna work.”“The participants recognised the significance of being used to a system through having been trained in it as an undergraduate and using it commonly in practice. In relation to ICDAS this was a significant issue. None of the participants, and more importantly none of their dental nurses, had been introduced to ICDAS during their clinical training and this was expressed as a significant barrier. A participant revealed:I think the main barriers probably it’s something that I wasn’t trained [as an undergraduate] with and I am more as familiar with other techniques and the same for the staff that haven’t worked with it too …”“… Positive feelings expressed by one participant explained that dentists were given sufficient training during the research. But, some of the dental nurses were not trained enough to ingrain the ICDAS system and were not convinced about using it:I think because you have to have a trained nurse in it that’s very difficult, difficult to encourage some people certainly in the practice that I was in. Ahmm, so I think that would be probably barrier no.1, then after that it’s just getting used to it. Once you have had so much training in doing it [charting] one way, trying to do it in a different way is going to be quite difficult.”“Participants were not only concerned about training, but commented that on a personal level characteristics of dental nurses such as years of experience and training using older/ other systems was a barrier to accepting the ICDAS system, though sometimes this was not related to the ICDAS codes but to other features of the dental charting such as unfamiliar tooth notation:….every nurse is different in amount of experience and training. I think some were sort of maybe not so well experienced with charting. And, I think they struggled with this little bit. So, I think some found it difficult because they weren’t familiar with e.g. more familiar with upper left 7 and not FDI notations in 4, 7 and 4, 8.”“It was affirmed that the GDP’s may not consider a sudden transformation in practice, more focus on training was perceived important to alter their practices and accepting the system completely. This was evident when a participant stated:But I think, for most of us who are just trained in dentinal caries, that might be a big thing, we might not want a change in that system so much, so maybe simplify the codes a little bit. Give us much more training, much more time onto it. So, we actually understand the codes a bit more.”“.The above mentioned quotes demonstrate that lack of simplicity is a noticed barrier amongst the general dental practitioners.In addition to expressing their views on barriers of using the ICDAS system, participants suggested solutions or facilitators to help in uptake of the system by GDP’s in clinical settings and research. Most of the participants commented on making the ICDAS system computerised which would possibly lead to a wider majority willing to change their practice and increase their efficiency while using it.Because, it’s all on the paper the charting was a bit difficult to deal with. And, I think so with the new technology, even the ICDAS should be computerised.”“Yeah, I definitely think so because there are times that we had to start over with a new chart and things because there would be so many scribbles on it, which most of the time I had to redo in my spare time. Whereas, if it was computerized you could just double click and it would be sorted.”“The participants also revealed that providing more training and skills to the dental health professionals  from a grass root level on the ICDAS system would help in overcoming the barrier of not understanding and the disadvantage of spending a lot of time. Consequently, they would become more skilful and faster in using it; their mind-set would change:I think the main one is probably familiarization so I am used to doing it the way I was trained in university to do it. I do personally think that it takes longer, but I think if you did regularly have to use it you’d get quicker and you’d get more experienced in, that barrier maybe could be overcome….”“They also acknowledged that their use of the ICDAS system would have been a lot more efficient and pleasant in research, if they would have had additional instructions such as the use of a flow chart, pictures or having hands-on skills to make faster judgements of the caries codes. One of the participants stated:…I personally found that really helpful [when shown the ICDAS decision tree], like just working that logical way, so maybe having something like that available a flow chart that would help you, get you two numbers that you needed to so the result would be something I would have found useful…”“It was pointed out by a participant that if there was former teaching on the ICDAS system at an undergraduate level, it would have had a positive impact on their learning experiences and would have advantaged patients by using it early on in their dental practice:I would have liked to have had the training earlier because even in university because I found it quite helpful system for scoring caries and would have put it into my clinical practice earlier.”“Even though most of the participants had not recently used the ICDAS system because of the aforementioned barriers, they did mention how useful it is in providing better prevention and dental care; they would use it in their clinical practices if the barriers were to be overcome.I think it’s a really good way of charting because it is so thorough and it’s very systematic, so you know you are not missing any surface. Ahmm, which is a real benefit I think, it’s just so easy to skirt over the top of things. But, as I mentioned before there are some barriers to putting that in place.”“I think it would be easy, it would be definitely be easy, if everybody uses it.”“The aim of this study was to achieve a better understanding of the barriers and facilitators for GDP’s to use the ICDAS system in research and clinical practice. The literature review highlighted that there is a knowledge gap in the area of utilisation of the ICDAS system; therefore this study fills an evidence gap. There were four main themes identified as barriers complexity, cost implications, time disadvantage and training need and facilitators training, computerisation of the system, providing additional instructions that would lead to improving the utilisation of the ICDAS system in the future. The facilitators show that despite several barriers, the GDP’s want to provide prevention, better dental care and become more accustomed to the new.This is the first study to identify barriers and facilitators explained by the GDP’s who have used the ICDAS system. The barriers and facilitators identified by the GDP’s can be mapped to the theoretical domain framework (TDF) which aids the understanding of the findings and perhaps suggests next steps in the implementation of ICDAS. The TDF framework identifies the possible influences that are involved with the behaviour of health professionals, explanation of influences and their related domain; provides techniques to bring about a behaviour change . The TDFIn this study the quantitative measure of ‘knowledge’ was not explored; instead it looked at the ‘perceived knowledge’ of the GDP’s about the ICDAS system. These participants confirmed previous research findings that undergraduate level instructions and opinions from academic experts have strong influences on health care professional’s behaviour on their future practices . MoreoveThis study confirmed ‘cost’ is a crucial factor for dentists while considering a new diagnostic test into dental practice which was consistent with the findings from Berwick and colleagues . InsuffiMaterial investments play a role while utilising ICDAS too. Participants indicated that they would have found the ICDAS system more beneficial, if they were given additional materials for instructions such as pictures or flow charts that would aid them in making quicker clinical judgements in their busy working day.The fourth domain ‘social influences’ is a concept that states that individual thoughts are influenced by various factors such as peer pressure, opinions by colleagues, social norms and pressures from other groups . This noThe participants were also not aware of a much abbreviated version of ICDAS caries severity coding, Merged ICDAS, which has only 3 categories: initial, moderate and extensive caries severity . This moThe study sample comprised of GDP’s who took part in a research trial in the past, this might have influenced the respondents’ recall of certain elements of the ICDAS system. There may also be an element the information they presented being affected by others within the previous study. For example, either a positive or negative experience within the previous trail may have influenced the challenges and the ease to which ICDAS’s use was reported by the participants. Measures were taken to lessen the recall bias by the respondents by providing them with sample charts from the previous research to prompt their thoughts. To reduce the homogeneity between GDP’s in this study there was some purposeful selection of interviewees ensuring a balanced representation by gender (four men and four women), a spread of ages (25–55 years) and years of experience (3–20 years in practice as GDPs) and with different areas of dental interests . Regarding the sample size, GDP’s from a previous research study were chosen using convenient but purposive sampling, it may have been useful and the findings might have varied if the study was to have interviewed a larger sample size of dentists from various work settings e.g. community general dental surgeons, hospital based dental surgeons, specialist trainees.Moreover, the dependability of this study can be argued as the participants were GDP’s, they might not have wished to reveal their true weaknesses while using the ICDAS system for caries diagnosis process to the interviewer who was also a dentist. This bias might have influenced the findings in this study. However, the interviews were conducted in their personal dental practices, so that external influences could be avoided while they answered. If the participants would have been interviewed for a longer period of time, a more in-depth conversation with detailed findings might have been found. However, an attempt was made to reduce the influence of this drawback by conducting thematic analysis by Braun and Clarke’s method. An attempt was made to reduce this interviewer bias from this study by asking the same open ended questions from all the participants and increasing comparability between their answers; reaching a point of saturation. The investigator was given prior training on how to conduct neutral interviews without imposing their own pre-conception.Questions can be raised about the cause and effect interferences in this study due to the sample size not being large enough for the findings to be generalised. However, unlike quantitative research where a large sample size is needed for generalisability, in an qualitative research it is fundamental to explore meaning in depth rather than quantifying them . Most ofThis qualitative exploratory research study took place with UK-based dentists who had been working in general dental practice at the time of being involved in research utilising ICDAS; it was designed to explore the views and opinions of selected GDP’s regarding barriers and facilitators while using the ICDAS system in previous research. The findings reveal that GDP’s understand the important role the ICDAS system plays in caries prevention but there are various obstacles faced by the GDP’s while implementing the system into practice; also, there are possible drivers that are likely to overcome those barriers and increase the acceptance of the system. The research provided insights of the GDP’s, many facilitating factors such as making the ICDAS system computerised, giving additional training on the system to the GDP’s and dental nurses, ensuring undergraduate level teaching on the system (former teaching), additional incentives for using the system and for rewarding caries-preventive practices, could help in increasing the utilisation of the ICDAS system in clinical practice. Based on Rogers Diffusion of Innovation 2008, the adoption of new technologies is not a simple straightforward process it is a multi-level process that is influenced by contextual factors, national policies, organisational issues and adopter’s individual characteristics . Thus, tS1 File(ZIP)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


The accumulation of reactive oxygen species (ROS), lipid peroxidation and suppression of glutathione peroxidase (GPX) 4, which are characteristics of ferroptosis, were observed in glioblastoma cell after treatment of ALZ003. Furthermore, overexpression of AR prevented ferroptosis in the presence of GPX4. To evaluate the therapeutic effect in vivo, we transplanted TMZ-sensitive or -resistant U87MG cells into mouse brain followed by intravenous administration with ALZ003. In addition to inhibiting the growth of glioblastoma, ALZ003 significantly extended the survival period of transplanted mice, and significantly decreased AR expression in the tumor area. Taken together, AR potentiates TMZ resistance for glioblastoma, and ALZ003-mediated AR ubiquitination might open a new insight into therapeutic strategy for TMZ resistant glioblastoma.Drug resistance is the main obstacle in the improvement of chemotherapeutic efficacy in glioblastoma. Previously, we showed that dehydroepiandrosterone (DHEA), one kind of androgen/neurosteroid, potentiates glioblastoma to acquire resistance through attenuating DNA damage. Androgen receptor (AR) activated by DHEA or other types of androgen was reported to promote drug resistance in prostate cancer. However, in DHEA-enriched microenvironment, the role of AR in acquiring resistance of glioblastoma remains unknown. In this study, we found that AR expression is significantly correlated with poor prognosis, and AR obviously induced the resistance to temozolomide (TMZ) treatment. Herein, we observed that ALZ003, a curcumin analog, induces FBXL2-mediated AR ubiquitination, leading to degradation. Importantly, ALZ003 significantly inhibited the survival of TMZ-sensitive and –resistant glioblastoma   •ALZ003, the curcumin analog, inhibits TMZ–resistant glioblastoma.•ALZ003 induces AR degradation through FBXL2-mediated ubiquitination.•ALZ003 induces ferroptosis through blocking AR-mediated GPX4 expression.•ALZ003 increases ROS and lipid peroxidation through reducing GPX4 expression. The standard care protocol for glioblastoma is temozolomide (TMZ)-mediated chemotherapy conjugated with radiotherapy . The tumAndrogen receptor (AR), a steroid hormone receptor, mediates physiological functions through binding to its steroid ligands including dehydroepiandrosterone (DHEA), testosterone and dihydrotestosterone . Particuin vitro and in vivo, whereas it exhibited non-cytotoxic effect on normal astrocytes. Moreover, ALZ003 downregulated glutathione peroxidase 4 (GPX4) expression, leading to lipid peroxidation and abundant ROS accumulation. As caspases activation and lipid peroxidation are known to induce apoptosis and ferroptosis, respectively, ALZ003 is a potential therapeutic drug for primary and recurrent glioblastoma.Curcumin is a well-known tumor suppressive compound, and is considered to exhibit therapeutic effect on neurodegenerative disease because of its ability to cross blood-brain-barrier ,10. In t22.1Oncomine website (https://www.oncomine.org/resource/login.html), and TCGA lower grade glioma dataset was analyze as shown in Oncomine were also analyzed as shown in SurvExpress website (http://bioinformatica.mty.itesm.mx:8080/Biomatec/SurvivaX.jsp) [The comparison of AR mRNA level between normal and glioblastoma was performed using the vaX.jsp) . ParticuvaX.jsp)  was sele2.2U87MG and A172 cells were purchased from ATCC  and maintained in DMEM containing 10% fetal bovine serum (FBS), 100 μg/ml streptomycin and 100 μg/ml penicillin G . Luciferase-expressed U87MG cells were provided by Dr. Kwang-Yu Chang , and cells were maintained in DMEM containing 10% FBS, 100 μg/ml streptomycin and 100 μg/ml penicillin G. TMZ-resistant cells were established as described previously ,19. TMZ 2.3The use of human specimens was approval by the Institute Review Board (IRB)/Ethics Committee from the office of human research in Taipei Medical University . The patient consent and isolation protocol were described previously . The cul2.46 cells/cm2, and cultured in DMEM containing 10% FBS. After maintaining 72 h followed by the removing suspended cells, adhered cells were used for further experiments until 4th passage.Brain cortex was excised from post-natal Day 0 mice, and homogenized by 10 U/ml trypsin at 37 °C for 30 min. After mixing with DMEM containing 10% FBS , the cell suspension was filtered by a 70-μm nylon mesh filter . Cells were seeded onto culture plates at a density of 0.5 × 102.4.1The experimental procedure followed the protocol as described previously ,18. Brie2.4.2The protocol of previous study was followed. Cells were harvested in protein lysis buffer containing protease inhibitor cocktail . Five hundred microgram of protein was incubated with the anti-AR  or anti-ubiquitin (MerckMillipore) antibody followed by the precipitation using protein A/G agarose (MerckMillipore). After washing 4 times by lysis buffer, the complex was mixed with 2X SDS sample buffer containing β-mercapto-ethanol (MilliporeSigma Corporate), and subjected to western blotting.2.5After electrophoresis, proteins on the SDS-PAGE were transferred to polyvinylidene difluoride membrane and blocked by 5% non-fat milk. Blocked membrane was incubated with the primary antibody overnight at 4 °C followed by incubating with the secondary antibody against rabbit or mouse IgG for 1 h. After mixing with ECL chemiluminescent substrates,  were captured using the ChemiDoc™ Touch Imaging System . Primary antibodies were listed in the 2.6Caspase-Glo® 3/7, 8 and 9 assay kits were purchased from Promega Inc. , and were used according to the manufacturer instructions. After treatment, the cultured medium was collected, and mixed with reagents provided by Caspase-Glo® assay kit. The signal representing caspases was detected by the luminometer (Promega Inc.).2.7Cellular ROS was estimated using dihydrorhodamine 123 . After incubation with DHR for 30 min at 37 °C, DHR-derived fluorescence was measured by the flow cytometry and analyzed by the GuavaSoft software (MerckMillipore).2.82O2-Glo®  and GSH/GSSG-Glo®  assay kits were purchased from Promega Inc. and used according to the manufacturer instructions. For these 2 experiments, cells were seeded into 96-well plates. To detect H2O2 and evaluate GSH/GSSG, cell medium and lysates were prepared, respectively. After mixing with reagents provided in the assay kit, concentration of H2O2 and GSH in the mixture was determined by the luminometer (Promega Inc.). GSSG and the ratio of GSH/GSSG were calculated according to the instruction.The H2.96) were lysed for centrifugation, and supernatant was mixed with assay buffers provided in the kit. The absorbance of mixture was determined by ELISA reader  at OD 405 nm.The assay kit for GSHR activity was purchased from Biovision Inc. , and used according to the manufacturer instructions. Cells (1 × 102.10BODIPY™ 581/591C11 (Thermo Fisher Scientific), a commercial lipid peroxidation sensor, was used according to the manufacturer instructions. After drug treatment and siRNA-mediated gene knockdown, cells in the dishes were stained by adding BODIPY reagent and DAPI staining solution  into media. After 30 min, cells were monitored under fluorescence microscope (Leica).2.115) and U87MG-R cells (2 × 105) were injected into the cortex, respectively, at the depth of 3 mm using stereotactic guidance and microprocessor single syringe . After 10 days of transplantation, TMZ (15 mg/kg) [NOD-SCID male mice (8-week-old) were purchased from BioLASCO Taiwan Co., Ltd. , and maintained at the National Health Research Institutes . For intracranial transplantation, experimental procedure followed the previous study , and was5 mg/kg)  and ALZ02.12The protocol published previously was followed . The who2.12.1Total RNA was extracted using RNA Isolation Kit . After reverse transcription (Thermo Fisher Scientific), the mixture containing 1 μg of cDNA and SYBR Green (Thermo Fisher Scientific) was subjected to qPCR by PCR machine (Thermo Fisher Scientific) using primers targeting AR: F: CGGAAGCTGAAGAAACTTGG; R: ATGGCTTCCAGGACATTCAG.2.13t-test. To compare the difference in effect of TMZ with multiple doses on viability between control group and knockdown/overexpression group  and ASC-J9 are potent enhancer for AR protein degradation ,20, we p3.3ALZ003-mediated global protein ubiquitination was obviously increased in the time-dependent manner A. Additi3.42O2 level in parallel with the reduction of GSH/GSSG ratio (Disturbance of ubiquitination homeostasis has been shown to induce ROS production or impair redox reaction ,29. HencSG ratio B and C. SG ratio D. IntereSG ratio A which iSG ratio , suggestSG ratio C–a. We fSG ratio C–b and hSG ratio C–c. Thes3.5Although ALZ003 decreased both AR and GPX4 expression, whether AR regulates GPX4 is still unknown. In U87MG cells, knockdown of AR obviously decreases the protein level of GPX4, not GPX1, and dramatically induced lipid peroxidation A and B. 3.6in vivo, we established a mouse model intracranially transplanted with glioblastoma cells. In (Left Panel ofRight Panel ofin vivo(To clarify whether ALZ003 exhibits tumor-suppressive effect in vivo. The TMZin vivoA, and dein vivoB and C. in vivoD, suggesin vivoE. Under in vivoF and G, 4in vitro and in vivo. Through decreasing AR expression which regulates GPX4-mediated redox reaction, ALZ003 induced apoptosis and ferroptosis (Glioblastoma remains hard to be defeated by drug-mediated therapy because of very high frequency to develop drug resistance. In addition, most chemotherapeutic drugs, such as doxorubicin and cisplatin, are failed in brain tumor therapy due to the poor efficacy of crossing BBB ,32. Therroptosis , Fig. 5,Herein, although we found that FBXL2-mediated ubiquitination induces AR degradation , the proin vivo was not always significant with that in vitro [in vitro and in vivo through decreasing AR expression. The 48 h IC50 of ALZ003 in U87MG and Pt#3 is approximate <5 and 2 μM, respectively (Induction of AR degradation or repression of AR-mediated transcription is a well-known strategy to inhibit tumor development in prostate cancer ,38. Howein vitro , suggestectively , which iectively . Our resPreviously, we showed that DHEA, which is one kind of androgens or neurosteroids, induces TMZ resistance through enhancing DNA repair capacity . We hypoAlthough AR expression is important for redox homeostasis , the rolin vitro and in vivo. In multiple glioblastoma cell lines, ALZ003 potently suppresses cell survival. Additionally, ALZ003 inhibits tumor growth and extends the survival period of mice received intracranial transplantation with glioblastoma cells. We conclude that the strategy targeting AR will be potential for treating glioblastoma, and ALZ003 will benefit patients’ life in the future.Based on our study, AR is a promising target in glioblastoma, driving us to study effect the inhibitor targeting AR, including BBB-penetrating enzalutamide. However, disruption of ligand-induced AR activation is probably not efficient to inhibit AR-mediated transcription due to ligand-independent activation . CompareTCC, CHY and HC are employed by Allianz Pharmascience Ltd. when this collaborative study was conducted.


Drawing on social identity theory and positive psychology, this study investigated women’s responses to the social environment of physics classrooms. It also investigated STEM identity and gender disparities on academic achievement and flourishing in an undergraduate introductory physics course for STEM majors. One hundred sixty undergraduate students enrolled in an introductory physics course were administered a baseline survey with self-report measures on course belonging, physics identification, flourishing, and demographics at the beginning of the course and a post-survey at the end of the academic term. Students also completed force concept inventories, and physics course grades were obtained from the registrar.Women reported less course belonging and less physics identification than men. Physics identification and grades evidenced a longitudinal bidirectional relationship for all students (regardless of gender) such that when controlling for baseline physics knowledge: (a) students with higher physics identification were more likely to earn higher grades; and (b) students with higher grades evidenced more physics identification at the end of the term. Men scored higher on the force concept inventory than women; although no gender disparities emerged for course grades. For women, higher physics (versus lower) identification was associated with more positive changes in flourishing over the course of the term. High-identifying men showed the opposite pattern: negative change in flourishing was more strongly associated with high identifiers than low identifiers.Overall, this study underlines gender disparities in physics both in terms of belonging and physics knowledge. It suggests that strong STEM identity may be associated with academic performance and flourishing in undergraduate physics courses at the end of the term, particularly for women. A number of avenues for future research are discussed. First, women may experience lower levels of social belonging within the environment due to their female social identities. Belonging is a fundamental need : (1) the presence of positive emotion such as happiness and life satisfaction; (2) engagement in activities and tasks that help us reach a state of flow  living a life with meaning (subjectively defined); and (5) the accomplishment of goals for their own sake.A key question is how women in male-dominated STEM fields such as physics can have their belonging needs met and achieve strong academic performance and positive psychological health through positive and inclusive university experiences where students successfully actualize meaningful lives and careers. We propose that a meaningful step in this direction is to operationalize students’ well-being through a new conceptual framework called Flourishing is aligned with the goals of higher education in that universities are no longer charged with just graduating people with degrees, but with graduating individuals who are critical thinkers and life-long learners who seek to grow and make positive and innovative contributions to twenty-first century society , we define STEM identity as a type of social identity in line with Kim and colleagues  and see themselves and others in terms of specific prototypes of the STEM field   to what extent does physics identification at the beginning of the term in an introductory physics course predict various achievement outcomes (outlined below) at the end of the term? (research question 4); (b) To what extent does academic achievement predict STEM identification at the end of the term? (research question 5). Drawing on previous research, the following hypotheses were tested: controlling for pre-term physics knowledge (FCI scores), higher physics identification at the end of the term will be related to higher scores on a physics knowledge test (FCI scores) (hypothesis 4a) and higher physics course grades (hypothesis 4b) at the end of the term. Additionally, controlling for pre-term physics knowledge (FCI scores), higher post physics knowledge scores (hypothesis 5a) and physics course grades (hypothesis 5b) will be related to more physics identification at the end of the term.1A robust body of research underlines the profound mental and physical benefits of social connection that are associated with valued social identities  to examine gender differences social belonging in the physics classrooms; (2) to examine gender differences in physics identification and physics academic achievement; and (3) to assess the relationship of physics identification and gender on academic achievement and flourishing change. This longitudinal study was conducted in an introductory physics course for STEM majors (except life science majors). During required laboratory sections, students who agreed to participate in the study completed surveys on “how to improve physics education” at the beginning of the term and at the end of the term along with physics knowledge assessments. Participants provided consent for the research team to access their course grades. Overall, it was predicted that gender differences would exist in course belonging, physics identification, and academic achievement. It was also predicted that physics identification would affect achievement outcomes and that the influence of physics identification on flourishing change would be moderated by gender.N = 160) enrolled in an introductory physics course for STEM majors (except life science majors) and a co-requisite lab at a university in the Western United States were recruited for the study. Participants were sampled from three sections of the same course; two sections  were taught by a female professor and one section was taught by a male professor (n = 63). Participants in the sample consisted of the following STEM majors: engineering (67.4%), computer science/computer information systems (17.5%), mathematics (7.5%), biochemistry/biotechnology/chemistry (6.3%), geology (.6%), and physics (.6%). Males constituted the majority of students (72.5%) and females were in the minority (27.5%). The student sample was ethnically diverse: 47.5% White/Caucasian, 39.4% Hispanic/Latino, 20% Asian/Asian American, 17.5% did not indicate their ethnicity, 6.2% consisted of 2 or more races, 4.4% African American/Black/African, 3.8% Pacific Islander, and .6% Native American/Alaska Native. Participant ages ranged between 18 and 34 years , with 76.3% of participants in their first year of college, 6.9% second year, 9.4% third year, 2.5% fourth year, and 5.1% indicated being in their fifth year or beyond or did not indicate their year in college (refer to Table Undergraduate students (tudents 7.5% and fThe first survey was distributed at the beginning of the quarter (week 1) in 12 different sections of physics labs by the research team (three of the authors and research assistants). Approximately 15 min after a lab started, a research team member recruited participants by asking students to participate in a study designed to “improve physics education” and emphasized the confidentiality of participant responses. Participants who elected not to participate were asked to do a quiet activity for the duration of data collection. Participants took approximately 10–15 min to complete the survey. Once consent forms and surveys were completed, they were collected by the research team and participants were thanked for their time. Close to the end of the quarter (week 9), participants once again completed surveys during lab sessions. One lab section did not complete the second survey because the lab session was canceled. Most students in the physics labs and in our sample volunteered to complete both surveys (88.8%). The female professor’s two course sections had a response rate of 54.8% and 70% respectively, of students who completed both surveys. The male professor’s course section had a response rate of 73% of students who completed both surveys.physics identification  on a scale a scale ranging from 1  to 7 (very true) (α = .74). Physics identification was measured at the beginning and end of the term. The beginning of the term measure was employed in all analyses.Two items tapped into Course belonging consisted of two items that asked participants to rate: (a) I am not sure if I belong in this course, and (b) I belong in this course (α = .65). Belonging to the university included four items: (a) I belong at this university, (b) I feel like this university is a good fit for me, (c) I feel welcome on this campus, and (d) I am satisfied with my experience at university (α = .87).Two measures of social belonging were employed on response scales ranging from 1  to 7 (very true). force concept inventory  (α = .89) . Physics course grade was measured on a scale ranging from 1 (fail) to 4 (A—excellent), which is in line with widespread grade point average calculations in the United States.Physics course grades were obtained from the registrar’s office. The research team asked students permission to access their end-of-term physics course grades and most participants agreed .Intercorrelations and descriptive statistics for all continuous variables are listed in Table t tests supported this hypothesis. Women reported marginally lower course belonging  than men , t(67.42) = − 1.69, p = .09, 95% CI . No significant gender differences emerged for university belonging, n.s. Results for hypothesis 1 approached marginal significance with a Mann-Whitney U non-parametric t test (p = .10).It was predicted that women would report less belonging in the courses than men (hypothesis 1), but no significant gender differences would emerge for belonging to the university. Results from two sets of independent samples t tests provided support for the idea that: (a) men reported significantly higher physics identification at baseline  than women at baseline , t(59.87) = − 2.56, p = .01, 95% CI ; and (b) men reported significantly higher physics identification at the end of the term  than women at the end of the term , t(66.99) = − 2.21, p = .03, 95% CI . Results were replicated with Mann-Whitney U non-parametric t tests.Hypothesis 2 predicted that men would evidence higher physics identification at baseline and at the end of the term than women. Results from two independent samples t tests only yielded significant gender differences for FCI scores. Specifically, men  scored significantly better on the FCI at the end of the term than women , t(48.60) = − 2.18, p = .03, 95% CI . Following the suggestion of one of the manuscript reviewers, we conducted some additional analyses to shed additional light on gender differences on the FCI. Results from an independent sample t test examining gender differences on FCI at the beginning of the term showed that men  also scored significantly higher on the FCI at the beginning of the term than women , t(49.29) = − 2.89, p = .01, 95% CI . However, results showed no gender differences on the difference score of FCI (post FCI-baseline FCI), n.s. This suggests that men began the physics course with significantly more physics knowledge than women. However, over the course of the term, males and females made comparable gains in physics knowledge, but women still significantly lagged behind men in physics knowledge by the end of the course. In fact, women’s mean FCI score at the end of the term (M = 13.70) was comparable to men’s FCI at the beginning of the term (M = 13.52).Hypotheses 3a and 3b posited that men would score higher on knowledge test (FCI scores) in physics and earn higher grades than women. Results from two independent samples ). Additionally, controlling for pre-term physics knowledge (FCI scores), higher post physics knowledge scores (hypothesis 5a) and physics course grades (hypothesis 5b) would be related to more physics identification at the end of the term. A series of four step-wise hierarchical regressions were conducted to test these hypotheses. In all the step-wise hierarchical regressions, the first step (model A) involved regressing pre-term FCI scores on the dependent variable, which consisted of either post FCI scores (hypothesis 4a) or physics course grades (hypothesis 4b). The last step (model B) added either physics identification (hypotheses 4a–b) or post FCI scores (5a–b) to the regression equation. Results showed significant effects only for the regression analyses involving physics course grades (hypothesis 4b and hypothesis 5b), but not those pertaining to FCI scores (hypotheses 4a and 5a). Tables Hypotheses 4–5 predicted that controlling for pre-term physics knowledge (FCI scores), higher physics identification at the end of the term would predict higher scores on a physics knowledge test (FCI scores) (hypothesis 4a) and higher physics course grades (hypothesis 4bt(135) = 2.68, p = 0.008). On the other hand, results from simple slope analyses for men showed that higher physics identification was (surprisingly) associated with less flourishing over time  = − 2.17, p = 0.03).Hypothesis 6 predicted that all high identifiers would report gains in flourishing over the course of the quarter than low identifiers, particularly for women. This hypothesis was tested with a step-wise regression. We began the analyses by regressing a dummy-coded gender variable  on the flourishing change variable (difference score of baseline and post course flourishing) outcome (model A). In the next step, we added baseline physics identification into the regression equation (model B). In the final step, we examined whether physics identification was moderated by gender by adding the product term of gender and physical identification (model C) into the regression equation. Table The goal of the current study was to examine the longitudinal influence of STEM identity for women and men in an introductory physics university course for STEM majors from a positive education perspective. Various insights emerged from this research with important implications for STEM education. First, results showed that the social psychological experiences of women in physics classrooms may be different from those of men. Women STEM majors reported marginally less course belonging after taking an introductory physics course. Women also identified less with the field of physics both at beginning and at the end of the term than men. Of note, there were no gender differences reported in belonging at the university level, which suggests that the university environment may be relatively inclusive of women but that the situation inside the classroom is different. This underlines the need for additional research efforts to further examine classroom social environments and shed light on any aspects of the social environment within physics classrooms that may be contributing to gender differences in belonging and identification. Classroom climate may be a critical component of helping retain women in STEM, particularly for universities that have a predominantly commuter student population such as the current sample, where only 9% live on campus.Second, the current study showed that men scored higher on the FCI at the beginning and at the end of the term than women, but no significant gender differences emerged in FCI difference scores in FCI or for course grade. Gender differences on the FCI have consistently emerged in previous research . Due to the small sample size, these results should be interpreted with caution and replicated in other samples, but they do suggest that exposure to a female prototype of a physicist may have provided female students with hopes of success for minorities “like me,” which may positively influence women’s changes in flourishing levels. In other words, the presence of a female professor may help to buffer some of the negative experiences of being in a male-dominated field for women. This idea is supported by previous research. Undergraduate women who take math and science courses with women professors are more likely to report interest and anticipate success in STEM fields, and to more strongly associate women with leadership on implicit measures  and high identifiers may also take into account the gender of the professor in their overall perception of physics prototypes. In a male-dominated field such as physics, male professors may be perceived as more closely aligned with the physicist prototype at the beginning of the term than female professors. Since prototypes are context-specific and malleable , but the same pattern did not emerge for males with a male professor . This suggests that changes in prototypes may negatively affect members of the dominant group. Prislin and colleagues’ research consistently shows that in the aftermath of social change, there is considerable tension between majority and minority factions, and previous majority group members respond unfavorably to a relegated status  from the current study support this idea. Male students enrolled in the physics course with a female professor showed negative changes in flourishing at the end of the term . It is possible that STEM identity processes may differ for those who intend to major in a given field. As such, the results of the current study should also be replicated in samples of physics majors.all types of students from all walks of life to ensure success for all.The current study investigated responses to the social climate of physics classrooms by examining gender differences in course belonging. Next, the study examined STEM identity and gender disparities on achievement and changes in flourishing in an introductory physics course for STEM majors. Results highlighted that women report less belonging in physics courses, less physics identification (baseline and post) and less physics knowledge at the end of the term. It seems that the experience of women in physics classrooms is different from those of men. Another insight that emerged from the current study is that a strong STEM identity at the beginning of the term in a discipline is associated with stronger academic performance and positive gains in flourishing in at the end of the term for women. As such, the current research highlights that interventions that strengthen STEM identification for women may signal one promising approach to reduce gender disparities. Additionally, we outline a number of areas for future research inquiry, including more closely examining the social identity complexity of STEM identities , studying the malleability of STEM-based prototypes in education, investigating how to develop education interventions that increase STEM identification and alter the content of identity to boost students’ feelings of belonging, academic performance, and positive flourishing. With further work on STEM identity in undergraduate settings from a positive education perspective that includes a focus on both academic achievement and well-being, the STEM experience can blossom into an inclusive experience for