Acquiring optimal motor skills in preschool children presents a key element for proper psychomotor development. Therefore, the aim of this study was to determine gender differences and the effects of a multi-year exercise program on the level of motor skills in 161 preschool children (5–6 yo). Participants were divided into one control and three experimental groups. Motor skills were assessed with the Bruininks-Oseretsky Test of Motor Proficiency (BOT-2). To determine the difference in scores for each BOT-2 test between control and experimental groups, one-way ANOVA was used for girls and boys separately, while two-way ANOVA was used to determine the difference between the genders in the overall BOT-2 score. The results indicate that a 1-year multilateral exercise program has a positive effect on the level of motor skills in preschool children. Interestingly, additional years of participation in exercise programs yielded the maintenance of acquired motor skills level. Additionally, the exercise program affected preschool girls more than boys considering both individual and composite BOT-2 scores. According to the findings of this study, the presented exercise program could have potential benefits on multilateral development of the motor skills in preschool children, which could facilitate the balance of locomotor and manipulative skills. Therefore, the integration of multilateral programs intended for preschool children could be considered for implementation within the kindergarten curriculum. Early childhood presents a sensible period for cognitive, emotional and motor development . TherefoA period between two and seven years of age is considered as the period of the significant development of the FMS. This period is divided into three developmental stages: initial, basic and mature ,8. AccorStill, to the best of the authors’ knowledge, none of the studies investigated the effect of multi-year physical exercise programs on children’s motor skills level. Therefore, this study aimed to determine the effect of the multi-year physical exercise program on the children’s motor skills level. Additionally, this study will explore whether this program equally affects boys and girls considering the motor skills levels.This is a cross-sectional study that included preschool children from a kindergarten in the city of Zagreb, Republic of Croatia. A total of 161 children participated . The total sample was divided into four groups: three experimental and one control group. The experimental groups participated differently in exercise program in terms of time duration: group one (G1) exercised for 1 year (n = 38); group two (G2) exercised for 3 years (n = 36); and group three (G3) exercised for 4 years (n = 38). Participants from the control group (CG) participated in the standard preschool educational program (n = 49). This educational program was organized in the same kindergarten according to the State Pedagogical Standard of Preschool Education. All children from experimental groups were tracked and involved in the program. However, only final measurements were taken into consideration for this study. Because the program ended at the same time for each group, this study examined the difference between groups by the end of exercise programs. Consequently, all measurements were taken at the same time point in 2019 see .In addition, only healthy children were included in the study, and out of the total sample of 170 children, 9 subjects dropped out. Thus, the total number of participants was 161. This study was completed according to the declaration of Helsinki, and the Ethical Committee of the Institution verified that this investigation complied with all ethical standards for scientific investigations involving human participants .2 (kg/m2) was used, and BMI was calculated accordingly. Bruininks-Oseretsky Test of Motor Proficiency (BOT-2) [Body height was measured with an anthropometer where the participants stood barefoot on a flat surface, in an upright position, with relaxed shoulders and joined heels. Bodyweight was measured with a medical scale where participants stood barefoot in an upright position. To estimate the weight status, the formula body weight/body height (BOT-2)  was used (BOT-2) .The physical exercise program in the kindergarten has been implemented since 2015/2016. The experimental groups were actively involved in the physical exercise program, and they were included in the program as follows: G3 starting from 2015/2016, G2 from 2016/2017 and G1 from 2018/2019. One pedagogical year was defined as the period of 10 months and included the physical exercise program of 40 weeks, containing 120 training units (hours). This means that when the measurement took place, the total number of hours in the physical exercise program for G1 was 120, followed by the G2 with 350 h and the G3 with 470 h. The exercise program was organized three times per week (three sessions) with a duration of 60 min per each session, while two physical education teachers were responsible for the realization, control and assigning of the workload for the children. Two physical education teachers systematically planned, programmed and conducted physical exercises with the children. Specific training sessions were carried out so that everyone followed the same protocol. The physical exercises program was focused on the multilateral psychophysical development of the child. Additionally, within the physical exercises program, a schedule with the distribution of the workload through program contents was defined. Within one school year, the participants were introduced to basic gymnastics exercises, various jumps and runs, polygon obstacles, manipulation of various props and learned the basics of team sports such as football, handball, basketball and volleyball . During p < 0.05.First, descriptive analysis for basic anthropometric variables was completed for girls and boys separately. Second, to determine difference in scores for each BOT-2 test between control and experimental groups, one-way ANOVA was used for girls and boys separately. To investigate difference between the gender in the overall BOT score, two-way ANOVA was employed, using gender and years of experience as fixed factors. The level of the statistical significance was set at 2 for the total sample, respectively. A two-way ANOVA did not reveal a significant interaction between gender and the years of exercise (p = 0.48). However, analysis has shown a statistically significant difference in the overall BOT-2 test score among both girls and boys between most groups  117 ± 6.2 cm, 21.8 ± 4.1 kg and 15.8 ± 2.1 kg/moups see .p = 0.0004–0.0001), ambidexterity (p < 0.0001), hand coordination (p = 0.01–0.0003), balance (p = 0.001–0.0009) and strength (p < 0.0001).In girls, a one-way ANOVA has shown a statistically significant difference between CG and G1, CG and G2 and between CG and G3 in the following variables: fine motor precision . However, no significant difference was evident in other variables between CG and other experimental groups (see Among boys, a significant difference between CG and G1, CG and G2 and CG and G3 was observed in three variables (fine motor precision: oups see .The results of this study indicate that a 1-year multilateral physical exercise program has a positive effect on the level of motor skills in preschool children. Moreover, our results show that additional years of participation in exercise programs yielded the maintenance of the acquired FMS levels. Additionally, evident sex difference was observed in the effects of exercise programs on FMS level. More specifically, the findings of this study suggest that the program affected preschool girls more than boys considering both individual and composite BOT-2 scores.Regarding sexual dimorphism, differences were found between CG and experimental groups in all eight components of movement skill among girls, while among boys, these differences were evident in only three tests see . The eff‘Why did the additional years of exercise program not result in more significant effects on FMS level in preschool children?’ The explanation for such findings in the current study may be due to the fact that the participants did not exercise daily. Consequently, it would be necessary to increase the weekly frequency of physical activity, which could potentially have an effect on FMS level. This approach is in accordance with the recommendations of several scientific institutions [Although this program affected both sexes, one question still remains: itutions ,20, whicitutions . HoweverThe results of this study differ from the results of a more recent study  that incFurthermore, Guo et al.  found a This study has three advantages. First, this study included three experimental groups with different previous motor experiences and different exercise volumes, which can allow better conclusions about the effects of the exercise program on the measured motor skill parameters. Second, this study followed children for 4 years through a sensitive period of development. Lastly, given the long duration of the physical exercise program, a relatively large number of participants remained throughout the study, which contributes significantly to the quality of this study. However, this study has several limitations. In this study, we did not control for other variables such as children’s cognitive status, family’s socioeconomic indicators and parents’ physical activity. Additionally, physical activity of the children was not measured outside of the school environment. This could potentially affect the results and conclusions drawn. Additionally, this research has a cross-sectional design, which limits causal conclusions. This study did not include pretest measurements and did not control for the above-mentioned variables, and therefore, these limitations need to be considered while interpreting the results of this research. Lastly, the sample is not representative, which may influence the generalizability of the results in the context of the general preschool children population.According to the results of our study, authors support that preschool children take up regular exercises with the multilaterally directed exercise program as covered within this study. In order to increase the effectiveness of motor skills, further researches are needed regarding the validation of new methods and the construction of multilateral exercise programs intended for preschool children. Conclusively, the presented exercise program could have potential benefits on multilateral development of the motor skills in preschool children, which could facilitate the balance of locomotor and manipulative skills. Therefore, the integration of multilateral programs intended for preschool children could be considered for implementation within the kindergarten curriculum.


The availability of clinical decision support systems (CDSS) and other methods for personalizing medicine now allows evaluation of their real-world impact on healthcare delivery. For example, addressing issues associated with polypharmacy in older patients using pharmacogenomics (PGx) and comprehensive medication management (CMM) is thought to hold great promise for meaningful improvements across the goals of the Quadruple Aim. However, few studies testing these tools at scale, using relevant system-wide metrics, and under real-world conditions, have been published to date. Here, we document a reduction of ~$7000 per patient in direct medical charges  in Medicare Advantage patients (≥65 years) receiving benefits through a state retirement system over the first 32 months of a voluntary PGx-enriched CMM program. We also observe a positive shift in healthcare resource utilization (HRU) away from acute care services and toward more sustainable and cost-effective primary care options. Together with improvements in medication risk assessment, patient/provider communication via pharmacist-mediated medication action plans (MAP), and the sustained positive trends in HRU, we suggest these results validate the use of a CDSS to unify PGx and CMM to optimize care for this and similar patient populations. Pharmacogenomics (PGx) has been shown to play an important role in certain prescribing situations by identifying medication responders and non-responders to avoid specific adverse events and optimize drug dose ,2. IndeeLikewise, comprehensive medication management is an established mechanism by which a patient’s medications  are assessed by pharmacists to ensure that a given regimen is appropriate, safe, and effective. Many improvements in patient care have been observed by this approach to jointly consider medical history, conditions, comorbidities, medications, extrinsic factors, and desired outcomes ,24. ThusHowever, combining PGx testing and CMM services, empowered by a unifying clinical decision support system (CDSS) , promiseIn 2017, the Teachers’ Retirement System of the State of Kentucky (TRS) partnered with Coriell Life Sciences (CLS) and the Know Your RX Coalition (KYRx) seeking to improve member health by reducing medication-related adverse events and increasing the success of medication therapies used by their Medicare Eligible Health Plan members. Consequently, CLS was engaged to offer TRS members the opportunity to participate in a PGx + CMM program that included member engagement and education, genetic testing, and pharmacist review by KYRx of broad medication risks.While long-promised, few large-scale, longitudinal datasets have been published evaluating the real-world impact of PGx and CMM on healthcare delivery. The particular PGx + CMM intervention implemented with TRS addressed practical limitations mentioned above, and also facilitated system-wide implementation by supporting a wide variety of activities  includinIn this retrospective study of a real-world implementation, we document the direct outcomes of this program on direct medical costs and healthcare resource utilization in patients ≥65 years of age in the first 32 months after program initiation. We conclude with a discussion on the impacts in this population and further application in more diverse population samples.Members of the Teachers’ Retirement System of the State of Kentucky (TRS) were invited to enroll via a mailed announcement which included educational material tailored for an older adult population and a letter from TRS’s Executive Secretary explaining the goals and benefits of the program. TRS membership consists of, and is restricted to, Kentucky’s public school teachers who are retired and Medicare eligible, and their spouses. Enrollment in the PGx + CMM program consisted of a brief phone call, web-based survey, or in-person event to collect contact, medication, diet, and lifestyle  information. Additional mailings, phone outreach, webinars, an information website, and newsletters were also used as part of the ongoing recruitment process.Upon enrollment, a DNA collection kit was sent to the participant for in-home saliva collection. The sample was then shipped to and analyzed by a CLIA- and CAP-licensed DNA sequencing laboratory . The variants assayed were chosen based on documented clinical utility and their relationship to medication use outcomes . ResultsPharmacists  utilized GeneDose LIVE™ , a comprehensive clinical decision support system (CDSS) to evaluate risks associated with a participant’s medication regimen. This evaluation involved detailed information on medications and indications, genetic and non-genetic sources of patient-specific risk, as well as mechanisms for the validation of regimen adjustments. The results were distilled by a clinical pharmacist to create a medication action plan (MAP) that summarized the proposed changes for the patient’s prescribing physician.®, Food and Drug Administration (FDA) black box warnings, and pediatric-specific risks , and added any missing patient-specific information to the CDSS. A comprehensive medication risk assessment was then performed by analyzing the prepopulated CDSS Risk Chart—a data visualization of the magnitude of medication risks across nine potential sources. These distinct but not mutually exclusive risks include pharmacogenomics, drug–drug interactions (including phenoconversions), contraindications, anticholinergic burden, lifestyle factors , pregnancy and lactation warnings, AGS Beers Criteriasee also . Next, tIn this retrospective study of a real-world implementation, deidentified administrative medical and pharmacy insurance data were used to establish baseline statistics and analyze changes over time in healthcare resource utilization and medical costs for members of TRS. Specifically, data for the 12 months pre-program period and for the first 32 months after program start were extracted and prepared for analysis. Continuously insured members—defined as those with a medical insurance claim in the 12 months prior to the start of the program, and at least one claim in the last six months of the program evaluation—aged 65 and greater were included in the economic analysis. Data from TRS members who did not enroll in the program functioned as a contemporaneous comparison cohort  while TRS members who completed the program  were explored as the “intervention group”. De-identified retrospective chart reviews of the medication risks and pharmacist recommended regimen changes were conducted for the intervention cohort to evaluate clinical impacts of the intervention.This study was approved by the Biomedical Research Alliance of New York Institutional Review Board . While it was determined that the retrospective chart review did not constitute research involving human subjects, written consent for the use of the patient’s de-identified sample and information for research was nonetheless requested as part of the sample collection process and data from TRS members not consenting to research use were excluded from the retrospective chart review.Invitations to participate in the PGx + CMM program  were senDemographic information was captured from insurance claims to calculate basic statistics prior to program enrollment including age, sex, Charlson Comorbidity Index (CCI) , and nump values less than 0.05 were considered statistically significant.A similar approach was utilized to calculate healthcare resource utilization (HRU) rates. Specifically, claims for each category of service   were aggn = 4716) were analyzed.All active TRS members were invited to participate in the PGx + CMM program. Of those invited, 28,619 were determined to be continuously enrolled in their medical insurance plan . A totalp < 0.0001), with more participants prescribed 11 or more medications than the non-participant group. Additional member-specific demographic information was not available to this study, but publicly-accessible data suggest that fewer than 5% of the Kentucky teachers population are non-white [At baseline—defined as the 12 calendar months preceding the program start—the characteristics of the intervention and control cohorts were similar. Specifically, the average age of the intervention and control cohorts was 73.7 versus 74.0, respectively, and the distribution of sex and Charlson Comorbidity Index (CCI) was 67% female and CCI = 3.6 in both cohorts . The inton-white  and descon-white . As suchn = 4716) was AGS Beers Criteria® with 90% of participants prescribed a medication with the associated risk . The most prevalent risk in this population (ted risk . This wated risk . The nexp = 0.0151) compared to the control group, whose costs continued to increase linearly. The intervention group realized a calculated cumulative reduction in direct medical charges of $37,027,769 relative to the control during the first 32 months of the program ; 63.00 fewer ED visits on average, a decrease of 34.41 visits ; 2664.95 more OP visits on average, a decrease of 420.64 visits . We also compared healthcare utilization by type of service  visits, emergency department (ED) visits, and inpatient (IP) days to explore the impact of the PGx + CMM program on the HRU, a proxy for patient health and wellness . At basevice see  during tvice see .Here, we document multiple real-world economic and clinical outcome improvements, as measured by direct medical costs and healthcare resource utilization, produced by the deployment of a voluntary pharmacogenomics-enriched comprehensive medication management program in an older adult population. Comparing members that opted into the program to those who did not , we observe an average calculated $218.34 PMPM savings. This equates to a cumulative $37 million savings in direct medical charges to the plan over the first 32 months post-implementation in the 5288 intervention members. In addition, we observed a 1.9%, 6.8%, and 14.9% reduction in outpatient, emergency department, and inpatient events, respectively, compared to expected healthcare resource utilization (HRU) . Thus, wOne potential source of the large improvements observed in this analysis is the comprehensive nature of the intervention which combined best-of-breed components into a unified process of medication management. This program embraced the complexity and competing priorities inherent in healthcare delivery by bundling patient education and engagement, pharmacogenomic testing, comprehensive medication risk evaluation, pharmacist intervention, efficient clinical decision support tools, and dissemination of information to physicians.Of particular note is the high quality of the pharmacogenomic information included in this intervention. As has been described elsewhere, it is the result of a rigorous selection process for the inclusion of genes, polymorphisms, and variants that have emerged from evidence-based evaluation using established criteria which is independent of the population at hand ,33,40,41Another likely source of the strong trends we observed is the unique nature of the implementation that combined the science of PGx with the systematic evaluation of regimens provided by CMM, and empowered by a comprehensive CDSS. This provided the opportunity to examine outcomes at a high level, bridging the fractured nature of the healthcare system which includes generalists, specialists, outpatient, emergency care providers, and inpatient hospital facilities. By looking at administrative health insurance claims and measuring outcomes over the first 32 months of a combined PGx + CMM program, we were able to detect significant changes in economics and healthcare resource utilization. In addition, the program was designed with intrinsic scalability  and actioned by trained pharmacists communicating to program-naive prescribers to maximize potential benefits. There are few, if any, constraints on implementing this solution in other contexts as there is minimal disruption in the existing workflow of care delivery.In addition to the effective engagement of participants, the unique structure of this program places pharmacists in a key role and can provide value even prior to a medication being prescribed. Such pre-emptive testing arises naturally through genome-informed approaches and represents a key enhancement in the CMM process. Moreover, PGx + CMM delivered by trained pharmacists creates an opportunity for these members of the healthcare community to use their training, knowledge, and experience in an especially strategic and impactful way. Furthermore, the time spent with the patient is highly impactful and imparts the potentially synergistic effects of improved adherence on outcomes and so savings. While recent literature highlights the challenges of implementing PGx or CMM programs, pharmacists are an under-utilized component of the healthcare delivery apparatus for integrating meaningful change into clinical practice. In environments where collaborative practice agreements exist, PGx + CMM bolsters the tools available to directly ensure more precise prescribing. Even here, where there was not a collaborative practice agreement, the plain and direct language of the pharmacist-created medication action plan (MAP) allowed for efficient communication of medication change recommendations.The complexity of the healthcare delivery process and the science behind evidence-based decision-making requires the bundling of established PGx recommendations with the best practices in CMM, and the facilitation of patient and prescriber education. The CDSS serves this purpose in this implementation. This streamlining of several aspects, each of which can become rate-limiting under certain circumstances, makes it possible to efficiently deliver improved healthcare even in a disjointed healthcare system. Uniting the fragmented system’s tools, expertise, know-how, professional guidances, and data provides ample opportunity to increase system-wide confidence and scalability.Finally, an oft-overlooked part of the story in healthcare implementations is the patient experience. In this unique real-world implementation, factors such as the confidence participants gained from close interaction with pharmacists, their understanding of the potential impact of genetics on their response to medications via educational outreach, the ability to complete the entire process without leaving home, as well as the security, privacy, and ease of the program appeared to be advantages. Indeed, moving the point of care to the patient’s home also has the potential to improve healthcare delivery for patients with substandard access to healthcare. These factors are especially important in an older adult population, for whom technology can be a barrier rather than a convenience.Interestingly, while participant usage of prescription drugs is very well captured in the claims data, usage of over-the-counter (OTC) medications may also have been impacted by participation in the program. Several OTC medications also show some evidence of genetic factors influencing their safety and efficacy profiles. Thus, this implementation, through the use of the CDSS, provides the opportunity to improve the safety and efficacy of their usage. Common examples of such medications in this population include ibuprofen ,43 and oIt was also notable that our intervention group was prescribed more medications on average than the control group. This suggests that those who could benefit from this intervention were more likely to enroll. A follow up study could provide additional insights into the outreach and education process and how it impacted participation.In light of the disparate nature of healthcare delivery in a population such as TRS, which is unified by a common payor rather than a common provider, our approach was to implement a combination of best practices and to provide a unifying CDSS that is independent of the particular systems where patients receive their care. The CDSS thus became the critical tool to deliver precision medicine successfully at scale in similar scenarios, and the MAP became the output that drives outcome improvements.However, while pharmacists delivered all of the MAPs to members’ prescribing physician of choice, we were unable to assess specific physician–patient interactions in our retrospective study design. Thus, the significant net effects observed at the level of systemwide billing cannot be attributed directly to any one of these unmeasured activities. Nevertheless, our real-world implementation of PGx + CMM demonstrated meaningful economic and clinical impacts. Ultimately, future studies focusing on the subtleties of program population, process, and delivery will be needed to investigate the specific causal factors that drive the observed outcomes.As this ongoing PGx + CMM program continues, we expect to see continued downward bending of the cost curve—a sustained decrease in healthcare costs—and improved patterns of HRU for participants. This may even accelerate due the advancing age and healthcare burden of the participating members who were not prescribed any medications at the start of the program. In addition, these benefits are expected in any similar population dealing with issues of polypharmacy, and the more controlled usage of HRU ensures efficient use of healthcare dollars and other resources overall.We document the impact of a large real-world implementation of a pharmacogenomics-enriched comprehensive medication management (PGx + CMM) program in the US. By deploying these tools, supported by a robust and efficient clinical decision support system, patients, healthcare providers, and the system as a whole benefited in synergistic ways. The observed reduced costs, meaningful shifts in the patterns of patient healthcare resource utilization, as well as other encouraging trends suggest that wide-spread adoption could significantly advance the goals of the Quadruple Aim in health systems globally.


Previous studies address posttraumatic stress disorder (PTSD) following disasters as a public health issue. However, few studies investigate the long-term effect of disaster exposure on PTSD among hospital medical workers (HMWs).This study aimed to study the prevalence of ICD-11 PTSD and complex PTSD (CPTSD) among exposed and non-exposed HMWs 11 years after the Wenchuan earthquake in China, to identify the factors associated with PTSD and CPTSD scores, and to examine the factor structures of PTSD and CPTSD models.A cross-sectional study was conducted using a self-administered online questionnaire. Two thousand fifty-nine valid samples were collected from four hospitals in 2019. Descriptive statistical analysis, multivariate regression models, and confirmatory factor analysis (CFA) were performed.The prevalence of PTSD and CPTSD was 0.58 and 0.34%, respectively. The unexposed group reported higher PTSD and CPTSD scores than the exposed group. The type of workplace and marital status were significantly associated with the PTSD and CPTSD scores of HMWs. The CFA results indicate that both the correlated first-order model and the correlated two-layer model were a good fit to explain the structure of PTSD and CPTSD.These findings suggest that few HMWs who were exposed to the Wenchuan earthquake suffered from PTSD or CPTSD 11 years following the disaster. However, psychological support was still necessary for all HMWs, especially for unmarried HMWs who were Working in smaller hospitals. Further research is required to analyze mental health status using ICD-11 PTSD and CPTSD to provide ongoing evidence to help HWMs cope effectively with the challenges of future disasters. Posttraumatic stress disorder (PTSD) is one of the most common types of psychopathologies experienced after a mass traumatic incident caused by a disaster . The disAccording to a report released by China's Ministry of Civil Affairs in 2008, the Wenchuan earthquake event of May 12, 2008, claimed 69,227 lives and injured 374,643 people, whereas 17,923 people were missing at the time of the report and 46,240,000 people were affected , 9, 10. The prevalence of PTSD was relatively high directly following the Wenchuan earthquake, but its prevalence declined over time for the majority of people, according to a systematic review, which analyzed 58 original studies of PTSD prevalence in the 10 years after the earthquake among community populations . The preThe World Health Organization has published the 11th version of the International Classification of Diseases (ICD-11), which was approved by the World Health Assembly in 2017 . Brewin Therefore, this study investigated ICD-11 PTSD and CPTSD among hospital medical workers (HMWs) 11 years after the Wenchuan earthquake. Specifically, the objectives were to examine (1) the prevalence of PTSD and CPTSD among earthquake-exposed and non-exposed HMWs, (2) the factors associated with their PTSD and CPTSD scores, and (3) the factor structure of PTSD and CPTSD models. The findings are significant as they demonstrate the long-term effects of PTSD and CPTSD. This helps us understand the effect of disasters on healthcare professionals' well-being and provides evidence to inform policies on the prevention of mental health disorders in the long term.This was a cross-sectional study. Data were collected from four hospitals from June 2019 to October 2019. These four hospitals were selected based on the following criteria: (1) geographic location as they were located either at the epicenter of the Wenchuan earthquake , near the epicenter , or a great distance from the epicenter  and (2) the size of the hospital, including the total number of beds and HMWs and the number of potential participants. Hospital B was the largest hospital , and Hospital D was the smallest hospital  medical doctors or registered nurses working in one of the four selected hospitals and (2) willing to participate in the study and complete the online questionnaire. Those who were taking prolonged leave due to illness or maternity were excluded from the study. The sample size was large enough to ensure that parameter estimates were within the acceptable margin of error of 4% with a confidence level of 95%. Approximately 500 valid questionnaires were collected from each of the hospitals . HoweverThe International Trauma Questionnaire (ITQ)  was usedAppendix I).The ITQ measurement assesses six symptoms of PTSD distributed across the following three clusters: re-experiencing , avoidance , and a sense of threat . The six symptoms of DSO are distributed across the following three clusters: affective dysregulation , negative self-concept , and disturbances in relationships . All symptoms were assessed using a five-point Likert scale ranging from 0 = “not at all” to 4 = “extremely”  and DSO-related functional impairment , also in the domains of social life, work life, and other important areas of life, respectively. Scores ≥ 2 (“moderately”) indicated the presence of a symptom. PTSD and DSO prevalence requires the presence of at least one symptom in each PTSD/DSO cluster plus the presence of functional impairment associated with these symptoms. CPTSD prevalence requires a PTSD prevalence, one symptom in each DSO cluster, and associated functional impairment. For example, if RE1 or RE2 > 2 (“moderately”), RE criteria were met; if AV1 or AV2 > 2, AV criteria were met; if TH1 or TH2 > 2, TH criteria were met; if PTSDI1, PTSDI2, or PTSDI3 > 2, PTSDI criteria were met; and if RE, AV, TH, and PTSDI criteria were all met, a diagnosis of PTSD was made. The same criteria were used for CPTSD prevalence. PTSD and CPTSD were assessed using a self-reported measure rather than a clinician-administered assessment. For this reason, the term “probable” is used when referring to estimating the prevalence of PTSD and CPTSD.Wenchuan earthquake exposure: The participants were divided into the following three groups according to their level of exposure to the Wenchuan earthquake: unexposed HMWs, earthquake survivors (who were adolescents in 2008 and were exposed to the earthquake as survivors), and earthquake relief workers (who were adults in 2008 and were exposed to the earthquake as HMWs).Workplaces: Four hospitals participated in the survey . They had different geographical locations and different numbers of HMWs and beds .Sociodemographic factors: The sociodemographic data that were collected included gender , educational level (college or below/university or above), marital status (unmarried/married), profession , age, and length of service in years.p-values (p), and 95% confidence intervals (CIs) were reported for each independent variable in the regression models. An analysis of variance test was performed, and p- and R2-values were reported to evaluate the performance of each regression model.First, a descriptive analysis was performed to understand the characteristics of the sample and determine the prevalence of PTSD and CPTSD. Second, the PTSD and DSO scores were calculated for different groups of HMWs. Multivariate regression models were established to examine the effect of Wenchuan earthquake exposure, the type of workplace, and sociodemographic factors on the PTSD and DSO scores of the HMWs. All analyses were performed using SPSS software, version 25.0  with the threshold for statistical significance at a two-tailed α value of 0.05. The beta coefficient (β), the associated two-tailed Third, confirmatory factor analysis (CFA) was performed using Mplus 7.11  to examine the structure of PTSD and CPTSD in HMWs. Four different models were established for CFA based on data from multiple trials –26. ModeThe comparative fit index (CFI), root mean square error of approximation (RMSEA) with 95% CI, and Tucker–Lewis index (TLI) were used to assess the fit of the hypothetical model. Cutoff values of 0.95 for CFI and TLI  and 0.06 for RMSEA  , 28 wereOf the 2,059 HMWs in the study, 9.9% experienced the Wenchuan earthquake as survivors , 15.4% experienced the earthquake as disaster relief workers , and 74.7% did not experience the earthquake  and 0.34% , respectively. All participants with PTSD  and most of those with CPTSD  had not been exposed to the Wenchuan earthquake. Approximately equal numbers of participants were recruited from the four hospitals. However, Hospital A reported the highest prevalence of PTSD  and CPTSD . Moreover, male participants reported a higher prevalence of both PTSD (1.1 vs. 0.5%) and CPTSD (0.4 vs. 0.3%) than their female HMW counterparts. The participants with an education at the university level or above reported a higher prevalence of PTSD (0.7 vs. 0.5%) and a lower prevalence of CPTSD (0.1 vs. 0.5%) than their counterparts with lower educational levels. A higher prevalence of PTSD (0.7 vs. 0.5%) but a lower prevalence of CPTSD (0.3 vs. 0.4%), was reported by unmarried participants compared with married participants. Medical doctors reported a much higher prevalence of PTSD (1.5 vs. 0.4%) than registered nurses, whereas all cases of CPTSD were found in registered nurses. The mean age was lower and the length of service as an HMW was shorter in those with CPTSD than in those with PTSD and those without CPTSD or PTSD.F(2) = 17.281, p < 0.001] and DSO  scores. Unexposed HMWs reported the highest PTSD  and DSO  scores.Participants, who were probable with CPTSD, reported the highest PTSD  and DSO  scores, followed by those with PTSD . ParticiF(3) = 11.985, p < 0.001] and DSO  scores. Participants from Hospital A reported the highest PTSD  and DSO  scores, followed by those from Hospital C . Moreover, participants with different marital statuses had significantly different DSO scores .Participants from different hospitals showed significantly different PTSD [According to the multivariate regression models , experieThe type of workplace also had a significant effect on the HMWs' PTSD and DSO scores. Specifically, working in Hospital D (located at the epicenter) was associated with a decrease in the PTSD score of 0.859 SDs  and a decrease in the DSO score of 1.279 SDs  while holding Wenchuan earthquake exposure and sociodemographic factors constant. Similarly, working in Hospital B (close to the epicenter) was associated with a decrease in the PTSD score of 1.521 SDs  and a decrease in the DSO score of 1.335 SDs  while holding Wenchuan earthquake exposure and sociodemographic factors constant.In addition, being married was associated with a decrease in the DSO score of 0.697 SDs  while holding Wenchuan earthquake exposure, type of workplace, and other sociodemographic factors constant. Finally, none of the associations of HMWs' PTSD or DSO scores with gender, educational level, profession, age, and length of service was statistically significant .The CFA results are presented in p < 0.001) with coefficients ranging from 0.693  in Model 4 to 0.930  in Model 4. All correlations between the six latent factors were significant with coefficients ranging from 0.508  to 0.875  in Model 2. In Model 4, the latent factors of RE, AV, and TH constructed the second-layer latent factor of PTSD, while AD, NSC, and DR constructed DSO. PTSD was significantly correlated with DSO with a medium-sized effect .All of the factors loaded in Models 2 and 4 were significant  11 years after the Wenchuan earthquake. Moreover, none of the participants who reported Wenchuan earthquake exposure were probable for PTSD. After such a long time, many cases of PTSD may have been treated or spontaneously resolved. In some cases, the rescuers who may have had PTSD had left the HMW labor market and could not be recruited for this study. Moreover, in general, the prevalence of PTSD is lower after natural disasters than after manmade disasters . For exaThe factors that had a significant and positive association with HMWs' PTSD and CPTSD scores were not being exposed to the earthquake and working in a hospital that was distant from the epicenter . These findings confirm that geographical location has a significant role in an individual's adaptation following a disaster as previously reported . AnotherIn addition to earthquake exposure and the type of workplace, sociodemographic factors were also associated with HMWs' PTSD and CPTSD scores. An unmarried status was positively associated with PTSD and CPTSD scores, which is consistent with the findings of previous studies . This maThe results from the CFA indicate that both the correlated first-order model (Model 2) and the correlated two-layer model (Model 4) had a good fit to explain the structure of PTSD and DSO. The correlated first-order model, which was constructed with three latent variables  representing PTSD and three latent variables  representing DSO, was the model with the best fit. The correlated two-layer model included two second-order latent variables, PTSD and DSO, to explain the covariation among the six first-order factors. These results are consistent with previous studies showing that these two models are generally the best-fitting models –44.First, this study was limited by its sampling methodology and the unbalanced ratio between medical doctors (18.2%) and registered nurses (81.1%) from the four selected hospitals. This may limit the generalizability of the finding that being a medical doctor predicted the development of PTSD and CPTSD. Second, this study only assessed HMWs' exposure to the Wenchuan earthquake, which occurred 11 years prior to the survey. There may have been other traumatic events during that period that caused PTSD or CPTSD. Therefore, a life event checklist for the past 11 years may be warranted to evaluate the effect of other traumatic events during this period. Third, we used an online self-reported survey, and we did not conduct interviews to make the diagnoses. Finally, this was a cross-sectional study with a non-random sampling strategy. With the recent increase in the number of disasters globally, a long-term longitudinal study is needed to serve as a guide to advocating for the protection of the mental health of healthcare professionals working in hospitals of different sizes.The findings of this study suggest that HMWs who were exposed to the Wenchuan earthquake rarely reported PTSD or CPTSD 11 years following the disaster. However, psychological support is still necessary for all HMWs, especially for single HMWs working in smaller hospitals. Further research is required to evaluate the mental health status of HMWs using the ICD-11 criteria for PTSD and CPTSD to provide ongoing evidence to help HMWs cope effectively with the challenges of future disasters.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Human Subjects Ethics Sub-Committee in the Hong Kong Polytechnic University (Ref.HSEARS20190416035). The patients/participants provided their written informed consent to participate in this study.SL and SC designed and performed data collection. CG, SL, and SC analyzed the data. CG and SL drafted the manuscript. All authors contributed to the article and approved the article submission.This study was supported by Dean's Reserve Grants (FHSS and FENG) from the Hong Kong Polytechnic University (Project ZZHH).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Despite improved survival due to new treatments, the 10-year survival rate in patients with breast cancer is approximately 75%. Lymphovascular invasion (LVI), a prognostic marker independent from histological grade and stage, can only be fully determined at final histological examination. Lipid composition is deregulated in tumour via de novo lipogenesis, with alteration in lipogenic genes in LVI. We hypothesise alteration in lipid composition derived from novel non-invasive spectroscopy method is associated with LVI positivity.Thirty female patients (age 39–78) with invasive ductal carcinoma were enrolled, with 13 LVI negative and 17 LVI positive. Saturated, monounsaturated, polyunsaturated fatty acids and triglycerides  were quantified from ex vivo breast tumours freshly excised from patients on a 3 T clinical MRI scanner, and proliferative activity marker Ki-67 and serotonin derived histologically.p = 0.0189) in LVI positive : 0.25–0.64) than negative . There were significantly lower TRG (p = 0.0226) in LVI positive  than negative . There was no significant difference in SFA (p = 0.6009) or PUFA (p = 0.1641). There was no significant correlation between lipid composition against Ki-67 or serotonin, apart from a borderline negative correlation between PUFA and serotonin .There were significantly lower MUFA (Lipid composition might provide a biomarker to study lymphovascular invasion in breast cancer.• Monounsaturated fatty acids in lymphovascular invasion (LVI) positive invasive breast carcinoma were significantly lower than that in LVI negative.• Triglycerides in LVI positive invasive breast carcinoma were significantly lower than that in LVI negative.• Lipid composition from MR spectroscopy reflects the rate of de novo lipogenesis and provides a potential biomarker independent from histological grade and stage.The online version contains supplementary material available at 10.1007/s00330-020-07502-4. The 10-year survival rate in patients with breast cancer is approximately 75% . LymphovLipid composition can be measured using biochemical extraction methods for gas chromatography , but sufWe therefore hypothesise there is a difference in lipid composition derived from DQF-MRS and DQF-COSY between LVI status, and is independent from cell division and inflammation in breast cancer.We conducted a two-group cross-sectional study to quantify lipid composition in whole tumours freshly excised from patients with breast cancer, with further comparison against histopathological findings Fig. .Fig. 1StThe study was approved by the North West - Greater Manchester East Research Ethics Committee (REC Reference: n = 2), mixed phenotype (n = 5) or grade I (n = 2) on final histopathological examination of the excised tumour. Standard histopathological examination was performed to determine tumour size, grade, LVI, lymph node metastasis, receptors and Nottingham Prognostic Index (NPI) [Thirty female patients (age 39–78) with grade II or III invasive ductal carcinoma participated in the study. Patients over the age of 18, undergoing a wide local excision or mastectomy, and with a tumour size larger than 1 cm in diameter on mammography were eligible. Patients with previous breast malignancies or patients who had neoadjuvant chemotherapy or neoadjuvant hormonal therapy prior to surgery were excluded. In total, 50 patients consented to be included in the study from 53 consecutive patients in the Breast Unit at Aberdeen Royal Infirmary. However, from these 50 resected tumour specimens, 11 were not scanned due to theatre delays and MRI scanner unavailability. A further 9 tumour specimens were excluded due to lobular phenotype (ex (NPI) . Upon fuex (NPI) , while sex (NPI) .t1 increment of 1 ms, 256 increments, bandwidth of 1000 Hz and 256 points. The acquisition durations were 10 min 40 s for DQF-MRS and 9 min 45 s for DQF-COSY. Further details of the methodology can be found in the The freshly excised tumour specimen without formalin treatment was immediately transported to the Aberdeen Biomedical Imaging Centre. The lipid composition spectra were acquired from a single voxel snug fit to the tumour on a 3 T whole-body clinical MRI scanner  using a 32-channel receiver coil for high sensitivity detection and a body coil for uniform transmission. PUFA spectrum was acquired using DQF-MRS sequence  with a rDQF-MRS and DQF-COSY are complementary methods for lipid composition quantification, with respective strength. DQF-MRS targets a single metabolite through the manipulation of quantum states of coupled spins, with the resonance frequency at 2.8 ppm and 5.3 ppm for PUFA . The empPUFA was quantified using DQF-MRS and conventional MRS, demanding minimal discrepancies between the two acquisitions for accuracy. Both acquisitions adopted PRESS sequence backbone , 23 with1 and T2. PUFA, quantified as spectral peak amplitude at 5.3 pm from DQF-MRS, was referenced to 0.9 ppm from conventional MRS, while all the other lipid constituents, quantified as corresponding spectral peak volumes from DQF-COSY, were referenced to  ppm from DQF-COSY. The PUFA peak at 5.3 ppm from DQF-MRS is spectrally edited through coupling peak at 2.8 ppm [Lipid constituents were calculated as a ratio of the corresponding spectral peak against the methyl proton peak, without the quantification of absolute concentration using estimated T 2.8 ppm  equivale 2.8 ppm . The uns 2.8 ppm , 26.The peak amplitudes of PUFA (5.3 ppm) and methyl proton (0.9 ppm) were quantified using prior knowledge databases  and AMARU tests were used for MUFA, SFA and TRG while independent sample t test for PUFA  between LVI groups. Fisher’s exact tests were applied for categorical variables . Spearman’s correlation tests were performed between lipid composition against proliferative activity marker Ki-67 and serotonin, while Pearson’s correlation test between PUFA and serotonin in the entire cohort. A p value < 0.05 was accepted as being statistically significant.All statistical analysis was performed in the SPSS software . Normality was determined on all the collected data using the Shapiro-Wilk test. Mann-Whitney The patient demographics are summarised in Table z = 2.3470, p = 0.0189, Fig. z = 2.2810, p = 0.0226, Fig. p = 0.6009, Fig. p = 0.1641, Fig. There were significantly lower MUFA , occupying spectral peak at  ppm , typicalLipid composition did not show a significant correlation against proliferative activity marker Ki-67, indicating no evidence to support a direct link between lipids and tumour cell division within the framework of this work. Lipid composition did not show a significant correlation, apart from a borderline negative correlation in PUFA, against serotonin (5-HT), an epigenetically identified marker of poor 10-year survival rate . Hence, Lipid composition in the breast has been quantified using MRS in the adipose tissue from both patients and healthy volunteers. Conventional STEAM sequence has been used for the quantification of lipid composition in breast adipose tissue from patients at 3 T  and healThis study is the first to investigate the association between lipid composition and LVI positivity in whole human breast tumours. The quantitative nature of the novel spectroscopy method provides an objective approach for LVI evaluation, compared to time-consuming and experience-dependent histological approaches. The quantification at whole tumour estimates an overall degree of compromise in lymphatic vasculature , eliminaSFA and TRG are determined from diagonal peaks, with MUFA from cross peak, leading to a difference in scaling from underlying population in energy states. Hence, the comparison is performed on individual lipid constituent across patients, and comparison of nominal values between peaks will require further correction on the scaling factors. The overlap of the distribution in MUFA or TRG between groups may limit the diagnostic value of lipid composition as a biomarker in LVI. This work, as a precursor for consequent in vivo patient studies, attempted to augment the understanding of lipid composition in breast cancer with limited statistical power for multiple comparison correction. Future large cohort in vivo studies, supported by novel spectral analysis approaches , are reqIn conclusion, MUFA and triglycerides are reduced in positive LVI resulting from accelerated de novo lipogenesis. Lipid composition observed through novel spectroscopy method might provide a biomarker to study LVI in breast cancer.ESM 1(DOCX 4467 kb)


In the USA, numerous summer programs are available for undergraduate students that seek to increase the number of individuals from groups underrepresented in medicine (URM) that matriculate to medical school. These programs have typically been conducted at research-focused institutions, involving hands-on-research and various enrichment experiences. For 2020, the COVID-19 pandemic resulted in the suspension of on-campus student activities at American universities, necessitating a switch to a virtual format for these URM-focused programs. Outcomes, however, from these programs conducted virtually, necessitated by the COVID-19 pandemic, have not been reported. The Penn Access Summer Scholars (PASS) program at the Perelman School of Medicine (PSOM) targets URM undergraduates, providing two consecutive summers of mentored research and enrichment experiences, with the goal of enabling participants’ matriculation to PSOM. PASS has been an 8 week on-campus experience, but during summer 2020, virtual programming of 6 weeks was provided due to the COVID-19 pandemic. Participants in the 2020 virtual offering of PASS completed pre- and post-program surveys that included 5-point Likert-style and open-ended questions to determine the impact of the programing on self-assessments of research skills, familiarity with the physician identity, and preparedness to be a PSOM student. Post-program, participants also assessed program administration and content. With respect to program objectives, participants reported significant increases in their self-reported confidence in conducting research, understanding of physician identity, and sense of preparedness for medical school. The educational value of the program content, their level of engagement in the program and the overall quality of the program were rated as excellent or outstanding by large majorities of respondents. Content analyses of participant comments were consistent with these quantitative results. Therefore, a premedical summer enrichment program targeting URM undergraduates can be successfully conducted virtually to achieve program objectives and may increase the availability to these initiatives. Diversity in medical education and in the physician-workforce has been recognized as both societal and healthcare imperatives [1–5]. This has prompted, over the last 30 years, the establishment of summer programs that target undergraduate students from historically disadvantaged groups to expose them to research and other enrichment experiences with the goal of enhancing participants’ competitiveness for and/or increasing participant success in medical school . These pTo support its efforts in recruiting a diverse student body, the Perelman School of Medicine (PSOM) at the University of Pennsylvania, since 2008, has sponsored the Penn Access Summer Scholars (PASS) program. The program targets URM and first-generation and/or low-income (FGLI) undergraduate students, providing two summers of mentored research, didactic presentations and enrichment activities, with the goal of enabling participants’ successful matriculation to PSOM.Typical of programs of this type, PASS has been an on-campus experience, with housing provided and participants interacting directly with peers, mentors, lab personnel and program leaders. However, because of the COVID-19 pandemic, all on-campus, in-person activities were suspended by the University of Pennsylvania for the summer of 2020 for all university and visiting students. This necessitated the reconfiguration of PASS as a virtual program. Here, we describe the initial outcomes from our delivery of PASS as a virtual experience, findings that may have implications for increasing the availability of these types of summer enrichment programs to more students.The PASS program was launched in 2008 by the Office of Admissions of PSOM as an initiative to increase the diversity of students that matriculated to its medical school. It was originally conceived of as a program in which disadvantaged students  from partnering colleges and universities would spend two consecutive summers after their sophomore and junior years of college on the campus of the University of Pennsylvania doing mentored research and participating in pre-medical enrichment experiences. Students who successfully completed the two summers of PASS would be eligible to apply to PSOM without having to submit a MCAT score. Beginning with 3 partnering schools – Haverford College, Princeton University, and the University of Pennsylvania – the program has expanded to now include 6 additional institutions – Bryn Mawr College, Howard University, Morehouse College, Oakwood University, Spelman College, and Xavier University of Louisiana. As of September 2019, 50 students  had participated in the two summers of program. Of this group, 38 had matriculated to PSOM, 8 gained admission to another medical school, 1 had deferred application to medical school to conduct research, and 3 opted not to pursue a career as a physician. For the on-campus programing, students currently receive a stipend of 4000 USD and are provided housing (~ 1500 USD/student) free-of-charge. Funding for the program has come from institutional funds and extramural grants from the National Institutes of Health.Promote the development of physician identity and professionalism in the PASS participantsExpose PASS participants to high quality biomedical research as well as develop pertinent research skillsFoster the mentor/mentee relationship between the PASS participants and their supervising research facultyCreate a sense of community and cohesion among the cohort of PASS participantsFacilitate the participants’ transition into PSOMThe established on-campus program has involved two consecutive, 8-week summer experiences, the goals of which have been to:It has involved 40 hours/week of mentored research (25 hours) with various enrichment events and experiences (15 hours) that include a weekly combination of didactics, physician shadowing, visits to student-led community clinics, physician career narratives, book club discussions, events with medical students and one-on-one meetings with program leadership.In adhering to institutional mandates, the PASS program for summer 2020 was delivered as a 6-week experience in a virtual format, while still pursuing the five goals noted above. The virtual program was also a 40 hours/week experience for the participants, but in contrast to the on-campus experience, it was structured to be 15 hours/week of research-focused activities with 25 hours of enrichment recapitulating as much as possible the enrichment programing that was typically provided. In addition, the virtual program was modified to have two paid rising second-year medical students to help coordinate and facilitate the online experience for the PASS participants. The medical student coordinators also assisted in content development and program assessments. Comparisons of the on-campus versus the virtual offerings of PASS are summarized in 9:00 AM-1:00 PM: Research-related activities, preparation for future sessions and/or lunch1:00 PM – 3:30 PM: Enrichment and didactic activities done virtually as a group3:30 PM −5:00 PM: Flexible time for 1-on-1 meetings with program team and for preparationThe 2020 virtual PASS programing included four major components that are detailed in In addition, the medical student coordinators hosted ‘Game Nights’ in which the participants could come together outside of the required scheduled programing on evenings and weekends for fun activities. The schedule of programing for the first two weeks is presented in the S1 Supplemental Information. Video conferencing was provided using the Zoom platform.Participants completed a pre-program survey that included 5-point Likert-style and open-ended questions to determine baseline expectations as well as self-assessments of their research skills, familiarity with the physician identity, and preparedness to be a PSOM student. Students subsequently completed a post-program survey using 5-point Likert-style and open-ended questions to evaluate self-reported changes in research competence, sense of physician identity and preparedness for PSOM as well as the effectiveness of the program in achieving its goals. Finally, each event or activity was evaluated for its educational value, participant engagement, and overall quality by the participants using 5-point Likert-style questions and free text comments . All surveys were anonymous. At the start of the program, participants were informed that the data would be collected anonymously and used for program evaluation and improvement. Further, throughout the program, participants were encouraged to complete the surveys to provide their honest feedback to the program team.Mixed method approaches were used to analyze all survey data collected from the participants. Likert-style question results and other quantitative question results were analyzed using calculations of means and standard deviations (SD), using Microsoft Excel and R (Version 3.5.1). Percentages of ‘yes/no’ questions and gender differences were calculated. Free response question entries were qualitatively analyzed independently by three authors , searching for common themes across the participants’ responses and using a qualitative content analysis with inductive reasoning. This study was reviewed and approved by the Institutional Review Board of the University of Pennsylvania, and was deemed to be exempt given minimal risk to participants.The 15 participants of PASS 2020 were undergraduate students from 9 institutions  across 5 different states. Participants represented 10 different majors ranging from Mathematics to Sports Medicine, with a majority of students studying Biology or Biology-related majors. Participants are all URM, with 11 students self-identifying as Black/African American, 3 students self-identifying as Hispanic/Latinx, and 9 students self-identifying as FGLI. Six participants were returning students who had participated in the in-person PASS program the previous summer of 2019, while 9 students were first-time participants of the program. A summary of program participant characteristics is presented in Program activities/events were individually rated on a 5-point Likert scale  by participants for their educational value, level of participant engagement during the activity and overall quality. For the entire group of 40 program activities/events, their mean educational value was rated at 4.33 (SD = 0.78); mean level of participant engagement during activities was rated at 4.38 (SD = 0.70); and mean overall quality of activities was rated 4.41 (SD = 0.68). The mean response rate for each activity or event was 71.5%. Participants’ qualitative comments on the various categories of activities were consistent with these high ratings and are summarized in the S3 Supplemental Information.Participants were variously queried using a 5-point Likert scale  about the success of the program in achieving its explicit goals ; advocates for patients (‘advocating for those whose voice is silenced’), and translators of biomedical science research into care . Second, participants spoke of physicians as having a package of humanistic qualities including, possessing a willingness to ‘sacrifice [one’s] needs for someone else’s’ as well as being ‘humble,’ ‘compassionate and supportive,’ and ‘always willing to do what they can to help.’ Finally, respondents referenced the high level of competence required of physicians, emphasizing that the physician is someone who is ‘always learning and sharpening [their] clinical skills.’“Being a physician is having an in-depth understanding of medicine but also an understanding of the audience you’re serving. After this program I’ve come to realize that having a connection with your patients [and] empathizing with them really makes the biggest difference not only in the patient’s health outcome but in their overall day to day life.”“A physician should not only be technically competent and deeply committed to their craft, but they should realize that practicing excellent medicine includes taking into account the relational aspect. Overall, they should go above and beyond to provide excellent care, this includes what’s taught as medicine, but can also mean simply listening to what their patient is going through and caring about their whole being.”While respondents continued to reference advocacy and clinical competence, their descriptions of physician identity following the program focused more explicitly on the relational qualities of being a physician. In this regard students commented:Respondents’ self-reported competence in their ability to do research increased significantly after this summer of PASS   ; status as a racial minority (‘[adapting to] being a double minority in a predominantly white institution and profession’); and feelings of intimidation in attending a prestigious institution such as PSOM (‘Feeling empowered enough to see myself as a Perelman student’).“I was concerned about paying for medical school and being able to adapt to the fast-paced environment, but I feel a little more comfortable with both.”“I feel more confident in that when I encounter any problems during my time at Perelman, I have resources and people to connect with that will support me.”“The discussion about imposter syndrome really helped to reassure me about this natural occurring feeling and that I am fit and worthy of being a medical student at Perelman.”Participant comments after the program indicated that the PASS experience helped, at least in part, to alleviate these concerns as reflected in the following comments:Despite fears and concerns, participants following the program were excited for the opportunities in medical school to connect with their peers and with patients, and to further develop their identities and skills as future physicians. As one student commented, ‘I am really looking forward to honing into how I want to be a physician … I am also looking forward to continuing relationships with PASS students and creating new relationships with other students.’Despite being well-received, there were challenges and limitations to the delivery of PASS using a virtual format. The first were technical. Because of a lack of a consistently stable internet connection, or weather-related disruptions in electrical power, some students missed parts of a session, were unable at times to contribute fully to a session and/or were disrupted while making a presentation.Second, participants in their qualitative comments indicated a continued desire for on-campus experiences such as visiting the student-led community clinics, shadowing physicians, hands-on research in a bench lab, and physically engaging the other PASS students, program staff and research mentors. As one student who commented on a virtual versus an in-person experience said, ‘[I missed] shadowing [opportunities], physically having access to the campus and the surrounding community, being able to live with my peers and getting to know them in person.’ Even as respondents indicated that this cohort of PASS students achieved a high level of cohesion and community, they also acknowledged that the ‘in-person experience is irreplaceable to a degree’ and that ‘it would have been easier to get to know my fellow PASS scholars if we lived together.’During the program, feedback was solicited from the participants to make real-time adjustments. We were particularly attentive to ‘Zoom fatigue’ ), which In addition to the real-time feedback, respondents also provided feedback in the post-program survey on the various program activities and events to enhance future iterations of the program. This included specific suggestions regarding the facilitation, duration and/or content of the programing, which are further highlighted in S3 Supplemental Information.Racial and ethnic minorities continue to be underrepresented in medical school matriculants and in the physician-workforce, with this underrepresentation being particularly dramatic for Black males . EffortsThere are a number of processes that may disproportionately disadvantage URM students in either gaining admission to medical school or, once matriculated, getting off to a strong start ,3. FirstPASS was developed to address these issues, which are reflected in its five interconnected program goals. The program seeks to facilitate participants’ successful transition to medical school by fostering an understanding of physician identity, competence in doing research, a strong research mentor-mentee relationship and a sense of community, that together enable student confidence and self-efficacy as well as establish affirming relationships that can be drawn on for future support. Among respondents, this virtual offering of PASS was seen as very successful in achieving the goals of the program. Specifically, respondents rated the educational value, participant engagement and quality of programing as excellent to outstanding and strongly agreed that PASS had achieved its program goals and thus would recommend it to their peers  were all rated as excellent to outstanding at the end of the program (Finally, the with respect to the transition to medical school, participants felt significantly more prepared to be a PSOM student after this virtual offering of PASS (There is a growing body of literature that speaks to both the power as well as pitfalls of virtual learning, which has become essential during the COVID-19 pandemic ,38. ThusOur findings have implications, not only for the continuance of established programs such as PASS, but also for the wider availability of pre-medical summer enrichment programs that target disadvantaged students. First, in not having to pay for housing and facilities charges, along with potentially less administrative costs, virtual programing may make diversity efforts of this type more affordable to institutions who have been constrained in doing so because of the costs associated with on-campus programing. Second, the virtual programing allows for participation of a potentially wider and more regionally diverse pool of participants who would not be limited by issues of travel for on-campus programing. Finally, a virtual format allows for recruitment of research mentors beyond that of a single institution as well as cross-institutional partnerships that enable robust programing not possible if the institutions acted individually.There are several limitations to this study that should be noted. First, this study involved a small number of participants at a single institution and thus may not be generalizable to other institutions with a different mix of students. Second, the assessments of the program were all based on self-assessments. Thus, going forward, it be will necessary to evaluate the impact of the PASS program with more objective measures. Third, the assessments were done immediately after the program. Future studies should therefore assess the durability of what was learned and how PASS impacted participants’ medical school experience. Finally, we cannot exclude a selection bias for participants who would be much more favorably disposed toward the program.Despite these limitations, our data provide evidence that premedical summer enrichment programs targeting URM undergraduates, offered virtually, can continue to be effective during challenges such as the COVID-19 pandemic in pursuing their goal of increasing medical trainee and physician-workforce diversity.Click here for additional data file.


Saccharomyces cerevisiae. S. cerevisiae strain S1 accumulated the highest amount of ergosterol [7.8 mg/g dry cell weight (DCW)] among the wild-type yeast strains tested and was first selected as the host for subsequent metabolic engineering studies. Then, the push and pull of ergosterol biosynthesis were engineered to increase the metabolic flux, overexpression of the sterol acyltransferase gene ARE2 increased ergosterol content to 10 mg/g DCW and additional overexpression of a global regulatory factor allele (UPC2-1) increased the ergosterol content to 16.7 mg/g DCW. Furthermore, considering the hydrophobicity sterol esters and accumulation in lipid droplets, the fatty acid biosynthetic pathway was enhanced to expand the storage pool for ergosterol. Overexpression of ACC1 coding for the acetyl-CoA carboxylase increased ergosterol content from 16.7 to 20.7 mg/g DCW. To address growth inhibition resulted from premature accumulation of ergosterol, auto-inducible promoters were employed to dynamically control the expression of ARE2, UPC2-1, and ACC1. Consequently, better cell growth led to an increase of ergosterol content to 40.6 mg/g DCW, which is 4.2-fold higher than that of the starting strain. Finally, a two-stage feeding strategy was employed for high-density cell fermentation, with an ergosterol yield of 2986.7 mg/L and content of 29.5 mg/g DCW. This study provided an effective approach for the production of ergosterol and other related terpenoid molecules.Ergosterol, a terpenoid compound produced by fungi, is an economically important metabolite serving as the direct precursor of steroid drugs. Herein, ergsosterol biosynthetic pathway modification combined with storage capacity enhancement was proposed to synergistically improve the production of ergosterol in  Saccharomyces cerevisiae is a promising host due to its generally recognized as safe (GRAS) status, industrial robustness, and ease of genetic manipulation  pathway has been successfully engineered for efficient production of heterologous and native terpenoids, such as artemisinic acid  and post-squalene biosynthetic module , most of which are involved in the post-squalene biosynthetic pathway  was used as the host to construct, maintain, and amplify plasmids.Yeast strains used in this study are listed in E. coli was cultivated at 37°C in LB medium and ampicillin at 50 μg/mL was supplemented when necessary. Yeast strains were grown in YPD medium . When necessary, 200 mg/L G418 sulfate or 100 mg/L hygromycin B was added to the growth medium. For ergosterol production, the 2% glucose in YPD was replaced by 5% glucose.Yeast and bacterial strains were stored in 25% glycerol at −80°C. DpnI  and transformed into E. coli. The suitable target sequence was selected using E-CRISPR online tool . To construct the donor DNAs for integration with inducible expression of ARE2, UPC2-1, and ACC1, the constitutive promoters in the plasmid containing the relative donor DNAs was replaced by GAL1 promoter via homologous recombination using ClonExpress II One Step Cloning Kit . The expression cassette of UPC2 with TEF1 promoter was first generated by replacing Cas9 in plasmid p42H-SpCas9, resulting in the construction of the plasmid p42H-TEF1-UPC2P. The pleiotropic mutation G888D of the regulation factor UPC2 was introduced by inverse PCR using plasmid p42H-TEF1-UPC2P as a template, generating p42H-TEF1-UPC2-1P. The plasmid p42H-ACT1-UPC2-1P was constructed by replacing the TEF1 promoter of plasmid p42H-TEF1-UPC2-1P using ClonExpress II One Step Cloning Kit.All plasmids and primers used in this study are listed in ine tool . Donor Dsion PCR  using PrCRISPR/Cas9 guided gene knock out, integration, and substitution were performed as previously described with some modifications . Yeast c2SO4, 8 g/L KH2PO4, 3 g/L MgSO4, 0.72 g/L ZnSO4.7H2O, 10 mL/L trace metal solution, and 12 mL/L vitamin solution) in a 2 L glass bioreactor . Fermentations were carried out at 30°C, and pH was controlled at 5 by automatic addition of 5 M NH4OH. Dissolved oxygen (DO) was maintained at >15% saturation by adjusting the agitation rate  and airflow rate (1–3 vvm). After the batch culture phase, a feeding solution containing 500 g/L glucose and 12 mL/L vitamin solution was used to achieve fast cell growth with the pseudo-exponential feeding model. For the last phase, ethanol was fed to the fermenter until the end of the fermentation to improve the intracellular accumulation of ergosterol. The feeding rate FS during the pseudo-exponential phase was determined by the following equations quations :X0, V0, and S were the initial biomass density [g dry cell weight (DCW)/L], the initial culture volume (L), and the glucose concentration (g/L) in the feeding medium; YX/S was the yield of the cell on glucose (g DCW/g glucose); μ was the specific growth rate (h–1), m was the maintenance coefficient (g glucose ⋅ g DCW−1 ⋅ h−1), and t was the time (h) after starting the feeding. A predetermined specific growth rate of 0.13 h–1 was used to avoid overflow metabolism. The values of YX/S and m were 0.5 and 0.05, respectively, according to the previous study (where us study . The fee600) using a visible spectrophotometer . DCW was determined from plots of OD600 and DCW. The total ergosterol and squalene in yeast cells were extracted as previously described . Then quantitative PCR (qPCR) reactions were performed in a StepOne Plus Real-time PCR System  using Luna Universal qPCR Master Mix . The ACT1 gene was selected as the internal control gene to normalize the amount of the total RNA in different samples. The relative transcriptional level for each gene was determined using the 2–ΔΔCt method  analysis was performed to confirm the overexpression of the genes integrated into the genome. The RNAiso Plus Kit  was used for the extraction of total RNA from the harvested yeast cells. The residual genomic DNA in RNA samples was digested by DNaseI . The cDNA templates were synthesized from the DNaseI treated total RNA using LunaScriptt method .S. cerevisiae, ergosterol biosynthesis is tightly controlled  was integrated into the chromosome of S1 at the HO site, resulting in the strain S1-tHMG1. As expected, its squalene production was obviously increased and reached to 10.8 ± 0.3 mg/L within 48 h flask fermentation, while the squalene content in reference strain S1 was too low to be detected  were inserted in the genomes of strain S1 and squalene accumulation strain S1-tHMG1, respectively, generating the strains S1-ARE2 and S1-tHA. Overexpression of ARE2 increased ergosterol content to 10 ± 0.4 mg/g DCW, which was 28% higher than that of the parental strain  was inserted to the genome of S1-ARE2 and S1-tHA, resulting in the construction of strains S1-AU and S1-tHAU. The growth rate of strain S1-AU was further decreased when compared with S1-ARE  and ergosterol production into two-independent phases . This caon media .GAL80 to eliminate the dependency on galactose induction. With the ARE2 and UPC2-1 overexpression cassettes controlled by GAL1 promoter integrated into the genome of S1-G and the native ACC1 promoter replaced by GAL1 promoter, S1-G-AUAC was constructed. As shown in GAL regulatory network as anticipated. In addition, higher ergosterol production with comparable biomass was achieved in S1-G-AUAC owing to the decoupling of cell growth and ergosterol accumulation.In the current study, S1-G was first constructed based on S1 by deleting It is noteworthy that in addition to the promoted production of ergosterol in S1-G-AUAC, ergosta-5,7,22,24-tetraene-3β-ol, the direct precursor of ergosterol, was also accumulated to a rather high level . The peaS. cerevisiae. However, when growing on ethanol, the rate of yeast biomass production is lower than that on glucose (The performance of the best ergosterol-producing strain (S1-G-AUAC) was evaluated with fed-batch fermentation in a 2 L bioreactor. Glucose and ethanol are commonly used carbon sources for production of terpenoids in yeast . Compare glucose . With th glucose .600 reached 142.05 after 34 h fermentation, while ergosterol content remained at a low level (8.7 mg/g DCW). During the ethanol feeding stage, the production of ergosterol was greatly improved while the cell growth rate was decreased. The final cell density reached to 101.4 g/L  after 122 h fermentation. At the end of fermentation, 2,986.7 mg/L ergosterol with a content of 29.5 mg/g DCW was obtained. Despite the significantly increased ergosterol yield, ergosterol content was lower than that using shaking flasks (40.6 mg/g DCW). This might be caused by the different growth conditions of cells in shake flasks and the fed-batch fermenter. During the whole fermentation process, about 101 g glucose and 372 g ethanol were fed, and the yield was 1.4 Cmol % . As shown in The profile of fed-batch fermentation indicated that the stages of cell growth and ergosterol accumulation were clearly separated by employing the two-stage feeding strategy . After 1S. cerevisiae. Ergosterol production was firstly increased via engineering of the pull and push of ergosterol biosynthesis by enhancing ergosterol esterification and systematically upregulating the transcription of ergosterol biosynthetic pathway genes. Then, ergosterol production was further improved by storage pool expansion through facilitating fatty acid biosynthesis. In addition, the decoupling of ergosterol production from cell growth further improved ergosterol production. A titer of 2986.7 mg/L ergosterol was eventually achieved using fed-batch fermentation by adopting a two-stage feeding strategy. The engineered yeast strains constructed in this work not only provides new ideas for enhancing ergosterol production but can also be used as a compelling platform for the production of other economically important terpenoids.In this work, combined metabolic engineering was established as an efficient approach to improve ergosterol productivity in an industrial The original contributions presented in the study are included in the article/Z-JS and J-PL conceived the study. Z-JS performed the experiments, prepared figures and tables, and wrote the manuscript. J-ZL participated in data analysis, discussion, and revision of the manuscript. LZ and H-LX participated in the establishment of quantitative analysis method of ergosterol and squalene. Y-QJ participated in the fed-batch fermentation. G-SL participated in the revision of the manuscript. M-BW and L-RY participated in the discussion and coordination of the study. J-PL supervised the whole research and revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.