IntroductionThe COVID-19 pandemic resulted in a transition to a virtual format for all medical residency and fellowship application processes. Previous studies have discussed the successful implementation of virtual interviews, but a deep analysis of how the application process has changed for orthopedic surgery fellowship programs during the pandemic is lacking. The purpose of this study was to assess how COVID-19 impacted the orthopedic spine fellowship application and selection process.MethodsA web-based survey was administered to the program directors of all 75 U.S. orthopedic surgery spine fellowship programs, which often can accept both orthopedic surgery and neurosurgery trained graduates. Questions focused on the changes from the 2019-2020 application cycle to the 2020-2021 cycle. We collected data on connecting with potential applicants, the general application process, and interviews offered by programs. Univariate analyses were used to compare data from the 2020-2021 cycle with the prior 2019-2020 cycle.ResultsTwenty-five of the 75 contacted program directors responded to our survey (33% response rate). The percentage of programs that offered virtual open houses/meet-and-greets increased from 20% in 2019-2020 to 52% in 2020-2021 (p=0.018). Social media use was unchanged . Compared to the prior year, the number of interviews offered by programs increased by 1.5 . There were no significant differences in the numbers of applications received by programs, interview dates available, or separate interviews each candidate completed during an interview day . The in-person interview was the most important factor in 2019-2020 for selecting applicants, whereas the virtual interview, letters of recommendation (LOR), and research were equally ranked as the most important factors in 2020-2021. Regarding interviews, 50% of respondents would “likely” consider virtual interviews as an option in addition to in-person interviews in the future, but most (55%) answered that it was “unlikely” that virtual interviews would entirely replace in-person interviews.ConclusionSpine fellowship programs were more likely to use virtual social events to recruit potential applicants, send out more interview invitations, and equally consider LOR and research with interview performance during an entirely virtual application cycle. Half of the program directors would consider offering virtual interviews as an option for future application cycles, which may help reduce costs associated with the process. The COVID-19 pandemic has resulted in unprecedented changes to graduate medical education at all levels. Learning through in-person clinical care has been largely supplemented with virtual conferences, telemedicine, and independent time for other professional opportunities, such as research . These cPrevious studies have discussed how the shift to virtual interviews has been successfully implemented by fellowship programs for various surgical subspecialties during COVID-19 -9. VirtuThis research was previously presented as a podium presentation at the 2021 Orthopaedic Research and Education Foundation Northeast Regional Symposium.Survey design and administrationsurveymonkey.com, Palo Alto, CA). Depending on the contact information publicly available for a given program, surveys were e-mailed to fellowship program directors or program coordinators with instructions for the program director to complete the survey. In total, the program directors of 25 out of 75 spine fellowship programs completed the survey (33.3% response rate). The survey consisted of 19 questions across three sections: (1) Connecting with potential applicants, (2) general application process, and (3) interviews. The survey was designed to detect changes in these categories from the 2019-2020 application cycle to the 2020-2021 cycle. Questions were worded to elicit responses from the perspective of the spine fellowship program rather than from the perspective of the applicant. This study was deemed exempt from institutional review board review by the study institution.A survey was administered to all 75 orthopedic spine fellowship programs participating in the San Francisco Matching Program (SF Match) using web-based software /, where x represents the response count for each answer choice and w represents the weight of the ranked position. This "average ranking" equation has been previously validated for interpreting rank-choice survey questions . StatistQuestions on connecting with potential applicantsCommunication via e-mail was the most common method used to inform potential applicants about a given program in both the 2019-2020 (56%) and 2020-2021 application cycles (68%)  did not have supplemental requirements  for applicants in the 2020-2021 cycle relative to the prior year  of programs thought that virtual interviews in the 2020-2021 cycle were about the same as in-person interviews in the prior year in terms of importance. Half of the respondents (50%) would “likely” or “very likely” consider virtual interviews as an option in addition to in-person interviews if COVID restrictions are lifted in the future. However, most respondents (55%) answered that it was “unlikely” or “very unlikely” that virtual interviews would entirely replace in-person interviews.Understanding how fellowship application practices have changed in response to COVID-19 is important for continuing improvements in the process. We found that compared to prior years, orthopedic spine fellowship programs more often connected with applicants via virtual open houses during the 2020-2021 cycle, but social media remained nearly unutilized. Programs also offered a significantly greater number of interviews while reweighing candidate metrics. Notably, half of the program directors would consider virtual interviews as a supplement but not a replacement for in-person interviews.In recent years, fellowship programs across medicine have experimented with virtual platforms to conduct interviews with favorable results -15, and Compared to the previous year, we found that a greater percentage of spine fellowship programs offered virtual open houses or meet-and-greets to connect with potential applicants during the 2020-2021 application cycle. This finding may illustrate a concrete action taken by programs to combat the known downsides of using a virtual format, as described earlier. Open houses and meet-and-greets provide opportunities for candidates to assess the people, culture, and "feel" of a program . Some ofInterestingly, spine fellowship programs did not utilize social media as a recruitment tool before or during the pandemic. A study by Wang et al. found that compared to the previous year, orthopedic surgery residency programs saw a 355% increase in social media use during the 2020-2021 application cycle . SimilarRegarding applicant selection, there was an increased importance placed on letters of recommendation (LOR) and research during the 2020-2021 application cycle relative to the prior year. For the 2019-2020 cycle, these two factors were ranked third and fifth in importance for candidate evaluation, with the in-person interview the single most important factor considered. This aligns with previous reports regarding selection criteria for orthopedic surgery fellowships, with the in-person interview noted to significantly impact applicants' positions on the final rank list . HoweverFinally, we found that while half of the responding program directors would consider virtual interviews as an option in addition to in-person interviews, most answered that it was unlikely that virtual interviews would entirely replace in-person interviews. Considering the doubts surrounding the use of virtual interviews, these findings are in line with our expectations and with the results of previous studies . It is iThe results of our study should be interpreted in the context of its limitations. First, there may be response bias amongst the programs that completed our survey, so results may not be generalizable to the entire national sample of spine fellowship programs or fellowship programs for other orthopedic subspecialties. However, our response rate of 33% is on par with prior survey studies administered to orthopedic program directors ,23,28. ADuring the 2020-2021 application cycle, spine fellowship programs were more likely to use virtual social events to recruit potential applicants, send out more interview invitations, and equally consider LOR and research with interview performance when evaluating candidates. Further, programs did not utilize social media as a recruitment tool and are unlikely to replace in-person interviews with video interviews. However, half of the program directors would consider offering virtual interviews as an option during future application cycles, which may help reduce costs and barriers for applicants with financial restraints. Fellowship programs should consider the benefits of a virtual application process as a method to decrease expenses and lost clinical time as well as increase diversity in spine surgery.


The lactosylated poly(1-vinylimidazole) (PVIm-Lac) with various lactosylated degrees has been synthesized for the co-delivery of zinc ions (Zn) and plasmid DNA (pDNA). The Zn/DNA/PVIm-Lac complex formation has achieved the specific delivery of zinc ions to HepG2 cells. Especially, the resulting hepatocyte-specific delivery of zinc ions has increased the number of insulin receptors on the cell surface. Consequently, the Zn/DNA/PVIm-Lac complexes have suppressed insulin receptor internalization on the surface of the HepG2 cells, expecting to offer unique therapy to inhibit hepatic insulin clearance. The resulting sample mixed with a 1.5 μL of loading buffer was loaded onto a 1% agarose gel in the presence of ethidium bromide (1 μg/mL). In TAE buffer , gel electrophoresis was performed at room temperature , followed by the visualization of the pDNA bands under UV irradiation. The mixture of a stock solution of each PVIm-Lac and the dilution of pDNA stock solution (300 ng of pDNA) with physiological saline (150 mM NaCl), adjusted to 13.5 μL at various positive/negative charge ratios ([Imidazolium and aminopropyl]2) at 37 °C for 1 h, the above electrophoresis experiments were carried out. In the case of the complex stability assay, the above agarose gel electrophoresis was run after the incubation of each sample at 37 °C for 10 min in the presence of dextran sulfate (DS: 2mM as sulfate group).For the Zn/DNA/PVIm-Lac complex formation assay, after preincubation of each PVIm-Lac with 1.5 mM zinc acetate (Zn(OAc)Dynamic light scattering (DLS) determined the size of each Zn/DNA/PVIm-Lac complex (pDNA: 4.5 μg per sample) at a positive/negative charge ratio of 8 in physiological saline, as well as corresponding controls, by an electrophoresis light scattering spectrophotometer . Moreover, the ζ-potential of the resulting sample was measured by the ELS with electrodes in physiological saline.4 cells/well in a 96-well plate (100 μL/well) and incubated overnight at 37 °C in a 5% CO2 incubator. Then, the Zn/DNA/PVIm-Lac complexes , as a representative cell, were cultured in tissue culture flasks in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated FBS. The cells were seeded at 1×102O were added to the cells. Subsequently, 100 μL of the resulting cell lysate was diluted to 1.4 mL with H2O containing hydrochloric acid. The atomic absorption spectrometry at 213.9 nm determined the concentration of Zn2+ in the resulting sample by the atomic absorption spectrophotometer AA-6200 . As above in As above As above in t-test. Statistical analysis was performed using a Student’s 3, estimated by GFC , PVIm-Lac(50), or PVIm-Lac(75).d by GFC . The modspectrum . After tatograms . Further lactose . Through lactose . These rAs shown in 2+ ions, no retardation of the naked pDNA (+/− = 0) was observed, suggesting no complex formation between the Zn2+ ions and pDNA under these experimental conditions. Conversely, more retardation of the pDNA was especially observed in the case of PVIm-Lac(20) at a positive/negative charge ratio of eight in the presence of Zn2+ ions, as compared to the absence of Zn2+ ions. These results suggest that the chelated Zn2+ ions with PVIm-Lac(20), as well as PVIm-Lac(50) and PVIm-Lac(75), worked as a polycation. Therefore, the Zn/DNA/PVIm-Lac complexes are considered to form. The particle size and ζ-potential of the resulting Zn/DNA/PVIm-Lac complexes were approximately 100–120 nm and +2–5 mV, respectively  retained pDNA more tightly, as compared to the DNA/PVIm-Lac(75) PIC. Furthermore, as shown in 2+ ions and pDNA with each PVIm-Lac. As shown in p > 0.1) against the naked pDNA (100% cell viability). Therefore, these results suggest there is negligible cytotoxicity of Zn2+ ions in the complexes. We examined the human hepatoma HepG2’s cell viability in the presence of Zn/DNA/PVIm-Lac complexes because no cytotoxicity is desirable for the co-delivery of Zn2+ ions through the Zn/DNA/PVIm-Lac complexes was examined because of no significant cytotoxicity in the Zn/DNA/PVIm-Lac complexes. In the case of delivery into the HepG2 cells, the highest amount of Zn2+ ions was determined by the use of the Zn/DNA/PVIm-Lac(50) complex at a positive/negative charge ratio of eight. Because the amount of Zn2+ ions determined by use of the Zn/DNA/PVIm-Lac(50) complex was higher than that by the use of the Zn/PVIm-Lac(50) complex (without pDNA), the DNA/PVIm-Lac(50) PIC formation may enhance Zn2+ chelation, due to a formed hydrophobic microenvironment around imidazole groups of PVIm-Lac(50) [2+ ions inside human cervical cancer HeLa cells without asialoglycoprotein receptors was statistically significantly lower than that inside the HepG2 cells (p < 0.05). These results suggest that the Zn/DNA/PVIm-Lac(50) complexes with β-galactose residues were a suitable structure for cell-specific Zn2+ delivery to recognize the HepG2 cells via asialoglycoprotein receptors on the cell surface [As shown in -Lac(50) . Notably surface .2+ delivery, we examined the pDNA gene expression mediated by the Zn/DNA/PVIm-Lac complexes. As shown in 2+ ions. Generally, PVIm-Lac(50) mediated the pDNA expression, whereas PVIm-Lac(20) did not mediate the expression. Furthermore, the pDNA expression in the presence of Zn2+ ions was higher than that in the absence of Zn2+ ions. These results suggest that the tightest retention of the pDNA in the Zn/DNA/PVIm-Lac(75) complexes (2+ ions are considered to up-regulate gene expression of the co-delivered pDNA [As a result of cell-specific Znomplexes C worked red pDNA . p < 0.01). The lower gene expression of the HeLa cells is considered to be almost the same tendency as the lower amount of Zn2+ ions inside the HeLa cells  or PVIm-Lac(75), as shown in p < 0.005, ** p < 0.01). The number of insulin receptors on the HepG2 cell surface treated with the Zn/DNA/PVIm-Lac(50) complex was higher than that treated with the Zn/DNA/PVIm-Lac(75) complex. The intracellular amount of Zn2+ ions delivered by the Zn/DNA/PVIm-Lac(50) complex was higher than that delivered by the Zn/DNA/PVIm-Lac(75) complex  experiments were carried out in this study, the delivery of Zn2+ ions to the liver in vivo (in whole organisms) by the Zn/DNA/PVIm-Lac complexes is expected to inhibit hepatic insulin clearance during the first liver passage for the unique diabetes therapy without insulin injection. Due to there being no side effects, based on the specificity of HepG2 cells compared to HeLa cells, the hepatocyte-specific delivery of Zn2+ ions in this study is considered to be safe for in vivo administration.Because the decrease in cell-surface insulin receptors is reported to be suppressed by co-treatment of ZnG2 cells , the incn2+ ions , accordin2+ ions . If intr2+ ions and pDNA. The resulting Zn/DNA/PVIm-Lac complexes have exhibited negligible cytotoxicity for co-delivery of Zn2+ ions and pDNA. In particular, the PVIm-Lac with 50 mol% lactosylated degrees, that is, PVIm-Lac(50), has delivered the highest amount of Zn2+ ions inside the HepG2 cells from human hepatocytes, and not the HeLa cells from human cervical cancer. Namely, the Zn/DNA/PVIm-Lac(50) complex has achieved the hepatocyte-specific co-delivery of Zn2+ ions and pDNA. Notably, the resulting hepatocyte-specific co-delivery of Zn2+ ions and pDNA by the Zn/DNA/PVIm-Lac(50) complex has increased the number of insulin receptors on the cell surface. Consequently, the Zn/DNA/PVIm-Lac(50) complex has suppressed insulin receptor internalization on the hepatocyte surface, expecting to offer unique therapy to inhibit hepatic insulin clearance. In summary, we have synthesized lactosylated poly(1-vinylimidazole), that is, PVIm-Lac, with various lactosylated degrees. Each PVIm-Lac has formed the polyion complex with the Zn


When human infants are intentionally addressed by others, they tend to interpret the information communicated as being relevant to them and worth acquiring. For humans, this attribution of relevance leads to a preference to learn from communication, making it possible to accumulate knowledge over generations. Great apes are sensitive to communicative cues, but do these cues also activate an expectation of relevance? In an observational learning paradigm, we demonstrated to a sample of nonhuman great apes  how to operate on a food dispenser device. When apes had the opportunity to choose between an effective and an ineffective method in the baseline conditions, the majority of them chose the effective method. However, when the ineffective method was demonstrated in a communicative way, they failed to prioritize efficiency, even though they were equally attentive in both conditions. This suggests that the ostensive demonstration elicited an expectation of relevance that modified apes’ interpretation of the situation, potentially leading to a preference to learn from communication, as human children do. The communicator’s capacity to produce ostensive cues together with the audience’s matching disposition to expect what is ostensively communicated to be of particular relevance to them can be a crucial component in social interaction. It allows one individual to influence the mental states of others by not only drawing their attention to some items in the common environment but also by causing others to interpret these items as more relevant than they would have done if their attention had been attracted to these items in a non-ostensive way.Seeking eye-contact or calling the other’s name are typical actions we use in order to initiate communicative interactions with others. These signals, also called ostensive cues5, and that this sensitivity to communicative signals is found across different cultures6. Infants do not only orient towards the sources of these signals, but they also actively search for further referential cues in order to identify the content of the communication, for instance they can infer the presence of hidden objects from referential gaze information7. Furthermore, already at the age of 18 months they interpret new information in the context of ostensive cues as relevant to them, therefore worth acquiring8.In the past decade, various studies provided evidence that human infants, immediately after birth, are ready to interpret some of these signals (such as eye-contact or infant-directed speech) as indicating communicative intentions9. Hand-reared gorillas tend to make eye-contact with their caregivers in order to direct their attention before making a request regarding a certain object or location10, and chimpanzees living in captivity use eye-contact and gaze alternations to objects during their interactions with conspecifics and humans12. Similar to chimpanzees, bonobos and orangutans also seem to use eye-contact, gaze alternations, and imperative gestures both with their conspecifics and humans16. Furthermore, similar to human infants, captive apes can be sensitive to communicative cues, such as eye-contact or calling their name19, and they also prefer direct eye-gaze over averted gaze20.As in human communication, there is evidence that under certain circumstances great apes can also produce and react to ostensive cues as signals of communicative intentions. For example, while eye-contact can be a sign of potential threat or attack in the animal kingdom, apes seem to use eye-contact also as an expression of their intention to communicate with others21. In a recent study, when 18-month-old infants observed two models operating differently on an unfamiliar device, they tended to repeat the action of the demonstrator who made an eye-contact with them and talked to them in infant-directed speech, and not the action of the demonstrator who provided no such communicative signals, even though the non-communicative demonstrator managed to achieve the expected outcome more frequently than the communicative one22. Thus, in the case of infants and young children, it seems that the attributed relevance of an ostensively presented action can override perceived efficiency.However, it remains unclear how apes interpret the information they receive in the context of ostensive signals. In the case of infants and young children, intentionally communicated information can activate an expectation of relevance, i.e. that the new information is relevant for them, therefore worth acquiring. For example, when young children observe action demonstrations, even if the communicatively demonstrated action is apparently not the most effective way to achieve the intended outcome, children tend to copy it27, Myowa-Yamakoshi and Matsuzawa 28. However, is it possible that similar to humans, apes perceive the communicatively demonstrated information as relevant, therefore worth copying, even at the expense of its efficiency? Considering that apes seem to show similar sensitivity to ostensive signals as human infants, it is possible that they also perceive the ostensively demonstrated information as being more relevant, which might lead to selective copying, similar to human infants. The current study aimed at investigating the hypothesis whether, similar to human infants, nonhuman great apes would also attribute relevance to the ostensively presented information. In an observational learning paradigm, we manipulated two factors: the efficiency of the demonstrated actions and the use of ostensive signals vs. some non-social attention-getter cues by the demonstrators . Since there is evidence that apes can successfully detect and copy the more effective method29, we put the efficiency of the method in conflict with the use of ostensive signals when demonstrating to apes how to operate a food dispenser device. If apes do not consider the ostensively demonstrated information as being more relevant to them, we predicted, they should copy the more effective method, irrespective of ostensive cues. However, if ostensive could trigger an expectation of relevance also in apes, then they should stop prioritizing efficiency when it is in conflict with the use of communicative cues.There is exhaustive evidence that great apes are also able to acquire novel information via observing the other’s behaviorPan paniscus, 12 chimpanzees, Pantroglodytes, 6 orangutans, Pongo abelii) participated in this study , accompanied by flashing lights and the release of a small food pellet accessible to the apes. During the ineffective trials, on the other hand, after the demonstrator had inserted the object nothing happened to the device nor did the ape get any reward. After completing the four demonstrations (two successful and two unsuccessful), the two objects were placed on a tray and pushed on the two sides by the two demonstrators towards the subject (at this point none of them made eye-contact with the ape) so that she/he could choose between the two objects and operate on the device by herself/himself. Each demonstrator sat behind the respective object that they had inserted during the demonstration phase. Apes’ actions (object insertions) were rewarded by a food pellet released from the device only if they chose the effective object. The Only-Ostensive condition served as a baseline to see whether apes would reliably choose the object that was successfully used by one demonstrator, as opposed to the object used by the other demonstrator and that had never worked.In every trial, apes were presented with a novel pair of objects. In the Only-Ostensive condition both demonstrators used ostensive cues, but while one of them managed to activate the device twice (i.e. every time) when inserting the object, the other demonstrator failed in both attempts with a different object. During this Only-Ostensive condition, the demonstrator first made eye-contact with the subject, said ‘hello’ while holding the object so that it was visible to the ape. Then she/he approached the device, said ‘hello’ again, clapped with her hands and showed the object again before inserting it into the device. We decided to use these cues because they have been considered as signals indicating communicative intention for the apesIn the Only-Non-Ostensive condition, on the other hand, neither of the demonstrators communicated with the subjects, but again one of them managed to activate the device twice, whereas the other demonstrator failed on both attempts. In this condition, both demonstrators first held the object in such a way that it was visible to the ape but, instead of making eye-contact with the subject, they only looked at the object and said ‘aha’. Then they approached the device, held again the object in a way that made it visible to the ape and said ‘aha’. Before inserting it into the device, they knocked the floor twice with the object. These actions served to elicit the same amount of attention from the apes as in the Ostensive condition, but did not include any communicative gestures. The rest of the Only-Non-Ostensive condition was the same as in the Only-Ostensive condition. This condition allowed us to compare the two conditions in order to see whether the presence of communicative cues would modify apes’ performance and, more specifically, would affect their ability to choose the effective object to operate on the device.Finally, in the Ostensive vs. Effective condition we pitted the presence of communicative cues against efficiency. For this, one demonstrator was effective on both trials without communicating with the apes and only using those non-social attention getter cues that were used in the Only-Non-Ostensive condition, whereas the other demonstrator, who was communicating with the apes failed to activate the device on both trials. This condition aimed at testing whether apes, similar to children, would rely more on the communicatively presented demonstration than on their own observation of efficiency Fig. .Figure 1Subjects participated in two trials in each condition. Each trial consisted of two demonstrations, performed by two demonstrators. While one demonstrator was performing her demonstration, the other demonstrator was standing in the corner of the testing room. Out of the two trials, one trial always started by a successful demonstration, followed by an unsuccessful demonstration, whereas the other trial started by an unsuccessful demonstration, followed by a successful one. The order of the successful vs. unsuccessful demonstrations was counterbalanced. Each trial ended by the object choice of the subject.In total, five demonstrators  provided the demonstrations across the different conditions. The demonstrators were counterbalanced in such a way that for each subject they performed the same communicative role (i.e. they were consistently either communicating or not across the different conditions).2 = 0.45; see ESM for details), we found no evidence that the species’ performance differed across conditions . We then fitted a reduced model without the interaction term to examine the main effects. We found that apes’ performance varied significantly across conditions . The trial number within each condition  and species  had no significant effect on the performance.Apes’ object choices were recorded (see the data in the Supplementary Data File). First, we calculated the percentage of successful object choices (i.e. when the ape chose the effective object). In the Only-Ostensive condition, we obtained data of 24 subjects . During this condition, the effective object was chosen on 67% of the trials, which was significantly better than expected by chance (one-sample t-test: t(23) = 2.14, p = 0.043, 95% CI ). In the Only-Non-Ostensive condition, we collected data of 20 subjects . During this condition apes chose the effective object on 65% of the trials (t(19) = 2.04, p = 0.055, 95% CI ). However, in the critical Ostensive vs. Effective condition, where we collected data of 20 subjects , apes chose the effective object only on 45% of the trials, which was not significantly different from chance level (t(19) = − 0.62, p = 0.541, 95% CI )  nor the main effect of rearing history (in a reduced model without the interaction term) were significant  = 4.737, p < 0.001, Bayes Factor for the alternative vs. the null hypothesis: 223.120) and when comparing the Only-Ostensive condition to the Only-Non-Ostensive condition (t(20) = 5.208, p < 0.001, BF: 592.687). During the object demonstration, however, we found no significant difference in looking time between the conditions (t(20) = − 1.895, p = 0.073, BF: 1.022; t(20) = 1.669, p = 0.111, BF: 0.745) . However, when we pitted efficiency against communication, apes stopped prioritizing efficiency and chose the ineffective, but communicatively demonstrated object equally often as the effective one. Even though the number of ineffective object choices was not significantly different from the hypothetical chance level in the Ostensive vs. Effective condition, the fact that in comparison with the baseline conditions apes clearly changed their behavior indicates that ostensive cues modified their initial preference towards the effective objects.29 , just like in the original study, the performance in this observational learning task did not lead to large effect sizes. Thus, the small effect sizes and the limited sample size led to marginally significant effects for the comparisons between the different conditions.A limitation of our study is the limited sample size and the resulting moderately powered analyses, compared to studies conducted with human subjects. The small sample size also limits the conclusions that can be drawn concerning potential species differences and the lack of evidence for such differences should be interpreted with caution. In addition, while we replicated here the principal finding of the study of Völter and Call One might also point out that since apes participated in the three conditions in a fixed order, this could have potentially affected their performance (for example leading to fatigue by the time they participated in the third condition). While we acknowledge that assigning them randomly to the three conditions would have been favorable, logistically this would have raised some difficulties, since we were careful not to present the same demonstrator in different (i.e. ostensive vs. non-ostensive) roles. However, the fact that the different conditions were tested on average with a 1.5 month long time gap in between two conditions, and one testing session did not last longer than 4–5 min, makes it unlikely that apes would have become tired or less motivated by the time they participated in the third condition.Another limitation that we should mention here concerns in general the limitation of the observational learning paradigm we used in the study. While these paradigms usually give evidence about preferences to copy certain models or types of behavior, it is rarely tested whether these preferences would have an effect also on long-term memory processes, i.e. whether subjects would encode the copied behavior in a long-term. Therefore, although from the study we can draw the conclusion that great apes, similar to human infants, tend to interpret the information communicated as being relevant to them, we cannot tell whether this attribution of relevance would also modify what apes would learn on a long term from communicatively transmitted information. Testing this question should be a next step in the investigation of apes’ social learning processes.33 do not satisfactorily explain subjects’ choices in the ostensive vs. efficient (non-ostensive) condition because subjects watched the object demonstrations equally in both conditions. Similarly, the notion of social enhancement does not fully capture the phenomenon we described here because even though subjects paid more attention in the communicative phase of the ostensive compared to the efficient (non-ostensive) condition , such attentional differences disappeared during the object demonstration phase. If social enhancement were the only process at work here, one would have expected that differential attention would have also been apparent during the object demonstration phase, which is precisely what one sees in cases of social learning strategies based on following dominant or prestigious individuals34.Traditional notions of stimulus/local enhancementOne might argue that the experience of our zoo-housed apes with human communicative signals could trivially explain the current findings: apes might have associated certain communicative signals with food rewards. However, the ostensive cues that we used in the present studies (hand-clapping and saying ‘hello’) were clearly distinct from the gestures and utterances that the caretakers typically use . Additionally, the cues zookeepers tend to use in order to signal feeding-time for the apes are usually related to the food itself (for example shaking a bucket containing the food), while in our case the demonstrators never performed any actions in relation with the food reward.Another possibility, however, is that the ostension-induced attentional processes indeed affected apes’ memory, but only for the ostensively highlighted object, and not for the action demonstration and the subsequent outcome (i.e. being rewarded or not). Such decoupling of the object from the action demonstration would be somewhat surprising; nevertheless we cannot exclude this possibility. However, if ostensive cues could potentially increase the saliency of the highlighted object, leading to a better encoding of it, shouldn’t we consider this mechanism as an attention-related precursor of relevance attribution for the ostensively presented information?35), compared to the other non-communicative attention-grabbing signals.Recent evidence seems to support the hypothesis that apes might react to ostensive cues with an enhanced attention and exploratory behavior. In the study of Kano and his colleagues (2018) chimpanzees watched an experimenter, who either made eye-contact with them and called their name (in the Ostensive condition), or was shown together with a salient, attention-grabbing cue (in the Control conditions) and then gazed towards one of two objects. Even though chimpanzees spent equal time with looking at the experimenter’s face in both conditions, when the experimenter first provided them ostensive signals, they tended to spend more time by looking at both objects in the ostensive condition. This indicates that even though they failed to understand what was the intended referent of the communicative signals (i.e. the gazed at object), ostensive cues still elicited an increased interest and attention, leading to longer exploration of the scene . The main finding of our study is that, similarly, apes seem to be capable of interpreting the same objects or events differently depending on whether they are ostensively presented to them or not. When apes are presented two objects in the same manner (either both ostensively or both non-ostensively) with evidence that one of the two objects can be used effectively to obtain a reward and the other cannot, they tend to choose the effective object. When, however, the ineffective object is presented ostensively and the effective object non-ostensively, this preference for the effective object disappears. This suggests that the ostensive highlighting of the object can modify its saliency, leading to a change in apes’ initial preferences, which would be otherwise solely based on the efficiency of the objects. We propose that this effect can be considered as an attention-related precursor of relevance attribution of apes for the ostensively presented information. In other words, our results indicate that similar to human infants, also apes might consider the ostensively demonstrated information as being relevant to them.It is a well-established fact that, from a very early age, humans are able to interpret ostensive cues as indicators of relevance  in order to demonstrate how to operate on a device. It could be that they were not just influenced in their immediate object choices by the ostensively presented demonstration, but that these choices actually reflected an understanding of ‘what is the correct way of making this device work’.38.However, the precise origins of this ability are unclear. One possibility is that sensitivity to some forms of ostension is part of the ape natural communicative abilities that they use with conspecifics. According to this perspective, humans and their primate relatives may have been selected to use ostensive cues as a way to modify the intended meaning of their communicative exchanges with conspecifics or simply use them to gauge the likelihood that a particular event might occur. For example, recent evidence suggests that in some populations, chimpanzees in the wild tend to actively facilitate learning by transferring termite fishing probes to less skilled individuals 39. Additionally, apes interacting with humans in laboratory settings acquire human gestures like deictic pointing to communicate with them about external entities41. Also, enculturated apes can be receptive to elements of intentional teaching, when they have been exposed to human-typical social experiences. For example, orangutans, who were raised by humans show better understanding of referential gesturing14 and they prefer to spontaneously imitate human models42. In a deferred imitation task, both human-reared juvenile chimpanzees and orangutans tended to imitate the action in the absence of any reinforcement, which raises the possibility that they might be intrinsically motivated to imitate the model, similar to human infants and children43.Another possibility is that apes have acquired their sensitivity to ostensive cues by interacting with humans. Human-induced effects on apes for some aspects of social cognition have already been described in the literature. For instance, enculturated apes (those raised by humans in the human home) pay much more attention to humans in general, than mother-reared apes, presumably because the former have experienced humans as a valuable source of informationThus, even though chimpanzees have not been selected to attend to humans or use their gestures to communicate with them, they can readily do so provided the necessary developmental inputs. These two possibilities that represent phylogenetic and ontogenetic influences, are not mutually exclusive. In fact, both are likely to contribute to the development of ostension in humans. But the paucity of ape data in this area prevents us from determining at this time how each of them contributes to the emergence of apes’ sensitivity to ostension.35 study). Therefore, without the capacity of being able to safely rely on the referential cues of the communicator, apes wouldn’t be able to profit intentional teaching either. Another possibility, however, that even if apes are sometimes able to copy the actions of models in the context of communication, they would fail to encode the imitated action on a long term, which might prevent them from real learning. Indeed, so far studies on apes’ imitation only investigated spontaneous imitative behavior, or imitation after a short delay (e.g. Refs.43).However, while these studies might indicate that being in close proximity of humans, also apes could acquire new information via teaching, we should still keep in mind that so far we don’t have sufficient evidence to assume that apes in the wild would intentionally teach their juveniles. Therefore, it seems that sensitivity to ostension—and possibly a precursor of relevance attribution regarding the ostensively presented information—per sé is not a sufficient prerequisite of the emergence of intentional teaching. From the learner’s side, one possibility is that while ostensive cues might lead to an increased attention and longer exploration, there is no clear understanding what would be the intended referent of the communication in the case of ambiguity (similar to the findings of the Kano et al. Thus, future studies should examine to what extent the short-term bias to preferentially copy the communicatively demonstrated information established in our study would also lead to enduring changes in behavior, i.e. to long-term learning processes. If it does, this could open new horizons in the research of apes’ evolutionary shared capacity to learn from teaching, and possibly also about their potential to accumulate knowledge through generations.Supplementary Information.


Uveal melanoma (UM) is a rare form of melanoma originating in the eye. Unlike cutaneous melanoma (CM), the role of ultraviolet radiation (UVR) in UM aetiology is still unresolved. UM has a high incidence in Australia. Epidemiological analyses revealed heterogeneity in UM incidence between two eastern Australian states, Queensland (QLD) and Victoria (VIC). It was found that QLD has a 21% higher incidence of UM than VIC and, in fact, has one of the highest incidences in the world. A weak south-to-north trend in incidence along the eastern Australian coast is seen, and rural areas have a 24% greater burden than major city areas in both the states. The two states are similar demographically, but differ socially, industrially and latitudinally. This is important because it could indicate a minor UVR role in UM incidence, especially in QLD. Preventative measures by sun-protective behaviours may be important, especially in the northeastern Australian demographic. Uveal melanoma (UM) is the second-most-common melanoma in humans and has a high age-standardized incidence rate (ASR) in Australia. Regional patterns of UM ASRs in Australia are unknown. The aim of this study was to determine and compare UM ASRs in two geographically disparate eastern states, Queensland (QLD) and Victoria (VIC), by using cancer registry data that was obtained from 2001 to 2013. World-standardized UM ASRs and incidence-rate ratios (IRRs) were calculated. Higher UM ASR was also observed in anterior UM compared to posterior UM ASR. UM ASR remained unchanged from 2001 to 2013 in QLD but decreased in VIC. A south-to-north latitude trend in UM ASR along the east of Australia is weakly evident, and rural populations have higher UM ASRs than major city populations in both states. Differences in ultraviolent radiation (UVR) susceptibility, indigenous populations, social behaviours, chemical exposure, and socioeconomic status could all be contributing to differences in UM rates between QLD and VIC and between rural compared to major city areas. It is possible that a minority of cases in QLD and VIC might be prevented by sun-protective behaviours. This is important, because these findings suggest that QLD, which is already known to have one of the highest cutaneous melanoma (CM) ASRs in the world, also has one of the highest UM ASRs. Uveal Melanoma (UM) is the most common intraocular melanoma in humans, arising from melanocytes of the iris, choroid or ciliary body in the eye. UM age-standardized incidence rate (ASR) appears to be elevated in Australia, with world-standardized ASR ranging from 6.2 to 8.0 per 1,000,000 person-years in men and 5.2 to 6.1 per 1,000,000 person-years in women , the UniQueensland (QLD) and Victoria (VIC) are states with the most northern and southern latitudes of the Australian mainland, respectively, and are found at similar longitudes. QLD is 7.5 times the size of VIC ,7, with The comparison of two states with very similar populations, health systems, and governing variables provides a unique setting for the scientific investigation of population-wide factors associated with UM ASRs. Indeed, there already exist substantial differences in cancer burdens between the pair of states: QLD has one of the highest Cutaneous Melanoma (CM) ASRs in the world , and betThe aim of this study was therefore to use the unique characteristics of this state pair to compare UM ASR and incidence-rate ratios (IRR) patterns between 2001 to 2013 and to reflect on possible factors that drive differences in UM ASR. UM data from 2001 to 2013 was obtained from the QLD Cancer Registry (extracted 3 March 2016) and the Victorian Cancer Registry (extracted 8 March 2016). This encompassed all ICD-O-3 C69 sites (eye and adnexa), from 2001 to 2013, with ICD-O-3 morphology 872-879. Information on sex, year of birth, year of diagnosis, postcode inhabitancy at time of diagnosis, and site of origin were provided for both states. In this study, melanomas of the “conjunctiva” (C690) and “lacrimal gland” (C695), which have sometimes been classed as “ocular melanomas” in the past ,15, are In order to assess the relationship between UM ASR, latitude, and remoteness, the data on postcode inhabitancy at the time of diagnosis was used. Cases without postcode information were excluded from comparison statistics involving rurality and latitude. Rurality was based on the Australian Bureau of Statistics (ABS) Remoteness Area (RA 2011) level , i.e., mx-axis points were available as a result of insufficient case numbers (<25) for age standardization, per demographic, sex, time interval, and/or latitude band. Significance of correlations and t-tests were both determined using p values at the 0.05 significance level. The data was further analyzed by Poisson regression models to produce incidence rate ratios (IRRs) for characteristics such as rurality and latitude. Age and sex person-year exposure was included in every model to control for differences between the populations. IRRs were computed using per-sex, per-age-group, per-year population to account for increasing population over time. Statistics were performed using SPSS Statistics 26 or R 4.1.1.ASRs per 1,000,000 person-years and their 95% confidence intervals (95% CI) were calculated for different geographical groups from 2001 to 2013 using annual population data from the ABS , and thep = 0.03) in VIC (62.8 years old) than in QLD (60.9 years old). From 2001 to 2013, there were 432 UM cases in QLD and 434 cases in VIC see . Nine cap < 0.001) and females . When assessing individual sites of origins, QLD had a statistically higher ASR of anterior (ciliary body or iris) melanoma than VIC in both genders . QLD also had a higher ASR of posterior  melanoma in men (5.4) compared to VIC (4.2) per 1,000,000 (p = 0.05) but not in women .Summaries of ASRs in Australia, VIC, and QLD are presented for UM in p = 0.005) higher incidence rate of UM in QLD than VIC when controlling for age, remoteness, and sex (p = 0.018) higher incidence than females when controlling for age and latitude and 17%  higher incidence than females when controlling for age, state, and remoteness  only when the northernmost third of QLD is excluded . When looking at the incidence rate we see that Central Queensland (QLD band 2) has a 74% greater incidence of UM  than in VIC when controlling for age, sex, and year of diagnosis  but decreased in VIC . Comparisons of ASRs for younger and older VIC and QLD populations from 2001–2007 and 2008–2013 are presented in p = 0.03). In contrast, the ASR in older VIC males was lower in 2008–2013  than in 2001–2007 . In younger cohorts (<55 years old), a lower UM ASR was seen in 2008–2013 than in 2001–2007 in QLD females  but was not statistically different in QLD males . In VIC, the UM ASR in younger individuals was not statistically different from 2001–2007 and 2008–2013 in either sex.UM time trends are presented in p = 0.04). This is also seen in VIC . When controlling for age and sex, the ASR of UM was still higher in QLD than in VIC in major city areas  and rural areas . This is largely due to the higher rate of UM in QLD males in major cities and in QLD females in rural areas (see p = 0.002) when accounting for age, sex, and state . The age of diagnosis is also lower (p = 0.03) in QLD (60.9 years old) than in VIC (62.8 years old). These differences in site of origin and age of diagnosis could implicate distinct aetiological drivers of UM in QLD and VIC.This is the first time that the ASRs of UM in QLD and VIC have been directly compared. The (world-standardized) ASR of UM from 2001 to 2013 was higher in QLD  than in VIC . The UM ASR in QLD (but not VIC) is also higher than the Australian average reported previously  observed from VIC (latitude −39.1° to −34.0°) to the more populated parts of QLD (−29.0° to −21.6°) also suggests a role for UVR in pathogenesis. However, UM ASR falls in the remote, most UVR exposed areas of northern QLD. This, surprisingly, could also be argued to be supportive of a role of UVR in UM ASR along eastern Australia. This seems counterintuitive, but while UM ASR has not shown a south to north increase across the entire continent previously [The weak positive correlation (eviously ,36,37. Aeviously , the redeviously , includieviously  and in Qeviously , and lowQLD has a higher ASR of UM than VIC even when considering rural and major city areas. Analysis of the state pairs showed that the ASR of UM is higher in rural areas than in major cities for VIC and for QLD. Higher ASR of UM in rural areas compared to urban areas has been reported in Australia-wide data previously . Differences in ASR between rural and urban areas could be due to differences in UVR, pollution, occupational exposure to carcinogens, or socioeconomic status. In dense urban areas, the increased pollution and built environment can cause the absorption and scattering of UV light, reducing UVR exposure . DiffereThis study observes the ASR of UM in two eastern Australia states, VIC and QLD, from 2001 to 2013 as recorded in the Victorian Cancer Registry and QLD Cancer Registry. These registries are reliable for a number of reasons. Firstly, reporting of all cancer diagnoses is mandatory in all Australian states and territories , and secDespite thorough case reporting, the ASRs of UM reported in this study may still be underestimations of the true values for several reasons. Nine QLD UM cases were excluded from the analyses due to a lack of postcode data, potentially creating a sampling bias in the latitude and rurality IRR investigations. In addition, the current study did not employ methods to investigate possible non-notifications by healthcare facilities. Despite mandatory reporting, 109 unnotified cases were discovered in Australia in the period of 1990 to 1998 , and theThis study relied on postcode at diagnosis for many of its analyses, and information on changes of inhabitancy of UM cases was not available. It can be observed through census data that high turnover or growth was greater in regional and remote areas over the study period than in major city areas, especially in QLD ,51,52, wUnfortunately, data on the molecular profiles and pathological features of the diagnosed UM is not reported in the QLD or VIC cancer registries. This may not be surprising given that, in contrast to most other cancers, UM is frequently diagnosed without a confirmatory biopsy, and, instead, diagnosis and subsequent treatment is solely based on clinical findings . This maOur study benefits from the comparison of two latitudinally polar states with relatively similar demographics, latitude, and healthcare systems. Thus, an association be-tween UVR on UM ASR may be commented on. However, caution must be taken in this ecological study to not overstate the evidence for the relationship between UVR radiation and UM ASR, as this and similar ecological studies can only establish weak evidence towards this finding.Finally, UM’s orphan status means that, statistically, a small change in the number of cases can have a large impact on the rate. Consequently, fluctuations in data should be interpreted with caution and more complicated analyses may not have their assumptions met in many samples of UM. QLD and VIC face different carcinogenic, demographic, and solar UVR burdens but represent grossly similar populations and living styles suitable for demographic comparison. Together, the identified factors may contribute to the higher ASR of UM in QLD, especially in men, in rural areas, and in central QLD. This is significant for 2 main reasons: Firstly, south-to-north latitude trends, higher rural ASR, and the substantially greater ASR of anterior UM in QLD than in VIC may all suggest a relationship between UM ASR and UVR in a proportion of cases. This agrees with anatomical, molecular, and epidemiological evidence that at least a small portion of UM cases are UVR-related . Secondly, rural areas typically have less access to healthcare than major cities. This is particularly a problem in UM, which is usually asymptomatic in the early stages and often diagnosed incidentally by an optometrist . This isOur data has shown that from 2001 to 2013, QLD had a greater ASR of UM than VIC resulting in a 21% greater incidence. The UM ASR in QLD is also higher than average Australian rates and represents one of the highest UM rates in the world. Differences in UVR exposure, occupation, chemical exposure, and rurality could all plausibly contribute to the higher rates of UM in QLD than VIC, in men than in women, and in rural areas than in major city areas of both QLD and VIC. This means that awareness and early detection regimes should be considered, especially for these demographics.


Deregulated metabolism is one of the characteristics of hepatocellular carcinoma. Sex hormone receptor signalling has been involved in the marked gender dimorphism of hepatocellular carcinoma pathogenesis. Oestrogen receptor (ER) has been reported to reduce the incidence of liver cancer. However, it remains unclear how oestrogen and ER regulate metabolic alterations in liver tumour cells. Our previous work revealed that ERα interacted with carbohydrate responsive element binding protein (ChREBP), which is a transcription factor promoting aerobic glycolysis and proliferation of hepatoma cells. Here, the data showed that ERα overexpression with E2 treatment reduced aerobic glycolysis and cell proliferation of hepatoma cells. In addition to modestly down‐regulating ChREBP transcription, ERα promoted ChREBP degradation. ERα co‐immunoprecipitated with both ChREBP‐α and ChREBP‐β, the two known subtypes of ChREBP. Although E2 promoted ERα to translocate to the nucleus, it did not change subcellular localization of ChREBP. In addition to interacting with ChREBP‐β and promoting its degradation, ERα decreased ChREBP‐α–induced ChREBP‐β transcription. Taken together, we confirmed an original role of ERα in suppressing aerobic glycolysis in liver cancer cells and elucidated the mechanism by which ERα and ChREBP‐α together regulated ChREBP‐β expression. Oestrogen and oestrogen receptor (ER) negatively regulate initiation and progression of HCC.ERα and E2 suppress lipogenesis by inhibiting mRNA and protein expression of carbohydrate responsive element binding protein (ChREBP) in insulin‐secreting INS‐1 cells.Our former work indicated that ChREBP‐α interacted with both ERα and its cofactor flightless I homolog (FLII), and FLII also interacted with ChREBP‐β.22.1The human ChREBP‐α and ChREBP‐β cDNA was generated as described.2 atmosphere. 17β‐oestradiol (E2) of 10 nM was used to treat cells.293T human embryonic kidney cells, human hepatocellular carcinoma cells HepG2 and SMMC7721, and HeLa human cervical cancer cells were cultured in DMEM containing 2 mmol/L L‐glutamine, 1 mmol/L sodium pyruvate, 10% FBS, 100 μg/mL streptomycin and 100 unit/mL penicillin at 37℃ in humidified 5% CO2.2HA‐ChREBP‐α and Flag‐ERα were transfected into 293T cells. E2 was used to treat the transfected cells. The preparation of buffer A and buffer B, and the operating steps were handled as presented previously.2.3Triton X‐100 buffer contained 1% Triton X‐100, 100 mM NaCl, 40 mM Tris‐HCl, pH 8, 1 mM EDTA and 0.5% NP‐40. Using Triton X‐100 buffer to lyse fresh cells, primary antibody incubation was performed overnight at 4℃, followed by an additional incubation for 2 hours together with protein A/G agarose beads (Santa Cruz) at 4℃. Triton X‐100 buffer was applied to wash the beads for four times and then to boil the beads in 2 × SDS protein loading buffer. Then, the Western blotting analysis was carried out.2.4Flag‐ERα, HA‐ChREBP‐α, HA‐ChREBP‐β, β‐galactosidase, ChREBP‐α‐Luc reporter and ChREBP‐β‐Luc reporter were transiently transfected into 293T cells using Lipofectamine 2000 (Invitrogen). Beta‐galactosidase was employed as a reference for normalizing transfection efficiency. pcDNA3 was used to normalize the total amount of transfected DNA. At 48 hours post‐transfection, cells were collected and analysed by means of Beta‐Gal Assay Kit (Clontech) and the luciferase reporter assay system (Promega) according to the reagent specification.2.5TRIzol (Invitrogen Life Technologies) was used to isolate total RNA from cells with or without E2 treatment in accordance with the manufacturer's recommendations. The PrimeScript™ RT Reagent Kit (Takara Bio Inc) with 10 mL assay mix containing 2 mg total RNA was applied to synthesize cDNA. The reaction mixture was kept at −20℃ until the PCR analysis. β‐actin was applied to the endogenous reference. The primer sequences of ChREBP‐α and ChREBP‐β were described previously.Real‐time PCR was analysed using a StepOnePlus™ Real‐Time PCR System (Applied Biosystems). Relative quantification of mRNA amount was obtained in the light of the user manual of Applied Biosystems.2.6Cell lysates were collected. BCA Protein Assay Kit (Pierce) was used to quantify protein concentration. Protein bands were visualized with an enhanced chemiluminescent solution (Millipore) using Amersham Imager 600 (GE).2.7Cells in 10‐cm dishes grew to about 80% confluence and then were trypsinized and resuspended in 3 mL of culture solution. An Oxytherm System (Hansatech) was applied to detect oxygen consumption.Glucose uptake and lactate production were measured at 48 hours after cells were laid in 6‐well plates or treated, and then collected and investigated culture medium. Lactate production and glucose uptake were analysed using Lactate Assay Kit (Sigma) and Glucose Assay Kit (Shanghai Rongsheng Biotech), respectively, according to product specification.2.82. A number of cells were measured at days 0, 2, 4 and 6 after plating.A total of 2000, 4000, 6000, 10 000 or 20 000 cells were laid in 6‐well plates in triplicate. After cell adherence, CCK8 reagent was added and incubated for 2 hours, followed by OD measurement at 450 nm. The standard curve was drawn according to cell numbers and OD values. The four groups are cells stably expressing GFP or ERα cDNA with or without E2 treatment. Each group of 4000 cells were plated and cultured at 37℃ with 5% CO2.9®, which were conjugated secondary antibodies, were added to the cells and reared for 30 minutes at room temperature away from light. In the final 5 minutes, DAPI (40728ES03) was added. The coverslips were mounted with nail polish after being washed twice in PBS, and images were taken using an LSM 710 laser scanning confocal microscope (Zeiss) and a confocal microscope .Cells were grown on coverslips, rinsed twice with phosphate buffer saline (PBS), each for 5 minutes. Then, cells were treated with 4% paraformaldehyde for 15 minutes, rinsed twice with PBS, permeabilized with 0.3% Triton X‐100 for 20 minutes at room temperature and blocked in 2% goat serum for 30 minutes at room temperature and primary antibodies were incubated overnight at 4℃. Alexa 488 and 555 Fluor2.10t test was used to analyse the difference between the treatment group and the control group. P value < .05 was regarded as statistically significant.Experiments were done at least three times independently; one representative experiment was displayed. Data were presented as mean ± standard deviation (SD) using Prism 5 (GraphPad Software). Student's 33.1It was known that ChREBP enhanced aerobic glycolysis and cell multiplication in tumour cells.3.2ERα and E2 can inhibit transcription and translation of ChREBP‐α.In order to examine whether ERα colocalized with either ChREBP‐α or ChREBP‐β, Flag‐ERα and HA‐ChREBP‐α, Flag‐ERα and HA‐ChREBP‐β were ectopically expressed in HeLa cells and their subcellular localization was detected using immunofluorescent staining. Our data showed that HA‐ChREBP‐α or HA‐ChREBP‐β colocalized with Flag‐ERα in both cytoplasm and nucleus, respectively Figure .Next, we further analysed which domains of ChREBP‐α interact with ERα. On the basis of the structural domains of human ChREBP‐α protein, we generated three ChREBP‐α truncates including Flag‐tagged N‐terminal NES and NLS domains of ChREBP (Flag‐ChREBP‐α 1‐251), Flag‐tagged Pro‐rich domain of ChREBP (Flag‐ChREBP‐α 252‐625) and Flag‐tagged bHLH/ZIP and ZIP‐like of ChREBP (Flag‐ChREBP‐α 626‐852). We discovered that only the Flag‐ChREBP‐α 1‐251 truncate co‐immunoprecipitated with Myc‐ERα Figure . As ERα 3.3We next analysed whether E2 affected the subcellular localization of ChREBP‐α and ERα. We overexpressed HA‐ChREBP‐α and Flag‐ERα in HeLa cells with or without E2 treatment and investigated the distribution of ChREBP‐α and ERα using immunofluorescent staining. Without E2 treatment, ERα was distributed in both cytoplasm and nucleus Figure . HoweverTo confirm the findings, we performed nuclear and cytoplasm separation experiment. Subcellular fractionation showed that Flag‐ERα and HA‐ChREBP‐α existed in both cytoplasm and nucleus. It is interesting that ERα increased ChREBP‐α protein level in the cytosol when there was no E2 present. However, ERα reduced ChREBP‐α protein level significantly no matter in the cytosol or nucleus when there was E2 Figure . In HepG3.4Herman et al reported that one isoform of ChREBP, ChREBP‐α, transcriptionally activated the other isoform, ChREBP‐β.We found that ERα weakened the interaction between ChREBP‐α and ChREBP‐β, and ERα with E2 treatment further weakened the association Figure . These rIn summary, ERα with E2 treatment reduced aerobic glycolysis and inhibited cell multiplication in liver cancer cells. One of the underlying mechanisms is that ERα suppressed ChREBP activity, including ChREBP‐α–mediated ChREBP‐β transcription Figure .4ERα and oestrogen negatively regulate liver cancer cell proliferation.ChREBP was down‐regulated in human breast tumour in comparison with vicinal normal tissues.ERα regulates ChREBP at both transcriptional and post‐transcriptional levels. ERα suppressed fatty acid and glycerolipid synthesis by inhibiting mRNA and protein expression of ChREBP in pancreatic islet β‐cells.Taken together, we uncovered a novel mechanism for ERα decreasing aerobic glycolysis and cell multiplication in liver cancer cells. The data showed that ERα, ChREBP‐α and ChREBP‐β coexisted in a complex, and ERα inhibited ChREBP‐α–mediated ChREBP‐β transcription. The ERα‐ChREBP axis might be a potential target in the therapy of liver cancer.The authors confirm that there are no conflicts of interest.Ying Lu: Conceptualization ; Funding acquisition ; Data curation (lead); Investigation (lead); Methodology ; Writing‐review & editing . Na Tian: Data curation ; Formal analysis ; Investigation . Lei Hu: Data curation ; Formal analysis ; Investigation . Jian Meng: Formal analysis ; Investigation ; Methodology . Ming Feng: Formal analysis ; Investigation ; Software . Yemin Zhu: Data curation ; Formal analysis ; Investigation . Ping Zhang: Data curation ; Formal analysis . Minle Li: Data curation ; Formal analysis . Qi Liu: Data curation ; Investigation . Lingfeng Tong: Formal analysis ; Investigation . Xuemei Tong: Conceptualization (lead); Funding acquisition (lead); Project administration ; Resources (lead); Writing‐review & editing (lead). Yakui Li: Data curation ; Funding acquisition ; Formal analysis ; Investigation ; Software . Lifang Wu: Conceptualization (lead); Data curation (lead); Funding acquisition ; Investigation (lead); Project administration ; Supervision (lead); Writing‐original draft (lead); Writing‐review & editing (lead).Fig S1Click here for additional data file.Fig S2Click here for additional data file.Table S1Click here for additional data file.


Although in 2020, there are more than 120 healthcare simulation fellowships established globally, there is a paucity of literature on how to design fellowship programs most effectively, to equip graduates with the knowledge, skills, and attitudes of a competent simulation educator. Offering a systematic structure to approach simulation fellowship programmatic design may aid in better achieving program goals. In this manuscript, we present the application of the 4-component instructional design model as a blueprint to the development of Simulation Education Fellowships. We offer examples used at the NYC Health + Hospitals simulation fellowship to illustrate how the 4-component model informs fellowship program design which promotes the development of a simulation educator. This manuscript will provide a roadmap to designing curricula and assessment practices including self-reflective logbooks to focus the path toward achieving desired skills and shape future conversations around programmatic development. Simulation fellowships have grown organically in many simulation programs throughout the world. Even with this growth, the literature offers little to understand what strategies the larger community of practice has applied to simulation fellowship program design. Several published manuscripts describe specific curricular components that are considered curricular standards during fellowship training –4, and oIn 2009, NYC Health + Hospitals (H+H), the largest municipal healthcare system in the USA, established a Simulation Center, focusing on deploying simulation to address enterprise-wide patient safety and quality priorities. As demand for the application of simulation grew across the enterprise, the program leaders agreed that more trained simulation educators were required to meet the needs of the approximate 46,000 employees of the health system.To address the issue of simulation capacity at H+H, a simulation fellowship was established in 2011. The year-long fellowship followed an apprenticeship type model, where fellows were paired with existing simulation educators and received “on the job” training. Fellows would shadow a simulation faculty member’s course facilitation and gradually begin to facilitate more parts of the course, until they were able to teach it independently. In the apprentice-style fellowship, there were no pre-defined competencies for the fellow to meet. Fellows gradually learned elements like scenario development, debriefing, and manikin operations by observing courses. There was also inconsistency in exposure to the wide range of simulation techniques and modalities available due to the specific needs of the system limiting the breadth of activities. In successive years, it became apparent that fellows were completing the program with varied experience and expertise. Knowledge and skill gaps became evident, as fellows were asked to implement programs or facilitate courses independently within their own institutions. The Simulation Center had to think differently about how to address the performance gaps in their graduating fellows.Most fellowships span a finite time ranging anywhere from 6 months to 2 years –4, 6. ThCLT distinguishes three main types of load: intrinsic, extraneous, and germane. Intrinsic load refers to the inherent complexity and elemental interactions of tasks and cannot be changed. Extraneous load is the working memory load experienced by learners as they interact with instructional materials. Lastly, germane load refers to the effort used by working memory to process and store information –10. The Recognizing the challenge of training proficient simulation educators, with all the requisite knowledge and skills required, the Simulation Center purposefully employed the four-component instructional design (4CID) approach that draws upon the principles of CLT to provide a robust framework to re-design its fellowship program , 15. TheMerriënboer and Kirschner’s 4-component instructional design (4CID) draws upon the principles of CLT and proposes a programmatic approach to achieve not only the concepts to be taught but their inter-relatedness, also known as a “holistic approach” over time . The fouAccording to Merriënboer and Kirschner’s approach, the instructional team must determine the skills, knowledge, and attitudes necessary to achieve competency, as a simulation educator, prior to initiating the 4CID approach. Simulation fellowship literature offers some guidance with core curricular elements –4. PartnThe 4CID model was applied to these domains as a strategy to focus fellows’ exploration of these elements.When designing individual learning experiences for each fellow, careful attention is placed on focusing the individual’s working memory to create new schema and impact existing schema. In other words, the fellow’s ability to think, troubleshoot, and problem solve differently upon completing the program is dependent on long-term memory created through the instructional design process delivered by the program , 17. As The simulation center environment and the number of interprofessional fellows set the context for the creation of dynamic learning experiences. A psychologically safe container was created at the start of the fellowship program on the first day. The lack of a psychologically safe context for learning may lead to distraction, or extraneous load, from learning the educational content provided , 21–23. The creation of immersive experiences is the cornerstone of experiential learning in simulation-based education. Merriënboer and Kirschner support learning tasks that mimic those that are reflected in real life. This can be achieved by attending to both fidelity and variability of experiences, which drives the learner toward effective translation into actual clinical practice . Real-liThe Fellowship Leadership purposefully allocated FUSE days in rapid sequence at the beginning of the year to lay down foundational knowledge, so that fellows have enough knowledge and skills to practice when assisting in facilitating simulation-based courses on non-FUSE days. This approach gives new fellows enough information to be competent assistant facilitators in simulation-based courses and to prevent negative transfer during teaching. From a programmatic perspective, a strategic approach was taken to help fellows navigate their learning constructively aligned with the 4-component model. By scaffolding concepts and delivering the content from simple to complex , 14, theTo highlight this concept, consider debriefing. Debriefing architecture is a foundational topic that is first introduced to the learner  through When considering fading guidance, a titration of feedback strategies is applied. Early debriefings may be coached using rapid cycle deliberate practice or pause-and-reflect , 34. RapEstablishing and maintaining psychological safety can be used as another example where scaffolding can support learning. The current approaches to psychological safety, how to set a safe container , 36 and Fellows participating in the program, depending on their prior schemata, exposure, and preference of learning approach, are at different levels of content mastery. As task complexity increases, there is the risk that certain learners may be overwhelmed. By choosing to teach complex learning tasks collaboratively, the processing of information can be carried over multiple working memories to lessen the load and maximize cognitive capacity –39. As aHaving constructs and theoretical approaches to these diverse skills is necessary for the learner to be efficient with their development. CLT suggests a mitigation strategy for intrinsic load’s task complexity by establishing theoretical frameworks for the creation of long-term memory prior to entering into a learning task , 14, 20.Some procedural tasks are prescriptive having a clear, stepwise approach that can become automated. These tasks have instructional approaches to create automation. Automation refers to cognitive processing that bypasses working memory. As a result, when confronted with a task, no working memory is used, and more cognitive capacity is available for execution . ProceduThe fellowship targets tasks that require automation by offering part-task practice of the skills. Redundant practice of these basic skills, such as debriefing architecture, certain manikin operations, and journal review approaches, frees up more working memory to mitigate complex skills such as a difficult debriefing situation, manikin troubleshooting, and unexpected learner actions during a scenario. The task complexity would increase exponentially if there were no automation in their tasks as there are more task elements to manage. By standardizing and automating certain parts of complex tasks such as manikin set up prior to every simulation experience, or incorporating the debriefing architecture into all post-event feedback situations, the fellow is exposed multiple times per day to these structures consolidating these approaches into their long-term memory for future applications. A breakdown of the 4 components and using debriefing as a skill to contextualize the components can be found in Table The 4CID approach creates a framework for a program to develop its teaching strategy to address the knowledge skills and attitudes of the simulation educator. For the faculty, understanding that they are achieving these goals is important so they can appropriately adapt to learner needs. Using assessment, both self-guided and faculty-driven, helps meet that need.The 4CID model supports the use of self-assessment. Some literature suggests that self-assessment is largely inaccurate, as most individuals tend to misconstrue confidence with competence . DespiteThe NYC H+H simulation fellowship incorporated self-assessment in a structured way by enforcing a digital logbook. The logbook serves as a personal reference used by the learner to record both the activities performed and individual’s reflections on each activity. The learner is then asked to propose personal future activities to address any gaps, and to reinforce and refine skills. The logbook is regularly reviewed by the team and used as a guide to tailor experiences to the fellow’s self-assessed needs. Tailored experiences reinforce that the team is invested in the fellow’s growth and reinforces greater utilization of the logbook. The logbook also serves as a record to appreciate the variety of simulation experiences in which the fellow has been engaged. Review of the logbook provides guidance for the team to assign fellows to experiences that they may have missed and to ensure exposure to the widest range of health simulation activities. Without monitoring, learners may self-select out of certain activities that are required for the fellow to meet their development needs.Within simulation fellowships, there are very few validated tools that are specific to fellow development and ensure acquisition of all required skills . The afoTo meet the gap, the NYC Health + Hospitals Simulation Center reviewed tools developed by other programs such as the milestones developed by the Accreditation Council of Graduate Medical Education (ACGME). The ACGME uses milestones as a means of offering summative assessment, across pre-determined core competencies. The tool is used by ACGME in Clinical Competency Committee Meetings for the purpose of tracking progress of trainees. The milestones use a numerical system, anchored by specific knowledge, skills, and attitudes to track the development of a competency. Although it is not plausible to include every element of the competency, the NYC Health + Hospitals team designed an assessment tool based on these milestones to meet the need of summative assessment in healthcare simulation educators. The milestones consist of 8 competencies that are listed in Table An example of the milestone created for debriefing is presented in Fig. Formative assessments can be offered through feedback from multiple sources (including peer feedback from other fellows and from mentors) and in a variety of settings. Regular feedback is an integral part of training for the program as a means of coaching and providing support for emerging skills. Formative assessments, such as the DASH , can alsThe NYC Health + Hospitals simulation fellowship program has become more robust, and the graduates since the change in the program structure have begun to establish quality simulation programs in their home facilities since the introduction of the 4CID instructional design approach. The challenges for the simulation fellowship faculty team are many. These include the markedly increased hours of curriculum development time to ensure the fellowship curriculum is embodying the 4CID model. There is also regular follow-up for each fellow by the faculty team to ensure they are recording in their logbook and working on capstone projects. Finally, each fellow receives individual feedback on their milestone progress twice throughout the fellowship to ensure they are meeting requirements and to provide opportunities to focus learning opportunities for their growth. These reviews also offer opportunities for the fellow to give the program feedback so it too can evolve and adapt. Although these challenges are not insignificant, the increased expertise of the graduating simulation fellow and return on investment from their training cannot be ignored. We look to explore this return on investment through a study that is currently underway surveying how the fellowship has impacted both the trajectory of the fellows’ career and the projects that they have undertaken to impact patient care.In this manuscript, we have applied the 4CID model as a strategy to design the NYC Health +Hospitals simulation fellowship curriculum and given examples to help guide how a program can be crafted and honed. The framework is supported by evidence-based approaches, best practices, and expert opinion. By relying on such a framework, the program is considering the cognitive load associated with simulation education knowledge, skills, and attitudes and suggested mitigation strategies. By applying such a roadmap, the goal is to develop reproducible and high-quality simulation fellowship curriculum, by which simulation fellows can create impactful simulation experiences and maybe move the dial on improved clinical outcomes. Further research into the constructs of programmatic design and development of assessment tools would be recommended to continue the maturation of simulation fellowships toward improved consistency and quality. We hope that through description of the model underpinning our program and how it has been applied, it will promote further conversation within the simulation community to shape future directions of simulation fellowships.


The geriatric population is rapidly growing, and this growth is beyond the pace of increase in the number of healthcare professionals who are qualified to care for and tend to the various needs of this significant subgroup of the population. The current university curricula have not been sufficient in terms of quantity as well as their ability to address the ageism inherent in the perspectives of students from across the educational spectrum. In recognition of the absence of standardized geriatric guidelines, medical associations across Canada and the United States have established geriatric learning competencies for medical programs. Nevertheless, there are exiting gaps regarding the development and evaluation of geriatric-focused didactic programs that adequately train and build competency among the students interested in pursuing careers with geriatric-specific elements. A university-wide program was developed to enhance aging education and build competency through sparking interest, providing better education related to aging, and building better relationships between future healthcare professionals and older adults. To evaluate the impact of this program, a logical framework was developed a-priori and revised through constant iterations and following discussion with the program’s multidisciplinary stakeholder group. Quantitative measures are being augmented with in-depth qualitative interviews to explore elements influencing students’ experiences with the program and the effect on their interests in and attitudes towards geriatrics. The results will inform our conclusions regarding program effectiveness in enhancing interest in geriatric-focused education among the students and trainees and assist with recommending future directions regarding impact and large-scale dissemination and implementation.


This paper describes the initial development process of an eLearning continuing professional education program primarily for post-licensure physiotherapists –“Electronic Aerobic Exercise Recommendations to Optimize Best Practices in Care after Stroke” (eAEROBICS). Our objective was to develop an evidence-based, clinically relevant, user-friendly eLearning program for online delivery tailored to facilitate prescription of aerobic exercise post-stroke by physiotherapists. The Demand Driven Learning Model guided curriculum design, delivery, and evaluation. Based on previously identified gaps in physiotherapists’ knowledge of aerobic exercise, four learning modules were developed and delivered using an eLearning platform to maximize cost-effectiveness and flexibility. Five physiotherapists volunteered to pilot eAEROBICS, providing preliminary feedback on strengths and suggestions for improvement.Theoretical information and clinical applications addressed the learning objectives of each module in a logical manner. All technical or administrative issues encountered during program delivery were addressed. The feedback from the pilot end-users informed modifications to the eAEROBICS program.Processes used in developing eAEROBICS have the potential to serve as a model of electronic continuing professional education for other areas of physiotherapy practice. Further investigation of end-user perspectives and clinical impact of the program is warranted to determine the overall effectiveness of the program. According to the most recent available information, there are currently over 400,000 people living with the effects of stroke in Canada –3. ThereKnowledge translation (KT) strategies, including eLearning, have been shown to influence clinician self-efficacy and practice behaviors but the effects have been modest at best –23 The iRecognizing the lack of self-efficacy regarding AE screening and prescription among physiotherapists in stroke rehabilitation, and the need for ready access to evidence-based, AE-related knowledge, we developed a novel CPE program entitled eAEROBICS. Five stages in the development and evaluation were identified by the eAEROBICS team: Stage 1: Design of a curriculum to prepare physiotherapists for safe and effective implementation of aerobic training in stroke rehabilitation; Stage 2: Delivery of the curriculum in an efficient, interactive and cost-effective manner; Stage 3: Preliminary evaluation of end-user feedback on the curriculum, delivery, and services of the eAEROBICS program; Stage 4: A more comprehensive evaluation of end-user perspectives of the program, using the four domains of the Kirkpatrick model of learning evaluation – reaction to learning, learning , clinician behavior (recommendation adherence), and patient results ; Stage 5Given the importance of documenting the processes used to enhance clinical uptake of recommendations, , 34 thisImportant barriers to AE implementation in practice include lack of access to relevant knowledge and low therapist confidence , 19, 20.Development of the eAEROBICS Program: We chose the Demand Driven Learning Model (DDLM)  as the tThe first two critical dimensions of the DDLM framework – structure and content – were of foremost concern in designing the eAEROBICS curriculum.To establish a foundational structure for the curriculum of the eAEROBICS program, we turned to previous literature reporting gaps in knowledge of practicing physiotherapists that could adversely affect clinical implementation of AE in stroke rehabilitation , 37. AmoTo optimize knowledge translation the eAEROBICS curriculum was designed to be learner-centered, evidence-based, and clinically relevant. Specific learning objectives were identified for each module. Subject matter experts (SMEs) – a physiotherapist with expertise in stroke neurotherapeutics, focusing on aerobic exercise and its application to aerobic capacity to reduce morbidity; a physiotherapist with expertise in exercise physiology; and a health psychologist with expert knowledge in health behaviour change associated with cardiovascular risk – were consulted to develop content relevant to the learning objectives. Each of these SMEs had doctoral degrees in the subject matter. Information presented in each module was comprehensive yet concise, and grounded in accessible, empirical evidence from the evidence-based AEROBICS recommendations  and otheThe SMEs were consulted on an ongoing basis throughout the curriculum design, delivery, and evaluation processes. Their feedback was utilized to ensure that each content area was presented authentically, in a clinically relevant manner, and was reflective of key elements of the DDLM Framework.The third and fourth critical dimensions of the DDLM framework – delivery and service – were important considerations in this stage of development of our eLearning CPE program.The mode of delivery of an eLearning curriculum must be carefully selected to ensure functionality and interactivity . A standInteractivity within eAEROBICS was regarded as key to both self-assessment and tailoring learning experiences to the needs of the individual end-user. To achieve these objectives, reflective questions and short quizzes using multiple choice, true or false, and matching formats, developed by the SMEs, were interspersed throughout the program, with feedback provided on the learner’s performance on the quizzes.Provision of administrative and technical assistance is fundamental in eLearning environments to ensure accessibility and responsiveness , 44, 45.The fifth and final critical dimension of the DDLM framework was the focus of Stage 3. Evaluation by end-users is a critical component in the on-going development of eLearning modules. An iterative process is recommended in order to improve the program over the course of development , 46, 47.Upon completion of each module, email feedback was solicited from each participant to the open-ended questions of ‘what worked’ and ‘what could be improved’ in terms of (i) content and structure of the curriculum related to each learning objective, (ii) delivery of the program, and (iii) services available to aid navigation of the module. After completion of all four modules, one of the researchers interviewed each participant by phone using a format similar to that used in the email feedback. The interviewer took notes of each participant’s responses, which were later entered into a spreadsheet. One researcher (MT) coded the responses by participant number (1-5) and grouped the responses under two main headings – strengths and areas for improvement – and 3 subheadings – content, delivery, and service. A second researcher (MM-L) checked the groupings. Disagreements were resolved by discussion. In the Results section that follows, the feedback from the participants is summarized.Module one (Behaviour Change) 21 min (m) 50 sec (s)Module two (Exercise Physiology) 26 m 50 sModule three (Screening for AE) 27 m 02 s plus 2x10 m case studiesModule four (Prescription of AE) 27 m 40 s plus 2x12 m case studies totalling 150 m 47 s (2 h 30 m 47 s)Five female physiotherapists volunteered to participate in the pilot evaluation of the eAEROBICS program. Three had a Bachelor of Science degree in Physiotherapy and 2 had a Master of Science degree in Physiotherapy. Two worked in the area of general rehabilitation providing mostly one-on-one therapy and 3 worked in community rehabilitation providing in home therapy. All had over 5 years of clinical experience treating patients post stroke. All treated patients that were predominantly stroke but saw a mix of neurology cases. Two of the therapists worked with under 5 stroke patients per week, 3 worked with 5–10 patients per week. All worked in medium to large sized cities. The total running time of the individual modules were:Actual time spent on each module by participants is difficult to determine, as they could leave and return to the module without limit. The goal of 30 min total to complete each module was an important component to making it accessible to busy therapists.In terms of delivery and service, no insurmountable technical or administrative issues were encountered in the delivery of the eAEROBICS program. Response time to email inquiries from participants seeking assistance was within 24 h, on average. Each participant provided feedback on every module. The DDLM framework was used to categorize perceived strengths and suggestions for improvement into three themes -content, delivery, and service. Table Although the participants were largely positive about the content and delivery of the modules, they provided several constructive suggestions for improvements, including: (i) adding a program navigation guide in the introduction to the program, (ii) reducing the content in the introductory section, (iii) adding a progress bar to mark the percentage of the module completed, (iv) inserting on-screen buttons to allow feedback and request technical support, (v) re-taping the voiceover, (vi) including additional learning resources such as references and downloadable documents, and (vii) providing of program completion certificates, which could be used for CPE credits. Each of the recommendations has been incorporated in a revised eAEROBICS program, which is currently being evaluated using a larger sample (Stage 4).Translating new research and guidelines into practice is an essential aspect of health care services . eAEROBILearning strategies must be carefully selected during the planning and development of CPE programs  The DDLMTo foster life-long learning, CPE programs should encourage learner involvement and provide meaningful educational experiences . We embeProviding opportunities for formative feedback has also been highlighted as a fundamental component of adult learning by theorists who develop CPE , 54, 55.The small sample size is a limitation, although it is acceptable given the pilot and qualitative nature of this phase of the project . Also, iFurther investigation of the impact of eAEROBICS on physiotherapists’ learning as well as on actual implementation of AEROBICS recommendations in clinical practice is warranted.Development of eAEROBICS offers an example of the application of best practices in digital learning to create an eLearning program for physiotherapy post-licensure education. The eAEROBICS program was designed to be an effective way to facilitate evidence-based knowledge translation and encourage self-directed learning relevant to the clinical application of aerobic exercise. The processes used in curriculum development, delivery, and formative evaluation, guided by the DDLM framework, have the potential to serve as a model of electronic CPE for other areas of physiotherapy practice. Further investigation of the impact of eAEROBICS not only on perceptions of physiotherapists but also on actual implementation of AEROBICS recommendations in clinical practice is warranted.