EPCAM, was up-regulated, while the mesenchymal markers vimentin and fibronectin were down-regulated after intervention with selenium. This implies an inhibitory effect of selenium on the epithelial-to-mesenchymal transition (EMT). Moreover, selenium was associated with down-regulated expression of genes involved in wound healing and inflammation; processes which are both related to EMT. In conclusion, our explorative data showed that selenium affected expression of genes implicated in EMT in the transition zone of the prostate.In parallel with the inconsistency in observational studies and chemoprevention trials, the mechanisms by which selenium affects prostate cancer risk have not been elucidated. We conducted a randomized, placebo-controlled trial to examine the effects of a short-term intervention with selenium on gene expression in non-malignant prostate tissue. Twenty-three men received 300 μg selenium per day in the form of selenized yeast (n=12) or a placebo (n=11) during 5 weeks. Prostate biopsies collected from the transition zone before and after intervention were analysed for 15 participants . Pathway analyses revealed that the intervention with selenium was associated with down-regulated expression of genes involved in cellular migration, invasion, remodeling and immune responses. Specifically, expression of well-established epithelial markers, such as E-cadherin and epithelial cell adhesion molecule  L-selenomethionine did not decrease the incidence of prostate cancer among men in the general population [Based on early observational and intervention studies, it has been suggested that an adequate status or intake of selenium may protect against prostate cancer –4. The Npulation , 9. For pulation . Similarpulation .The relatively high baseline selenium levels of the participants are often considered a plausible explanation for the unanticipated findings of subsequent trials as compared to the early NPC trial , 10, 11.L-selenomethionine per day differences exist in gene expression profiles in human prostate tissue as compared to a placebo group [L-selenomethionine itself did not show preventive effects for prostate cancer in the SELECT trial [In parallel with the inconsistency in observational studies and chemoprevention trials, the mechanisms by which selenium may affect prostate cancer risk have not been elucidated. Supplementation with selenium resulted in increased levels of selenium in the prostate as shown in previous studies . Furtherbo group . WhetherCT trial . Other fCT trial , 2, howeThe aim of the current study was to obtain more insight into the molecular pathways affected by selenium in the prostate. Therefore, we examined whole-genome expression profiles in non-malignant prostate tissue before and after a 5-week intervention with selenized yeast in a randomized, placebo-controlled trial in The Netherlands. The collection of prostate tissue before and after intervention allowed us to compare changes in gene expression within individuals over time.From June 2007 to October 2010, 281 men were assessed for their eligibility to participate in this study. Of these, 23 men were finally enrolled; 12 participants were randomized to the selenium group and 11 participants to the placebo group . BaselinBaseline levels of serum (p=0.562) or toenail (p=0.449) selenium did not differ between the two groups Table . As compGood quality RNA from prostate biopsies from the transition zone without evidence of malignancy was available before and after the intervention for 15 participants, resulting in a total of 30 microarrays used in this study . After RTo obtain more insight into the role of the differentially expressed genes, pathway analyses were conducted. The top-5 regulated canonical pathways are presented for both the selenium and the placebo group in Figure A gene set enrichment analysis (GSEA) was performed to explore the consistency of our findings for the selenium supplementation. Gene sets enriched in the selenium group are visualized in Figure TGFB1) was identified as an upstream regulator predicted to be inhibited in the selenium group and activated in the placebo group .Based on the IPA Upstream Regulator Analysis, we identified regulators that may have been responsible for the observed expression changes. Predicted upstream regulators were considered relevant if the z-score was below -2 or above 2 and if the p-value of overlap was <0.05. The top-5 (based on z-score) of the predicted activated and inhibited regulators is presented in Table TGFB1 and a selection of other predicted upstream regulators in the selenium group, i.e. the SAM pointed domain containing ETS transcription factor (SPDEF) and WNT1 inducible signaling pathway protein 2 (WISP2), showed some common targets that are directly or indirectly controlled by these regulators (VIM), E-cadherin (CDH1), OB-cadherin (CDH11) and connective tissue growth factor (CTGF) are all considered to be regulated by TGFB1, SPDEF and WISP2. Interestingly, both TGFB1 as well as the down-stream genes CDH1, CDH11, VIM and CTGF are suggested to be involved in the epithelial-to-mesenchymal transition (EMT). EMT is a process characterized by the transition of an epithelial phenotype towards a mesenchymal phenotype and is implicated in embryogenesis, inflammation, wound healing, as well as cancer progression and metastasis.Focusing on the network of gulators . VimentiFN1 (fibronectin), VIM, and CDH11 was down-regulated, while ‘epithelial markers’, such as CDH1, SDC1 (syndecan-1) and EPCAM , were up-regulated after intervention with selenium.Based on literature , we obtaIn order to explore the consistency and robustness of our findings, we correlated the selenium-induced expression changes of all statistically significant  EMT-related genes amongst each other. The corresponding Pearson correlation matrix for selenium Figure  showed tOur study demonstrated that a 5-week intervention with selenium was associated with changes in expression of genes implicated in EMT in non-malignant prostate tissue. EMT is characterized by the transition of an epithelial towards mesenchymal phenotype with its accompanying properties such as the ability to migrate and invade , 17. BasEMT plays an important role during embryogenesis, inflammation and wound healing as cellular migration and invasion facilitates organ development, tissue (re)modelling and regeneration , 18. In The intervention with selenium was also associated with expression changes of genes involved in wound healing, cellular adhesion and extracellular matrix interactions. Based on our GSEA results, we identified a cluster of gene sets related to wound healing which were down-regulated after the intervention with selenium. The pronounced effects hinting towards wound healing in the placebo group may again refer to repair and remodeling of prostate tissue resulting from the (repeated) biopsies. Interestingly, in the selenium group expression of genes involved in wound healing was down-regulated. Based upon these data, we carefully hypothesized that selenium may contribute to accelerated or improved wound healing in the prostate.GAJ1 gene [GAJ1/Cx43 as well as GJC1/Cx45, whereas in the placebo group the expression of these connexins did not change. Interestingly, increased expression of GAJ1/Cx43 has also been linked to metastatic behavior of prostate cancer cells [So far, the role of selenium in wound healing has not been extensively described. Few small clinical studies or animal experiments suggest that selenium may improve and accelerate wound healing under various conditions , 20. OneAJ1 gene . ExperimAJ1 gene . Our inter cells , 24.TGFB1) is a major inducer of EMT [TGFB1 is significantly down-regulated after the intervention with selenium, while up-regulated in the placebo group. TGFB1 was also one of the main predicted upstream transcriptional regulators. Changes in protein expression of TGFbeta in serum of elderly men after selenium supplementation have been previously described [The potential mechanisms underlying the effect of selenium on EMT, with or without a direct relationship to wound healing and inflammation, have not been described in detail previously. It has consistently been shown that transforming growth factor β1  as an upstream transcriptional regulator. SPDEF is highly expressed in normal prostatic epithelial tissue [SPDEF is required for CDH1 expression in prostate cancer cells [SPDEF is a downstream target of TGFB1 [SPDEF expression which is in line with the epithelial phenotype and the increase in CDH1 expression. In previous studies, it was consistently shown that SPDEF inhibits cancer cell migration in vitro [in vivo [SPDEF in prostate cancer cells [We also identified the sterile alpha motif (SAM) pointed domain-containing ETS transcription factor  seemed to benefit from the intervention with selenized yeast . The medA limitation of our study refers to the relatively small number of participants. Recruitment of participants was complicated inherent to the design of our study which required prostate sample collection at two consecutive time points and the unanticipated negative outcome of the SELECT trial. To what extent sample size affects the power to detect gene expression changes is difficult to assess for these data-driven expression profiling studies. At baseline, differences in gene expression exist between the selenium and placebo group (n=2624 genes), which may be attributable to unanticipated variation or outliers and the relatively small sample size. A subset of these genes was also differently expressed upon the intervention with selenium . We cannot exclude the possibility that these baseline differences to some extent affected the responses observed. However, despite substantial variation and differences between the groups at baseline, gene expression changes were highly consistent within the groups after intervention. Furthermore, we examined effects of selenium supplementation at a transcriptional level and therefore we cannot rule out any potential post-transcriptional effects that may determine activity and functionality of other potential regulators, such as the selenoproteins.For the current study, we collected non-malignant tissue from the transition zone of the prostate. Approximately 20% of the prostatic adenocarcinomas arise from this zone, in comparison to 70-80% from the peripheral zone . The traIn conclusion, our explorative data imply that selenium is associated with the regulation of genes involved in cellular migration, invasion, tissue remodeling and the immune response in non-malignant tissue collected from the transition zone of the prostate. These processes are implicated in EMT and related events such as inflammation, wound healing and cancer progression. Taken altogether, these results suggest a preventive effect of selenium on prostate cancer progression rather than on prostate cancer development and future studies are warranted to confirm this hypothesis.For this study, we collected non-malignant prostate tissue before and after a short-term intervention with selenized yeast or yeast placebo. We recruited participants from the Radboud university medical center, an academic tertiary referral center in Nijmegen, The Netherlands. Men scheduled for diagnostic prostate biopsies, and subsequent treatment with radical prostatectomy (RP) or radiotherapy (RT) for prostate cancer, were invited for this study. Also, men scheduled for re-biopsies because of high-grade prostatic intraepithelial neoplasia (HGPIN) were eligible for inclusion. For practical and ethical reasons, the men included in this study were all at high risk for prostate cancer or diagnosed with prostate cancer, as collection of prostate tissue for the study had to be combined with standard invasive clinical procedures. However, for all participants only non-malignant prostate tissue, verified by histological examinations, was considered for the analyses.Exclusion criteria were current use of dietary supplements providing more than the recommended daily allowance of 55 μg selenium per day, any malignancy in the preceding five years (except for non-melanoma skin cancer), current hepatic or renal disease or inflammatory bowel disease, and neo-adjuvant therapies for prostate cancer.Overall, 23 participants were eligible for participation in the intervention study . Before clinicaltrials.gov with identifier NCT00446901.Participants were randomly assigned using a permuted-block design (blocks of four participants) to take 300 μg selenium per day in the form of selenized yeast  or a placebo . The non-selenized yeast tablets were based on yeast grown on selenium-deficient medium and smell, taste and appearance did not differ from the intervention tablets. Both intervention and placebo tablets were previously used in the PRECISE Trial pilot studies –51 and hTM, SERO AS, Norway) were included. All samples were analyzed in triplicate.For serum selenium analyses, blood was collected into 10-ml serum tubes (Becton Dickinson B.V.). Serum was collected after centrifugation and stored at -20°C until analyses. Serum selenium levels were measured using an atomic absorption spectrometer  coupled with a graphite furnace and using Zeeman background correction . The detBaseline toenail selenium levels, indicative of a long-term selenium intake and status , were asProstate tissue was collected using an 18-gauge biopsy needle (Bard Biopsy Systems) during regular 10-core prostate needle biopsy series guided by transrectal ultrasound (TRUS) or with a biopsy during RP. In both cases, the study biopsy was taken from the superior, ventral region of the left lobe of the prostate, which is specified as the transition zone. The study biopsy was directly embedded in Optimal Cutting Temperature (O.C.T.) Compound (Sakura Finetek Europe B.V.) and snapfrozen in liquid nitrogen. All samples were stored at -80°C.The biopsies embedded in O.C.T. Compound were sectioned at -20°C. Representative sections of 5 μm were used for histological examinations; at least two sections were used for a Haematoxilin-Eosin (HE) staining. The remainder of the biopsy was sectioned at 20 μm and used for RNA extraction. All HE-stained slides were reviewed for malignancy, HGPIN and inflammation by an expert uropathologist.Total RNA was extracted from the sectioned prostate biopsies using TRIzol Reagent according to the manufacturer’s instructions (Invitrogen). Isolated RNA was purified using RNeasy Micro columns (Qiagen). RNA integrity  and total RNA yield  were assessed for all samples. The mean RNA integrity (RIN) score was 8.2 (standard deviation: 0.66). Prostate biopsies with inadequate RNA yield  or histological evidence of adenocarcinoma (n=6) were excluded, leaving biopsies of 15 participants available for microarray analyses  .A total of 30 RNA samples were processed for microarray analyses. Briefly, 100 ng of total RNA per sample was converted to cDNA, labeled using an Ambion WT Expression kit and hybridized to Affymetrix GeneChip Human Gene 1.0 ST Arrays. Probe sets were redefined according to Dai et al.  using thwww.ingenuity.com). For these analyses, expression of genes was considered up- or down-regulated if the p-value for the within- and between-group comparisons was <0.05. Canonical pathways with a p-value of <0.01 in the Fisher’s exact test were considered as significant to the data. IPA Upstream Regulator Analysis was used to predict common regulators that may be responsible for the observed changes in gene expression. Prediction of regulators is based on two parameters; the z-score and the p-value of overlap. The z-score reflects the consistency of the gene expression patterns of the downstream genes in the dataset, whereas the p-value of overlap is calculated by a Fisher’s exact test for the overlap between the genes in a dataset (expected) and the genes actually regulated by the potential transcriptional regulator (observed).Regulated pathways were identified through the use of IPA  [In order to further explore the biological relevance and consistency of our findings, we also conducted Gene Set Enrichment Analyses   for the dex.jsp)  was useddex.jsp) . Gene seGitools software was used to construct a correlation matrice showing the Pearson correlation coefficients for the individual expression changes expressed as signal-log-ratios of selected genes .Since levels of selenium after intervention, duration of intervention and a number of other participants’ characteristics were not normally distributed, data were summarized as median and interquartile ranges or numbers and percentages. Baseline serum and toenail selenium levels were compared between the selenium group and the placebo group using the Mann-Whitney U test. Serum selenium levels after intervention were compared to baseline values using the Wilcoxon Signed Rank test. All statistical tests were two-sided and p-values below <0.05 were considered as statistically significant. Statistical Package for Social Sciences  was used for all analyses unless otherwise stated.


During the reality monitoring task, subjects made judgments as to whether information was previously self-generated (self-agency judgments) or externally derived . During speech feedback monitoring, we assessed self-agency by altering environmental auditory feedback so that subjects listened to a perturbed version of their own speech. When subjects heard minimal perturbations in their auditory feedback while speaking, they made corrective responses, indicating that they judged the perturbations as errors in their speech output. We found that self-agency judgments in the reality-monitoring task were higher in people who had smaller corrective responses (p = 0.05) and smaller inter-trial variability (p = 0.03) during minimal pitch perturbations of their auditory feedback. These results provide support for a unitary process for the experience of self-agency governing low-level speech control and higher level reality monitoring.Self-agency is the experience of being the agent of one’s own thoughts and motor actions. The intact experience of self-agency is necessary for successful interactions with the outside world  and for responding to sensory feedback of our motor actions . Reality monitoring is the ability to distinguish internally self-generated information from outside reality . In the present study, we examined the relationship of self-agency between lower-level speech feedback monitoring  and a higher-level cognitive reality monitoring task. In particular, we examined whether speech feedback monitoring and reality monitoring were driven by the capacity to experience self-agency—the ability to make The intThe experience of self-agency, a necessary component of reality monitoring, enables accurate judgments that “I generated my own actions” Haggard, . This exThe experience of self-agency can also be observed during speech production experiments in which speakers monitor their auditory feedback while speaking . The predictions that participants make during speech feedback monitoring are largely automatic, unconscious and prospective, involving comparing incoming sensory feedback with predictions of that feedback  would be correlated with self-agency during reality monitoring (indexed by accurate identification of self-generated information). We had two specific hypotheses: (1) The magnitude of corrective responses only during minimal pitch perturbations would negatively correlate with accurate self-agency judgments on the reality-monitoring task. In other words, we predicted that speakers who make smaller corrective responses to compensate for their errors during these minimal pitch-induced perturbations would manifest a greater sense of self-agency, reflecting their increased reliance on internal predictions to guide their speech output, rather than reliance on external altered feedback to influence their speech output. (2) Inter-trial variability in the magnitude of corrective responses only during minimal pitch-induced perturbations would negatively correlate with accurate self-agency judgments on the reality-monitoring task. In other words, we predicted that smaller inter-trial variability in the magnitude of a subject’s corrective responses during minimal pitch-induced perturbations would also indicate increased confidence in the subject’s sense of self-agency, and would correlate with enhanced accurate self-agency judgments on the reality-monitoring task.In the present study, we recruited 19 healthy participants . This study was approved by the Internal Review Board at the University of California San Francisco (UCSF). All participants gave written informed consent and then completed in the pitch perturbation task. Inclusion criteria for healthy participants were: no psychiatric/neurological disorders, no substance dependence or current substance abuse, good general physical health, age between 18 years and 60 years, right-handed and English as first language. One participant was unavailable to complete the reality-monitoring task.Subjects wore an over-the-ears microphone  and a pair of circumaural headphones. The microphone was connected to a pre-amplifier (specs) that was connected to a sound card  in a computer . The amplified audio signal was played back through the headphones.The pitch perturbation experiment consisted of two runs  with 74 trials per run. Each trial began with a large green dot that appeared on the screen of the computer. Subjects were instructed to start vocalizing the vowel /a/ when they saw the green dot. They continued phonation for 2.4 s until the dot disappeared while listening to real-time auditory feedback via headphones. There was an inter-trial interval of 2.5 s.In each trial, the phonation onset triggered a brief perturbation  in the pitch of the subject’s feedback  and the feedback audio data (headphones output) were recorded at a sampling rate of 11,025 Hz. Pitch data from all 19 subjects were included in the analysis. Using an autocorrelation-based pitch tracking method, time-courses of all trials were plotted Parsons, . Time in(t) = 1200 log2(Hertz(t)/HertzRef) where Hertz(t) is the pitch value in Hertz at time = t and HertzRef is the reference pitch in Hertz which is the mean pitch in a window spanning 50 ms prior to perturbation onset to 50 ms after perturbation onset.CentsParticipants responded to applied pitch perturbations by deviating from their baseline pitch track. For each participant, the mean of all pitch response tracks prior to perturbation was considered as the baseline pitch track. Responses in each trial were then computed as deviations from this baseline pitch track. To assay the magnitude of pitch deviations for each person, we computed the peak response in relation to the baseline pitch value  in each trial and then computed the average peak response across trials, as we have performed in earlier studies The  or was externally-derived as it was provided by the experimenter . Altogether, participants were given 100 sentences for subjects to generate the final word  sentences and 100 externally-derived sentences, presented in blocks of 20 trials per run. For each sentence, participants were told to pay attention to the underlined words and to vocalize the final word of each sentence , and had to identify with a button-press whether the second word was previously self-generated or externally-derived  with reality-monitoring performance . Effect sizes (Cohen’s d) were used to quantify the power of the linear relationships. Outliers were defined as values above/below 3 standard deviations from the mean. We did not find any outliers in any of the correlations. Mean RT was computed for correctly identified self-generated and externally-derived information.F = 8.92, p = 0.008; Figure F = 28.01, p < 0.0001). We did not find any difference between 100 vs. 400 cents pitch perturbations in the time to reach peak magnitude from perturbation onset  = 0.77, p = 0.39; see Table The behavioral responses to brief pitch perturbations in auditory feedback were mostly compensatory  on both 100 cents (13 out of 19 participants made compensatory responses on average) and 400 cents (14 out of 19 participants made compensatory responses) experiments. In the example shown in Figure F = 40.01, p < 0.0001) although not more accurate  = 1.14, p = 0.30) at identifying self-generated information when compared to externally-derived information (see Table r(16) = −0.46, p = 0.05; Figure In the reality monitoring task, participants were faster . These results are consistent with our hypothesis, in which we expected to only find correlations between self-agency judgments during reality monitoring with the peak magnitude of deviation responses during minimal pitch-induced perturbations experienced with the 100 cents pitch shift, as only these minimal deviations in perturbations are thought to maintain the sense of self-agency.We did not observe any significant associations between 100 cents pitch perturbation responses and accuracy on identification of externally-derived information on the reality monitoring task. We also did not observe and significant associations between 400 cents pitch perturbations responses with either self-generated or externally-derived identification on the reality monitoring task  = −0.51, p = 0.03; Figure p’s > 0.40). Also, the length of time to reach peak magnitude from perturbation onset was not associated with either self or external agency judgments during reality monitoring, suggesting that only the size and inter-trial variability of perturbation responses provided reliable markers of self-agency judgments, and only during the 100 cents pitch perturbation. Finally, Cohen’s d analyses indicated that the correlation coefficients between self-generated accuracy during reality monitoring with peak perturbation size (Cohen’s d = 0.59) and variability (Cohen’s d = 0.48) during the 100 cents shift yielded large effect sizes. Together, these findings across converging different types of analyses provide support for our hypothesis that there is a unitary sense of self-agency that results from the ability to make reliable predictions about the outcomes of self-generated actions.We observed an even stronger negative correlation between the inter-trial variability in peak response to the 100 cents pitch perturbation with self-agency  during the reality-monitoring task (We found that when participants’ pitch was perturbed by 100 cents: (1) their peak perturbation responses were larger compared to when their pitch was perturbed by 400 cents; (2) their peak perturbation responses negatively correlated with accurate self-agency judgments  on the reality-monitoring task; and (3) the inter trial variability in their peak perturbation responses negatively correlated with their self-agency judgments on the reality-monitoring task.Our findings are consistent with prior work indicating participants produce larger perturbation responses to 100 cents pitch perturbations than 400 cents pitch perturbations . This likely reflects their increased reliance on internal predictions to guide their speech output, rather than reliance on external altered feedback to influence their speech output. It must be noted that only the perturbations experienced during the 100 cents pitch perturbations (not the 400 cents perturbations) are interpreted as self-generated as only these minimal pitch perturbations are thought to maintain the sense of self-agency. As mentioned previously, larger pitch perturbations greater than 250–300 cents, are interpreted as non self-generated outcomes that are due to external changes in the environment rather than being reflective of judgments in self-agency , reflecting their increased reliance on internal predictions to guide their own actions (their speech output). reliable predictions about the outcomes of self-generated actions. This is why measures of self-agency during lower level speech feedback monitoring correlated and predicted self-agency judgments  during higher-level reality monitoring.Taken together, our findings across converging different types of analyses provide evidence for the existence of a unitary sense of self-agency, and this results from the ability to makeIn conclusion, the sense of agency is critical for human beings to interact with the outside world. This is a first-of-its-kind study in which we clearly demonstrate how internal predictions regarding the outcomes of self-generated actions can modulate the experience of self-agency. These findings show that self-agency during lower-level speech feedback monitoring  directly correlates with self-agency judgments during higher-level reality monitoring  who manifest severe deficits in judgments of self-agency. Future studies are needed to investigate both behavioral and neural responses to auditory feedback perturbations during speaking in healthy people as well as in patients with psychosis. This will help further validate the speech perturbation and reality monitoring paradigms as accurate assessments of the experience of self-agency.KS recruited all the subjects and designed the reality monitoring experiment; acquired and analyzed the reality monitoring data; and interpreted the data and wrote and edited the manuscript. HK helped to design the speech monitoring experiment, acquired and analyzed the speech monitoring data. DM helped with recruitment of subjects and helped with designing and acquiring the reality monitoring data. SSN edited the manuscript, and provided advice on the acquisition, analyses and interpretation of the data; developed the speech monitoring experiment, and provided advice on the acquisition, analyses and interpretation of the data. JFH edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study was to investigate the bond strength of ceramic restorationsluted using a self-adhesive resin cement  under differentdentin conditions.In the experimental groups, ceramic restorations were luted to bovine incisorswith RelyX Unicem under the following conditions: [Dry dentin]:surface was dried using air stream for 15 s; [Moist dentin]: excessdentin moisture was removed with absorbent paper; [Bonding agent]:Clearfil SE Bond (Kuraray) self-etching adhesive system was previously applied todentin. In the Control group, cementation was done using an etch-and-rinseadhesive (Excite DSC) and Variolink II resin cement (Ivoclar Vivadent).Photoactivation of the resin cements was performed with UltraLume LED 5 unit(Ultradent). The restorations (n=5 per group) were sectioned into beams andmicrotensile testing was carried out. Data were subjected to ANOVA and Tukey'stest (p<0.05). Failure modes were classified under Scanning ElectronMicroscopic (SEM) (×120 magnification).The bond strength was dependent on the moisture status of the dentin. Bondstrength in the "dry dentin group" was significantly lower than that of all othergroups, which showed similar results. A predominance of mixed failures wasdetected for the control group, while a predominance of adhesive failures wasobserved for the "bonding agent" and "dry dentin" groups. The "moist dentin" grouppresented predominantly cohesive failures within the luting material. The previousapplication of a self-etching adhesive showed no significant effect.Only excess dentin moisture should be removed for the cementation of ceramicrestorations with self-adhesive resin cements. In addition toetch-andrinse adhesives, self-etching systems are used with the purpose of eliminatingthe rinsing/drying steps and facilitating the bonding procedure. The self-etchingapproach also potentially reduces the occurrence of the postoperative sensitivity thatmay occur due to incomplete infiltration of the demineralized dentin19. Previous studies have reported similarbond strengths to dentin for some etch-and-rinse and self-etching systems depending ontheir composition and application steps20.The luting procedure of ceramic restorations requires several sequential steps, and theuse of adhesive systems associated with resin-based luting agents is verycommon16. The use of these materials should alsoprevent the incomplete infiltration of dentin and reduce the occurrence of postoperativesensitivity. Their adhesive properties are attributed to acidic monomers thatsimultaneously demineralize and infiltrate the tooth substrate, resulting inmicromechanical retention. Secondary reactions have been suggested to provide additionalchemical bonding to the dental hard tissues7.Self-adhesive luting materials were introduced in an endeavor to reduce the number ofcementation steps by eliminating the previous application of bonding agent or otherpre-treatment of the tooth14. The dominant setting reaction occurs via free radicalpolymerization, initiated either by light or a redox system that allows thepolymerization in an acid environment14. Water has a critical role in bonding effectiveness: water isgenerated during neutralization of the functional groups modified by phosphoric acid andreused to react with acidic functional groups and ion-releasing basic fillingbodies14. However, it is unknownwhether the amount of water generated during cement setting is sufficient for properbonding, or whether dentin moisture might influence the bonding mechanism.The basic inorganic fillers in self-adhesive luting agents are able react with thephosphoric acid methacrylates present in the materialThe influence of previous application of a self-etching adhesive system on the bondingof self-adhesive luting agents is still unknown. Literature is lacking of studiesevaluating the influence of dentin conditions on the performance of self-adhesive lutingagent. The aim of this study was to investigate the bond strength to dentin of ceramicrestorations luted with a self-adhesive resin luting agent under different dentinconditions: wet dentin, dry dentin or dentin previously treated with a self-etchingadhesive. The null hypothesis tested was that substrate moisture and application of aself-etching system do not interfere with the bonding to dentin.Rectangular specimens (10×8×2.5 mm) were made of leucite-reinforced glass ceramic, shade eTC 2, used inaccordance with the manufacturer’s instructions. Briefly, cylindrical patterns weremade with organic wax, invested with phosphate-based material  and heated at 850ºC for 1 h in an oven . The ceramic was then heat pressed into the molds,using the eP600 furnace (Ivoclar Vivadent). After cooling to room temperature, thespecimens were divested, polished with 1200-grit SiC papers, and ultrasonicallycleaned in water for 10 min. The internal surfaces of the ceramic blocks were etchedwith 10% hydrofluoric acid for 20 s, rinsed with water for 1 min, and received alayer of silane coupling agent .Bovine incisors were obtained and their crowns were sectioned 7 mm below the incisaledge with a double-face diamond disc  under air-water cooling. The surrounding enamel was removed using diamondburs , the dentin surfaces were wet-polished with 600-grit SiCpapers , and the root portions of theteeth were embedded in epoxy resin. The teeth were randomly divided into four groups(n=5) defined by the dentin condition:"Dry dentin" group: the dentin surface was dried with air for 15 s and theself-adhesive resin luting agent RelyX Unicem , shade A2,was used following the manufacturer’s instructions;"Moist dentin" group: only the excess dentin moisture was removed using absorbentpaper, and the same procedures described for the previous group were performed;"Bonding agent" group: the dentin surface was dried with air for 15 s and a two-stepself-etching bonding agent  wasapplied according to the manufacturer’s instructions, followed by application ofRelyX Unicem, as described for the previous groups.Control group: the dentin surface was dried with air for 15 s, etched with 37%phosphoric acid gel for 15 s, rinsed with water for 30 s, and blot dried leaving amoist surface. An etch-and-rinse adhesive system  and adual-cured resin luting agent , shade A2, were used,according to the manufacturer instructions.2), with afinal 40-s exposure from the top surface.In order to obtain specimens for the microtensile test, blocks (4 mm in height) ofself-polymerizing resin composite were built-up on the ceramic surfaces to increase the height of the sample. Thespecimens were stored in 100% relative humidity at 37°C, for 24 h. Thereafter, thecomposite-ceramic-cement-tooth sets were cut perpendicular to the bonding interfaceinto beam specimens using a water-cooled diamond saw . The cross-sectional area of the bond interface of each beam wasmeasured with a digital caliper  and themicrotensile test conducted on a mechanical testing machine  at a crosshead speed of 0.5 mm/min until failure. Bondstrength values were calculated in MPa. An average of six beams was obtained for eachtooth, and the mean value of the six beams was computed as the bond strength valuefor each specimen. Bond strength data were subjected to one-way ANOVA and multiplecomparisons were performed using the Tukey’s post-hoc test. Differences wereconsidered significant at p<0.05. In the event of spontaneous debonding during thesectioning procedures, the specimens were excluded from the statistical analysis.10 , as follows: adhesive failure (Mode1), mixed failure involving bonding agent, dentin and luting material (Mode 2); mixedfailure involving luting material and dentin (Mode 3); cohesive failure within thebonding agent (Mode 4); cohesive failure within the luting material (Mode 5).The fractured specimens were coated with gold and examined with a scanning electronmicroscope (SeM) , at a ×120 magnification.Their modes of failure were classified using a modified criterionResults for the microtensile bond strength test are shown in The failure analysis demonstrated that the mode 2 was the predominant mode of failurefor the control group. The bonding agent and the dry dentin groups showed apredominance of failure mode 1. In contrast, a predominance of failure mode 5 wasdetected for the group in which the bonding was performed to moist dentin. Thepercentage of failure modes in each group is shown in 14. However, the current results suggestthat the water present in the substrate might also play an important role on the bondingmechanism. The increased water availability on the dentin probably improved the acidionization and etching effect, enhancing the bond between the negatively chargedphosphoric acid groups to the Ca ions on dentin. This result is in line with a recentstudy9 , which observedincreased bond strength to dentin when a self-adhesive cement was applied undersimulated pulpal pressure.The null hypothesis tested in this study was rejected, as the self-adhesive cement hadlower bond strength to the dry compared with the moist dentin. RelyX Unicem needs waterfor ionization of the acidic monomers to modify the smear layer and interact with thedentin. The initially anhydrous cement bonds to the substrate via mechanisms of watergeneration and subsequent water recycling, as proposed by the manufacturer17. Although previous studies have suggested that the weaklink in self-adhesive luting systems lies in their lack of genuine hybridization of thebonding surfaces13 , similar bond strengths were observed for the "moistdentin" group compared to the "bonding agent" and control groups in the present study.Therefore, it seems that application of a self-etching adhesive prior to the use of theself-adhesive luting agent has no beneficial effect for self-adhesive cements.Nonetheless, it is difficult to predict whether similar long-term performances would beobserved among these groups, as the quality of the hybrid layer formed is related to theresistance to bond degradation over the course of time11.Adhesive systems promote better interaction with the dentin than self-adhesive cements,due to the infiltration of the bonding agent into the substrate and formation of ahybrid layerOn the other hand, different failure results were detected among the groups. Apredominance of adhesive failures was observed for the dry substrate, confirming theweak interaction between the self-adhesive cement and dry dentin surface. This can beexplained by the lower water availability, poorer ionization and, in association withthe high viscosity of the cement, insufficient monomer infiltration into the substrate.In contrast, cohesive failures within the luting agent were predominant in the "moistdentin" group. This result might suggest that the mechanism of bonding to moist dentinwas improved. However, as shown in 18. These unprotectedareas may have served as spots for stress concentration during the tensile test,generating failures involving not only the bonding layer, but also the dentin tissue. Incontrast, the predominance of adhesive failures for the self-etching system is probablyrelated to its lower ability in creating micromechanical retention compared to theetch-andrinse adhesive has, leading to failures mainly at the dentin-adhesiveinterface.In the control group, there was a predominance of mixed failures involving bondingagent, dentin and luting material. This might be explained by the in-depthdemineralization of the dentin by the phosphoric acid, leaving non-encapsulated collagenfibrils after bonding, because of the inability of the bonding agent to fully infiltratethe exposed meshinvitro study do not take into account the effect that the pulpal pressuremight have on dentin permeability9 ,which could potentially overcome the lower water availability. In addition, it isuncertain whether the previous application of bonding agent could affect the polymernetwork formation of the cement. Moreover, the long-term bonding performance of thematerials and techniques tested in the present study must be investigated. Therefore,further laboratory and clinical studies are necessary.The present study has clinical implications. Although in dental practice it is difficultto control the state of hydration of dentin for proper bonding, it is advisable to useabsorbent paper only to remove the excess water and not to over-dry the dentin surfacewhen using self-adhesive luting agents. However, the conditions of this The bond strength of the self-adhesive luting agent RelyX Unicem was dependent mainly onthe moisture status of the dentin. The findings of this study indicate that only theexcess dentin moisture should be removed during cementation of ceramic restorationsusing self-adhesive resin cements.


The change in firing rate immediately post-fatigue was also greater on the affected side than less-affected side . Mean MU firing rates increased following the recovery period on the less-affected side when compared to the affected side . MU action potential duration increased post-fatigue on both sides , and changes in action potential duration tended to be smaller in subjects with greater impairment (p = 0.04). This study presents evidence of both central and peripheral fatigue at the MU level during isometric fatiguing contraction for the first time in stroke survivors. Together, these preliminary observations indicate that the response to an isometric fatiguing contraction differs between the affected and less-affected side post-stroke, and may suggest that central mechanisms observed here as changes in firing rate are the dominant processes leading to task failure on the affected side.Enhanced muscle weakness is commonly experienced following stroke and may be accompanied by increased susceptibility to fatigue. To examine the contributions of central and peripheral factors to isometric muscle fatigue in stroke survivors, this study investigates changes in motor unit (MU) mean firing rate, and action potential duration during, and directly following, a sustained submaximal fatiguing contraction at 30% maximum voluntary contraction (MVC). A series of short contractions of the first dorsal interosseous muscle were performed pre- and post-fatigue at 20% MVC, and again following a 10-min recovery period, by 12 chronic stroke survivors. Individual MU firing times were extracted using surface EMG decomposition and used to obtain the spike-triggered average MU action potential waveforms. During the sustained fatiguing contraction, the mean rate of change in firing rate across all detected MUs was greater on the affected side (-0.02 ± 0.03 Hz/s) than on the less-affected side (-0.004 ± 0.003 Hz/s,  Over the past decade, marked advancements in the acute management of stroke have led to an increase in the number of stroke survivors living with neurological disabilities . One comIn addition to enduring muscle weakness, stroke survivors may experience increased susceptibility to muscle fatigue. Both central and peripheral factors can contribute to the overall manifestation of fatigue, which can be defined as a transient exercise-induced reduction in the force-generating capacity of muscle . The fewChanges at the level of the single MU during muscle fatigue post-stroke have not yet been investigated. In the present study, we examine the hypothesis that there will be a greater loss in central activation during a sustained fatiguing contraction, and directly post-fatigue, on the affected side in hemispheric stroke. If higher central fatigue is present on the affected side, subjects may experience greater difficulty maintaining MU firing during a fatiguing contraction, as well as a diminished capacity to regulate MU firing rate directly post-fatigue. Changes in the excitability of the surface and tubular membranes of the muscle fiber on the affected side may also be lower post-fatigue  which would manifest as smaller increases in the MU action potential duration .In the present study, surface EMG decomposition was used to identify individual MU activities from non-invasive surface recordings. This provides direct information on both the MU discharge rates and the action potential duration, which is more closely correlated with muscle fiber conduction velocity and changes in muscle fiber excitability than indirect estimates from the surface EMG power spectrum. Samples of simultaneously active MUs were detected during short contractions (at 20% MVC) before and directly after a sustained isometric contraction of the first dorsal interosseous muscle in chronic stroke survivors. MUs were also detected during the sustained contraction (at 30% MVC).The results of this study show that in chronic stroke survivors, MUs on the affected side displayed a greater decline in firing rate during sustained, fatiguing isometric contractions, than on the less-affected side. Furthermore, a greater change in MU firing rate was observed on the affected side immediately post-fatigue. Mean MU firing rates increased following the recovery period on the less-affected side but not on the affected side. A significant increase in action potential duration was observed on both sides post-fatigue. On the affected side, the magnitude of the change tended to be lower in subjects with greater impairments post-stroke. In combination, these results suggest that during sustained isometric fatiguing contractions in stroke survivors, central mechanisms play a greater role on the affected side, when compared to the less-affected side, and likely contribute to difficulties maintaining force reported post-stroke.Table 1. The force and EMG activity of the first dorsal interosseous muscle was examined during isometric abduction of the index finger, on both the contralateral and affected sides. The proximal phalanx of the index finger was fixed to a ring–mount interface attached to a load cell , and forces were recorded from the x (abduction/adduction) and y (extension/flexion) directions, Figure 1A. Surface EMG was recorded from the FDI using a surface sensor array  that consisted of five cylindrical probes (0.5 mm diameter) located at the corners and at the center of a 5 × 5 mm square  to participate in this study, and the experimental protocols were approved by the Institutional Review Board at Northwestern University, m square , and a rThe experimental procedure was similar to that performed in healthy subjects, outlined in detail in Discriminable MUs were extracted from the surface EMG signal using the decomposition EMG system . The decomposition algorithm is outlined in detail in The identified firing times for each MU were used to spike-triggered average (STA) the surface EMG signal on each channel, resulting in four representative STA MU action potential estimates for each MU. MUAP duration was estimated as the time between the zero crossing before the first positive peak of the action potential and the zero crossing after the last positive peak. The variation of the spike-triggered averaged MU action potential template over time was quantified using a moving average window. A spike-triggered averaged MU action potential template estimate was calculated based on the firing events in each window and the window was shifted along the length of the surface EMG signal. The reliability of each detected MU was then assessed by comparing the STA template estimates across all windows, using two tests outlined in Motor units were required to have an average CV in action potential amplitude <0.3 and CC (between the STA MUAP estimate and the decomposition MUAP template) >0.7 across all four channels to be selected for further analysis. In addition, MUs were required to have a CC > 0.8 and CV < 0.2 (0.25 for the longer fatiguing contraction) on at least one of the two channels with the highest MU action potential amplitude. A moving average window of 2 s length and 0.5 s time step was used to obtain the MUAP template for the short contractions pre- and post-fatigue and a window length of 4 s with a 1 s time step was used for the long fatiguing contraction. A minimum average of five MUs was required over the three trials  for each subject. These units must also be recruited across a range of force levels, with a mean range of recruitment threshold defined as 5% MVC . MU data from 11 of the 12 subjects satisfied both criteria and was used in further analysis.Motor unit mean firing rates (MFR) were analyzed during the sustained fatiguing contraction and during the short duration contractions before and after fatigue. Firing rate analysis was restricted to periods of relatively steady force production (standard deviation ≤ 3% MVC). Any overshoot during the initial increase to the required force level was excluded from the analysis and firing trains were truncated at the point where the mean force was below 10% of the desired force level for 5 consecutive seconds. The change in firing rate during the sustained fatiguing contraction was examined for each MU by fitting a least-squares regression line to the instantaneous firing rate data. For each accepted MU, the slope and intercept of the line was obtained, describing the initial MU firing rate and the change in MFR over the course of the fatiguing contraction.For each subject, the median MU MFR and action potential duration were obtained in the pre-fatigue, post-fatigue, and recovery trials. To focus on the within-subject effect of fatigue and minimize the contribution of inter-subject variance to the visual representation of results, the median from the pre-fatigue, post-fatigue, and post-recovery contractions for a given subject was normalized by subtracting that subject’s mean for the three contractions minus the grand mean of all subjects before generating the boxplot figures .t-test.A two-way within-subjects (or repeated measure) analysis of variance (ANOVA) was conducted to compare the change in each parameter across the pre-fatigue, post-fatigue, and recovery states, and on the affected and less-affected sides. Mauchly’s Test of Sphericity was implemented to check the assumption of sphericity, and if violated, a Greenhouse–Geisser correction was applied to the data. Pairwise differences between conditions were conducted using Fisher’s Least Significant Difference test with Bonferroni correction for the affected and less-affected sides. The changes in median MU firing rate and action potential duration from pre- to post-fatigue conditions, and from post-fatigue to recovery, were compared between the two sides with a two-sided paired t-statistic was used to test for a significant increase or decrease in motor firing rate. The relationship between initial MU firing rate (the intercept of the regression line) and the change in firing rate (slope of the line) was examined using a Pearson product-moment correlation. For each subject, the root-mean-squared (RMS) value of the EMG signal was calculated during the fatiguing contraction on the highest amplitude channel, using a 2 s time window with a 1 s time-step. The percentage change in RMS-EMG amplitude was then calculated by fitting a least-squares regression line to the RMS value of the EMG signal over time. The percentage change in the median frequency of the surface EMG power spectrum and in the CV of the force was obtained using the same window and time-step.The change in MU firing rate during the sustained fatiguing contraction was examined using linear regression, and the 2) for the two-way ANOVA and as Hedges’ G (g) for the paired t-tests.The relationships between Fugl-Meyer score and changes in median MU action potential duration, and between MU firing rates and action potential duration, were investigated with a Spearman’s rank-order correlation. An alpha level of 0.05 was used for all statistical tests, and the effect size is reported as omega squared . MU MFRs were similar on the affected and less-affected sides pre-fatigue , though the firing rate CV was significantly higher on the affected side . A significant correlation was observed between the ratio of the MFR on each side and the ratio of the MUAP duration , with subjects for whom MUAP durations were longer on the affected side tending to also have higher MU MFRs on the affected side.Motor unit action potential duration was not significantly different between the affected and less-affected side when examined across all subjects  = 21.8, p < 0.001, ω2 = 0.62], Figure 2. Lower MVC forces were recorded in each state on the affected side compared to the less-affected side . Post hoc tests revealed a significant decrease in maximum force post-fatigue on both sides . The time to task failure varied greatly among subjects  and was not significantly different between sides (p = 0.07). After 10 min of rest, the MVC force increased but remained significantly lower than pre-fatigue values on both sides (p = 0.036 on the affected and p = 0.01 on the less-affected side), Figure 2.A two-way repeated measures ANOVA was used to compare maximum voluntary index finger abduction force in the pre-fatigue, post-fatigue, and post-recovery states. The results indicated a significant change in MVC across the three states , Figure 3. There was also a significant interaction between state and side (affected or less-affected) for MU firing rate , Figure 3. Post hoc tests revealed that MFRs were significantly higher following the 10-min recovery period than those reported pre-fatigue on the less-affected side .Motor unit MFRs changed significantly across the pre-fatigue, post-fatigue, and recovery states . An increase in MU action potential duration was observed post-fatigue on both the affected  and less-affected sides , Figure 5. Following the recovery period, MUAP duration recovered and did not differ significantly from pre-fatigue values . Subjects that were more impaired post-stroke, as evidenced by their Upper Extremity Fugl-Meyer scores, showed smaller changes in MU action potential duration following the fatiguing contraction , Figure 6. The percentage change in MUAP duration from pre- to post-fatigue did not differ significantly between the affected (8.2 ± 6%) and less-affected sides , though there was a greater range of changes in MUAP duration on the less-affected side, Supplementary Figure Motor unit action potential duration also changed significantly over the three states [Figure 1. A similar percentage of MUs exhibited a statistically significant decrease in MFR over time on the affected and less-affected sides . Only a small number of units exhibited a significant increase in firing rate . MU MFRs decayed faster on the affected side during the fatiguing contraction than on the less-affected side over all subjects , Figure 7. However, there was no significant difference in the absolute decrease in MU firing rate . When MUs were pooled over all subjects, lower threshold MUs with higher MFRs tended to show greater absolute decreases in firing rate during the fatiguing contraction on the affected side  and less-affected side , Figure 7.Motor unit firing times were obtained from the decomposed surface EMG signal during the fatiguing contraction at 30% MVC in 10 of 12 subjects, with data from an exemplar subject shown in p < 0.001, respectively), and increased on both sides as the contraction progressed . Force variability was also higher on the affected side during the short contractions pre-fatigue , and there was no significant change in force variability post-fatigue on either the affected or less-affected side . The RMS-EMG amplitude during the first quarter of the fatiguing contraction was lower on the affected side when compared to the less-affected side , with no clear change as the contraction progressed on either side . There was no difference in the median frequency of the surface EMG on the affected (146 ± 35 Hz) and less-affected sides  during the first quarter of the fatiguing contraction. However, there was a greater decrease in median frequency during the contraction on the less-affected side (-44 ± 15%) when compared to the affected side .The variability of the force (CV) during the fatiguing contraction was higher on the affected side than on the less-affected side . In this subset, two subjects were unable to sustain the fatiguing contraction on the affected side for more than 25% of the time obtained on the less-affected side. These subjects had shorter MU action potential durations and lower MU MFRs on the affected side compared to the less-affected side. This could indicate recruitment of a greater proportion of the MU population, including higher threshold MUs with higher muscle fiber conduction velocities and shorter duration action potentials to achieve the target force on the affected side. The lack of reserve MUs available for recruitment during the fatiguing contraction may have resulted in the early task failure. These subjects exhibited small changes (<10%) in MU action potential duration on the affected side post-fatigue, alongside a reduction in MU MFR . Of the remaining three subjects, two showed significant changes in MU action potential duration on the less-affected side (>25%), with smaller changes observed on the affected side (>15%) and the third subject showed little change in MU action potential duration on the affected and less-affected side (<5%). When changes in MU MFR during the fatiguing contraction were examined, all subjects with matched force levels exhibited a significantly larger rate of decrease in MU MFR on the affected side when compared to the less-affected side .In this study, changes in MU properties were investigated prior to, during, and directly after a submaximal, isometric fatiguing contraction in chronic stroke survivors. The ability to investigate adaptations in MU firing rate of many MUs using surface EMG decomposition gives a unique insight into the regulation of MU behavior and how this contributes to the overall manifestation of fatigue in stroke survivors. During the sustained fatiguing contraction, MUs on the affected side displayed a greater decline in firing rate than those on the less-affected side. Furthermore, a greater change in MU firing rate was observed on the affected side immediately post-fatigue. Mean MU firing rates increased following the recovery period on the less-affected side but not on the affected side. Changes in MUAP duration post-fatigue tended to be smaller on the affected side in subjects with greater impairment, indicating lower levels of induced peripheral fatigue. These observations suggest that central fatigue was more dominant on the affected side when compared to the less-affected side, resulting in greater difficulty maintaining or augmenting MU firing rates during and directly post-fatigue.No significant difference was observed in either action potential duration or MFR between sides pre-fatigue. Subjects with relatively longer MU action potential duration on the affected side tended to have higher MU MFRs on the affected side compared to the less-affected sides. Previous studies have reported slower muscle fiber conduction velocities in certain, but not all muscles , and lonFigure 7. The average magnitude of this decline was greater on the affected side, even in subjects that performed the fatiguing contraction at similar force levels on both sides. The mean time to task failure was lower on the affected side, though did not reach statistical significance due to the large variability across subjects. This is similar to the findings of previous studies that have used forces at the same percentage of maximal effort on each side to examine fatigue in various muscles post-stroke  and less-affected side (14 ± 17%) was considerably lower than that observed post-fatigue in young, healthy subjects using a similar protocol (25 ± 14%) . SubjectIn young subjects, lower MU MFRs were consistently observed post-fatigue. However, on the less-affected side, more than half the stroke survivors exhibited MU MFRs that were higher or unchanged post-fatigue. The reduction in MU firing rates and recruitment thresholds that accompanies the shift toward Type I fibers and slowing contractile properties with age  could acThe surface EMG decomposition method used in this study was chosen because its algorithm makes no assumptions about the characteristics of the MU action potential waveforms or the statistics of the MU firing instances , both ofIn the present study, the less-affected side was used as a control against the changes observed in MU firing rate and action potential duration on the affected side in stroke survivors. However, alterations in MU behavior are also observed on the less-affected side post-stroke, with higher MFRs reported when compared to healthy subjects . ChangesFor the first time in stroke survivors, this study presents manifestations of both central and peripheral fatigue by examining the activity of a large number of simultaneously active MUs. Mean MU MFRs decreased more rapidly during the sustained contraction on the affected side when compared to the less-affected side. The change in MU MFRs from pre- to post-fatigue trials was also greater on the affected side. Although the change in action potential duration was not significantly different between sides, changes in MU action potential duration tended to be smaller in subjects with greater impairment. These results suggest that central mechanisms are the dominant processes during fatigue on the affected side. The present study is the first to describe the specific changes in MU firing rates and action potential duration during a sustained fatiguing contraction to the endurance limit in chronic stroke survivors. These measures provide indices to assess the prevalence of central and peripheral fatigue from surface EMG recordings in stroke survivors. This opens-up the possibility of exploring fatigue in other pathological disorders using a non-invasive, stimulation-free protocol. These alterations in MU behavior provide insight into strategies employed by stroke survivors to compensate for impaired muscle force.Written informed consent was obtained for each subject to participate in this study, and the experimental protocols were approved by the Institutional Review Board at Northwestern University.LM, ML, and NS performed the conception and design of research. LM, XH, and NS performed experiments. LM, ML, and NS analyzed the data. LM, ML, WR, and NS interpreted the results of experiments. LM prepared figures. LM and ML drafted the manuscript. LM, ML, NS, and WR edited and revised the manuscript. LM, ML, WR, XH, and NS approved final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A bio-inspired approach was used to integrate an anti-tumor drug into nanocrystalline assemblies for sustained drug release. b-PLGA – within the emulsifier leads to the immediate nucleation of comet bundles, followed by a secondary nucleation to generate the comet head, which is an assembly of nanofibers aligned almost in parallel. The continuous manufacturing furnishes drug–excipient hybrid particles with high drug-loading and a sustained drug release profile. This simple and efficient bio-inspired approach led to a promising sustained local drug delivery system, and could be extended to the fabrication of other functional organic materials bearing mesoscopic structural units.Delicate mesoscopic architectures, bearing complex forms with multiple hierarchy levels, lead to significant functions in biogenic minerals. Herein, a bio-inspired approach was developed to fabricate comet-shaped assemblies of an anti-tumor drug – 10-hydroxycamptothecin (HCPT). The anti-solvent co-precipitation of HCPT and the excipient – PEG- Non-classical crystallization, usually a multistage process, not only dominates in biomineralization,2 but was overlooked in normal crystallization procedures for a long time. The precursors could have multiple forms. They could be non-crystalline species including clusters,6 amorphous nanoparticles,9 or liquid-like droplets.13 In addition, they can be crystalline in form including nanocrystals17 or mesocrystals.19 The transiently existing precursors pass through the coupled crystallization-assembly process before reaching the thermodynamically stable crystalline form.20 For instance, the precipitation of alanine within the proper pH range could pass through the porous mesocrystalline state, as detected using the small-angle neutron scattering technique.19 Soft ingredients can be actively involved via chemical modification with supramolecular side chains or the physical adsorption of surfactants or polyelectrolytes. Both possibilities enrich the multistage crystallization pathways and the structural outcomes by stabilizing the nanospecies and guiding the assembly behavior of the target compounds.24 For instance, supramolecular porphyrins can assemble into nanofibers, which continue to assemble spatially into microplatelets under the guidance of a block copolymer additive – Pluronic F127.22 In comparison, supramolecular porphyrin would grow into bulk crystals without the amphiphilic additive.24 The fabrication of functional materials via multistage crystallization pathways is promising to achieve advanced materials bearing rich mesoscopic forms and functions closely related to their mesoscopic structural forms.The delicate hierarchical architectures of biogenic materials across meso- and macroscales suggest the existence of an alternative non-classical crystallization route.25 Nevertheless, there exist two main hurdles in fabricating nanodrugs: the involvement of high amounts of surfactants as stabilizers and the long-term instability of nanodrugs. Hence, the manipulation of nanodrugs with high drug loadings and a high stability is of great importance. The organization of nanospecies into assemblies would be a promising option, where a small amount of stabilizers including surfactants,26 polyelectrolytes,30 and (block) copolymers31 could provide the colloidal forces to stabilize the assemblies and to prevent adjacent nanocrystals from merging into continuous architectures. This approach has been successful in fabricating a series of functional materials including metal oxides,32 organics,31 and metal carbonates/phosphates33 into assemblies of nanocrystals. Significantly, the assembly could be well-guided using external fields, which could induce the translational and orientational orders towards assembly at the initial assembly stage.35 Unlike their nanocrystalline counterparts, assemblies of nanocrystalline drugs, usually micrometers in size, are advantageous for the separation with current tools and could be a clever option to escape the nanotoxicity problems.For instance, multistage crystallization could have a profound and lasting impact on nanodrug manufacturing.via a multistage crystallization process. The anti-solvent co-precipitation of HCPT and the excipient PEG-b-PLGA38 (monomethoxy polyethylene glycol)-block-poly(lactide-co-glycolide)  leads to the nucleation of hybrid nanofibers with nanocrystalline HCPT as the core wrapped with different amounts of PEG-b-PLGA as the steric stabilizer, depending on the initial ratios of active pharmaceutical ingredient (API)/excipient. Simultaneously, the flow within the channels of the emulsifier induces the spatial alignment of nanofibers into assemblies of several micrometers in size. Delightfully, the continuous manufacturing of assemblies of nanocrystalline HCPT in this one-step process achieves HCPT–PEG-b-PLGA hybrid assemblies with an enhanced effect of sustained drug release for tumor treatments compared to both their nanocrystalline and bulk counterparts.Here, we report the fabrication of assemblies of nanocrystalline 10-hydroxycamptothecin (HCPT) – a promising broad-spectrum anti-tumor agent with remarkable success in early clinical trials – b-PLGA were first dissolved in acetone and stored in the dispersion phase. Afterwards, a high-pressure N2 flow pushed the dispersion solution through the SPG membrane to enter the bulk aqueous phase, which is a poor solvent for both HCPT and PEG-b-PLGA. Due to the good miscibility of water and acetone and the existence of a strong fluid force, the concentration gradient of the solvent mixture was readily achieved in the exit area of each channel. Hence, the nucleation of HCPT nanofibers and the accompanying co-precipitation of PEG-b-PLGA onto the growing HCPT nanofibers presumably occurred in the exit area where the steep increase of the water content immediately increased the local supersaturation of both components to generate co-precipitation. The dynamic nucleation and precipitation of the nanospecies within the channel, plus the active occlusion of the soft ingredient PEG-b-PLGA and the experimental conditions used, led to assemblies of the nanocrystalline HCPT with tunable morphological character and componential distribution.A key feature of these experiments is the performance of the anti-solvent crystallization within a SPG membrane emulsification kit . Both HCPT and PEG-b-PLGA molecules, due to their insolubility in water, coats a thin layer of the block copolymer onto the growing HCPT nanofibers, its unsymmetric distribution on the nanofibers still allows for the further growth of hybrid nanofibers on both ends, where the polymer density is relatively low. Considering that the crystallization depletes the amount of surrounding HCPT molecules, further growth of the nanofibers prefers to occur in the rear areas (the head of each bundle) to form the comet head, composed of almost parallel-aligned nanofibers, due to the high local supersaturation. The morphological details may vary according to the experimental conditions, which will be discussed together with the microscopic images. The assemblies are permanently preserved because of the strong depletion force between adjacent nanofibers in analogy to the porphyrin microplatelets22 and BaSO4 fiber bundles.39 The existence of an external fluid force is crucial for the morphological control in the multistage crystallization process. In comparison, the anti-solvent co-precipitation of HCPT and PEG-b-PLGA in the bulk phase under stagnant conditions led to spherulitic microspheres  are exposed to the water owing to their relative hydrophilicity and serve as the temporary steric stabilizer to stabilize the hybrid nanofibers from too fast and uncontrolled aggregation. This temporary stabilization was already utilized for inorganic nanocrystals using double hydrophilic block copolymers to allow a controlled superstructure formation.40 For these polymers, PEG blocks with a length of 3000–5000 g mol–1 were applied and striking examples of nanocrystal superstructures exist including BaSO4 fiber bundles.39The existence of PEG-2 pressure. Increasing the N2 pressure causes a decreased angle of the tail and an increased length of the comet head, as shown in 2 pressure keeps the HCPT supersaturation in the acetone–water mixture relatively high. Thus, a long comet head is achieved. In an extreme case, the tail area almost disappears when the N2 pressure is as high as 400 kPa  test was designed to optimize the formulation conditions , alongside with the nanorods collected from the sonication-assisted precipitation (15.0% content) and bulk HCPT powder, were performed using a dialysis technique. All samples were assayed using fluorescence spectrophotometry (excitation at 382 nm). The release profiles of HCPT are shown in 42The core–shell architectures of the hybrid particles allow the sustained release of HCPT. 43 Due to its practical insolubility in the aqueous phase, people use its soluble carboxylate form for clinical treatments, usually via intravenous administration. Nevertheless, the carboxylate form of HCPT shows a decreased activity compared to the underivatized molecule. Other approaches show difficulties in achieving high drug-loading formulations.41 Promisingly, the sustained drug release properties and the good colloidal stability of our core–shell hybrid particles allow a local injection of the particle dispersion directly around the tumor area for sustained release at relatively low concentrations. Our cytotoxicity experiments nicely indicate that the hybrid particles show an enhanced cytotoxicity (BEL-7402) compared with the commercial HCPT (hydroxycamptothecin injection) and the colloidal PEG-b-PLGA (see ESI materialThe manufacturing of core–shell hybrid particles provides an increasing number of opportunities for HCPT from the practical point of view. Importantly, the anti-tumor effect of HCPT is both dose- and time-dependent.b-PLGA particles are promising as sustained local drug delivery system for the treatment of tumors. Animal experiments are ongoing. The integration of nanodrugs into assemblies via bio-inspired crystallization approaches opens a door to grow mesoscopic architectures of nanodrugs, which allow for convenient separation using current separation tools and reduced side effects compared to dispersions containing kinetically-stable isolated nanodrug particles. Co-precipitation of the drug with the excipient provides an efficient continuous manufacturing strategy to produce a new generation of drugs with high stability.The study herein presents a simple and effective approach to obtain comet-shaped drug–excipient hybrid particles with a high drug-loading content and sustained release properties. The comet-shaped HCPT–PEG-


Anatomically distinct areas within the basal ganglia encode flexible- and stable-value memories for visual objects (Hikosaka et al.,  The caudate nucleus (CD) is involved in a variety of behaviors including voluntary movements, procedural learning and automatic behavior . We chose the injection sites based upon single unit recordings where we identified the locations within CDh and CDt that respectively had the strongest short-term, flexible value responses or long-term, stable value responses. For detailed methods and injection sites, see these previous studies  into the CDt and Diamidino Yellow  into the CDh of both monkeys. For the CDt injection, we used an injectrode comprised of a tungsten microelectrode (FHC) attached to a silica tube . This allowed us to identify the dorsal and ventral borders of the CDt and inject the tracer into the middle of the volume, thus minimizing tracer leakage. For the CDh injection, we used a 30-gauge stainless-steel needle. After each injection, we waited 1 h before retracting the needle or injectrode from the injection site to minimize tracer backflow along the injection track. The summary of these injections and their locations can be seen in Table After waiting 2 weeks for the tracers to be retrogradely transported, we deeply anesthetized monkeys SM and ZO and perfused with saline and 4% paraformaldehyde. After post-fixation and freezing the brains, we cut the brain into 50 μm sections using a sliding microtome. We collected every 250 μm-interval tissue section for plotting the fluorescently-labeled neurons and the adjacent tissue section for Nissl staining. The detailed histology method is described in a previous study  that allowed us to plot the locations of neurons with respect to the tissue section outline. After plotting the position of each neuron, we aligned the fluorescent neuron plots to the adjacent Nissl-stained section. For visualization purposes, we used Puppet Warp in Photoshop to adjust the fluorescent section to match the Nissl section and reduce anatomical distortions from mounting the tissue onto slides. We used two standard monkey atlases to identify and classify brain regions. To identify the anterior-posterior plane  and classify cortical areas, we used . After permeabilizing the sections for 1 h (0.25% Triton X-100 in 0.05 M TBS), we blocked the sections for 1 h using a TBS solution containing 3% normal goat serum, 2% bovine serum albumin and 0.3% Triton X-100. The sections were then incubated overnight at room temperature with mouse monoclonal SMI-32 antibody (1:1000). We then washed the sections in PBS (3 × 10 min) and incubated the sections for 2 h at room temperature in goat anti-mouse IgG antibody conjugated with Alexa Fluor 647 . Following three washes in PBS, the sections were air-dried overnight at room temperature and mounted with VECTASHIELD (Vector). Images were acquired using fluorescent and brightfield microscopes (Zeiss AXIO Imager M2 and Keyence BZ-X700). We adjusted the contrast and brightness of each color channel in Photoshop (Adobe) and ImageJ (NIH) to enhance the ability to differentiate fluorescently labeled neurons. This antibody has been shown to stain a subpopulation of pyramidal neurons in the neocortex  or exclusively CTB555-labeled neurons (CDt-projecting). The remaining regions contained a mixture of both CDh-projecting and CDt-projecting neurons, but the CDh-projecting and CDt-projecting neurons were often spatially segregated rather than intermingled. Less than 1% of all labeled neurons were double-labeled with DY and CTB555 tracers, indicating that CDh and CDt receive inputs almost exclusively from different neurons. Due to the low number of double-labeled neurons found, we could not identify any pattern to the distribution of double-labeled neurons. Dopaminergic inputs to CDh and CDt were described in previous study , caudal-lateral intralaminar thalamic nuclei (A5), amygdala A17–A23), caudal-ventral claustrum (A17–A23), pre/para subiculum (A3–A7) and DRN  to demarcate the cortical layer boundaries. This antibody has previously been shown to primarily label layer 5 neurons within STC  contained many CDt-projecting neurons Figures , confirmMany neurons in the ACC and PCC projected to ipsilateral and/or contralateral CDh Figure . This agSimilar mechanisms may be shared by the prefrontal and frontal cortices, which are shown to project to ipsilateral and/or contralateral CDh Figures , 6 in agPrevious studies showed that the PC did project mostly to the dorsal part of the CDh (Kemp and Powell, Within the thalamus, many of the nuclei projected in a topographic pattern to CDh or CDt. For example, CDh receives projections from the rostral-medial PF while CDt receives projections from the ventral-lateral PF and medial CM Figure , supportThe pulvinar contained many CDt-projecting neurons, but no neurons labeled from the CDh injection site. To the best of our knowledge, this is the first report of CDt projections from the pulvinar. A previous tracer study in squirrel monkey (Smith and Parent, To our knowledge, this is the first report of CDt-projecting neurons within the claustrum. We found that CDh received projections from the dorsal-rostral claustrum, consistent with previous studies in monkeys (Arikuni and Kubota, Within the amygdala, we found numerous CDt-projecting neurons in the BLA of both monkeys. We found no neurons labeled from the CDh injection site in any region of the amygdala. Our findings were similar to previous tracer studies in monkey (Parent et al., Our results support the previous findings that reported topographical projections from the STN to the caudate and putamen (Parent and Smith, To our knowledge, this is the first report of CDt-projecting neurons in the DRN of monkeys and suggests DRN may provide serotoninergic inputs to CDt Figure . Unlike Within the parasubiculum, we found many CDt-projecting neurons. To our knowledge, this is the first report of projections from the parasubiculum to the caudate in primates. Two previous rodent studies (Groenewegen et al., As with all tracer studies, damaged axons of passage near the injection site may have taken up tracer, especially given the numerous electrode penetrations needed to functionally identify each injection site. Uptake by axons of passage may have led to false anterograde and retrograde labeling, including false double-labeling of neurons. However, it is unlikely that this possible uptake heavily affected our results for several reasons. First, we only found anterograde labeling from the CTB555 injection site in the GPe and substantia nigra pars reticulata (SNr; Kim et al., Another possibility is that the tracer leaked into the STC above the CDt injection site and was retrogradely transported to nearby layer 6 neurons in the STC and dorsal ITC. However, such cortico-cortical connections are unlikely to originate selectively from layer 6 (Usrey and Fitzpatrick, Another limitation of this study is the small injection sites compared to the total volume of the CDh and CDt. Our injection sites were localized to the areas with the most flexible- or stable-coding neurons of the CDh and CDt respectively, but the tracer did not cover all of the CDh and CDt that respectively encoded flexible and stable value. This current study captured many of the differences between the inputs to the CDh and CDt, but may still have missed differences between these two areas of the caudate.Overall, we found anatomical evidence for distinct pathways encoding flexible and stable values. Many cortical and subcortical regions contained CDh-projecting and CDt-projecting neurons in anatomically segregated subpopulations. One significant example of this was in STC and ITC, where cortical layer 5 contained CDh-projecting neurons and layer 6 contained CDt-projecting neurons. The functional differences between these layers are unknown, but investigations into this layer difference may reveal important information about laminar organization. Another significant example of this anatomical segregation was in the claustrum. Rostral-dorsal claustrum contained CDh-projecting neurons while caudal-ventral claustrum contained CDt-projecting neurons. Although claustrum’s function remains unknown, this anatomical finding may guide future functional studies. This tracer study raises many new questions about the functional roles of each of the cortical and subcortical inputs to the CDh and CDt in the flexible and stable value pathways. As these areas are further explored, they may give us more insight into how value memories are created, stored and recalled.HFK performed recordings and injections. AG, WSG, HFK, MGC and KMW performed histology analysis. WSG analyzed the data with input from OH. WSG and OH wrote the article with input from other authors.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.