PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME downmodulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. In turn, PRC2-regulated genes were repressed in isogenic PRAME-KO lymphoma cell lines, and PRAME was found to directly interact with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects, leading to PRAME expression and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis and provide a preclinical rationale for synergistic therapies combining epigenetic reprogramming with PRAME-targeted therapies.PRAME is a prominent member of the cancer testis antigen family of proteins, which triggers autologous T cell–mediated immune responses. Integrative genomic analysis in diffuse large B cell lymphoma (DLBCL) uncovered recurrent and highly focal deletions of 22q11.22, including the   The current treatment standard of combined immunochemotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) has achieved significant improvement in patient outcomes in diffuse large B cell lymphoma (DLBCL) over the past 15 years . HoweverSeveral genetic landscape studies using next-generation sequencing have contributed to a near-complete description of the most prevalent somatic gene alterations and structural genomic changes in the context of transcriptionally defined DLBCL subtypes, such as the cell-of-origin classification –7. Howev+ and CD4+ T cells, both of which may be required for tumor elimination  that are presented by MHC on tumor cells is a promising therapeutic strategy for patients who experience treatment failure , 15. TAAmination , 17. Henmination . HoweverPRAME gene , as a clinically and genetically relevant CN alteration in germinal center B cell–like (GCB) type DLBCL. PRAME is a prominent member of the cancer testis antigen family of proteins that is expressed in various types of cancers but generally not in normal tissues apart from male germinal cells  analysis in a previously reported cohort of 347 DLBCL tumors from patients uniformly treated with R-CHOP , 19. We al cells , 21. PRAal cells –26. In oal cells . EZH2 mung DLBCL , 29. OurNFKBIZ, BCL6, CD274, NFATC1, and BCL2) and deletions , were found  of the cases. Of these, 10 cases were predicted to harbor homozygous PRAME deletions. These deletions were clustered in a narrow chromosomal region that included a very small number of genes  (PRAME deletions) were found significantly enriched in GCB type DLBCL (GCB type: 17% [31/180] vs. activated B cell (ABC) type: 8% [8/98], P < 0.01; PRAME deletions were significantly associated with poor treatment outcome in GCB-DLBCL. The 5-year disease-specific survival (DSS) was 84.5% in PRAME-CN-neutral cases versus 67.2% in PRAME-deleted cases (P = 0.03)  (https://doi.org/10.1172/JCI145343DS1). Using an independent de novo DLBCL cohort uniformly treated with R-CHOP .We performed CN analysis using high-resolution SNP6.0 arrays  in a prere found . NotablymiR-650)  and were = 0.03) . The 5-yype: 17% /180 vs. deletion . MoreovePRAME, which were involved in the minimally deleted region on chromosome 22q11.22, and correlated each mRNA level with deletion status. We found that only PRAME and ZNF280B mRNA levels were significantly correlated with 22q11.22 deletion status, whereas the other 3 genes were not significant  cases were positive (score +1 or +2) for PRAME protein expression, and results were concordant between PRAME expression and CN deletion status , A and BPRAME deletion–associated phenotypes, we next analyzed the corresponding RNA-Seq data from a subset of the de novo DLBCL cohort (n = 311), comparing expression profiles of the PRAME-CN-neutral and PRAME-deleted samples. PRAME deletion was significantly correlated with PRAME mRNA expression , Karpas-422, and SU-DHL-4  PRAME–isogenic KO cell line systems, using MHC I/II blockade conditions because donor PBMCs were not HLA matched. CD69 positivity and T cell proliferation were compared in cocultures with WT versus PRAME-KO cells. In HBL-1, the CD69+ T cell population was significantly reduced when cocultured with PRAME-KO cells as observed using flow cytometry (P = 0.0006). T cell proliferation assessed by CellTrace violet dye was also significantly reduced in the PRAME-KO group  assays with PBMC samples from 4 patients with PRAME-CN-neutral (2 of 4 HLA-A2+) tumors, 4 patients with PRAME-deleted tumors (2 of 4 HLA-A2+), and 6 healthy donors. We independently tested 4 PRAME peptides  using previously published peptide sequences , MYC, CD83, CD70 (P < 0.01), PIM1, HIST1H1E, and ZFP36L1 (P < 0.05) in PRAME-positive cases. In addition, we identified that EZH2 mutations were significantly less frequent in PRAME-positive cases (P = 0.00033)  in our DLBCL cohort. Using deep, targeted amplicon sequencing of the most common gene mutations found in DLBCL (0.00033) . Importamutation .EZH2 Y641 hotspot mutations with PRAME protein expression and CN status  and EZH2-mut DLBCL cell lines  using anti-H3K27me3 antibody. ChIP and qPCR analysis revealed that the amount of H3K27me3 in the PRAME promoter region was higher in EZH2-mut cell lines than in EZH2-WT cell lines . Upon application of EPZ-6438, 5 of 7 EZH2-mut cell lines showed significant PRAME restoration (1.5–20 times PRAME mRNA increase compared with vehicle), whereas all 5 WT cell lines did not show any significant PRAME restoration  develop GC-derived DLBCLs similar to the human disease with high penetrance .The association of EZH2 with the PRAME promoter and the transcriptional repression of PRAME in netrance . GeneratY641FEzh2 Cγ1-Cre mice and observed onset of splenomegaly occurring at 3 months after transplant. Upon onset of splenomegaly, mice were randomized to treatment with 250 mg/kg EZH2i EPZ011989 or vehicle for 28 consecutive days  for an additional 3 months without further treatment. Strikingly, we observed that EZH2i resulted in sustained suppression of lymphoma throughout the 3-month treatment-free window, manifesting as reduced spleen weights and size . NotablyPRAME-CN-neutral and PRAME-deleted patient samples. We found that PRAME KO induced an increased repression of PRC2 target genes in the EZH2-mutated cell lines Karpas-422 and SU-DHL-4  cells, and we performed GSEA using the same gene sets  by proximity ligation assays (PLAs). SU-DHL-4 and DB (EZH2 mut) as well as OCI-LY10 (EZH2 WT) showed strong colocalization signals, whereas the other cell lines showed only moderate colocalization patterns compared with negative control cells  during lymphoma pathogenesis. We observed that PRAME downmodulation was mainly caused by  biology . In partPRAME-deleted patient samples. CCL22 and CCL17 were significantly downregulated cytokines in PRAME-deleted tumor samples, emphasizing the role of PRAME in TME crosstalk. Specifically, these cytokines are known to contribute to increased infiltration of T follicular helper cells and/or Tregs in lymphoma and solid cancers  compared with PRAME WT (PRAME-KO cell line model with EZH2 WT (HBL-1) indicated that PRAME can act as a negative regulator of PRC2 independent of EZH2 mutation status.Addressing cell-intrinsic regulatory pathways, we focused on one mark , 42. Givone mark , we testPRAME WT . EZH2 inOur study outlines potentially new therapeutic choices for patients with intractable DLBCL. Recently, generation of cytotoxic T cells targeting PRAME antigens for the treatment of acute myeloid leukemia, chronic myeloid leukemia, and medulloblastoma has been reported , 24, 32.In summary, we have revealed dual functions of PRAME in DLBCL, providing therapeutic rationales for treatment of high-risk patient populations in DLBCL, other lymphomas, and cancers in general that rely on immune escape phenotypes.PRAME CN status was available from a published study , PFS (progression/relapse or death from any cause), DSS (death from lymphoma or acute treatment toxicity), and overall survival (death from any cause), with log-rank tests performed to compare survival curves. In this study, we mainly used TTP to reflect the influence of genetic features on tumor progression by removing the death event not caused by lymphoma. Univariate and multivariate Cox proportional hazard regression models were used to evaluate proposed prognostic factors.HLA typing was performed for cell lines using OptiType  and those with WT PRAME (n = 268), as well as EZH2 mutation (n = 46) or WT (n = 301). Significantly differentially expressed genes from this comparison were selected , and enriched pathways in these genes were identified using the Database for Annotation, Visualization and Integrated Discovery functional annotation tool . In addition, GSEA Preranked (v4.0.2) was used to assess enrichment of specific pathways using the log2 fold change between PRAME-deleted and WT cases for gene ordering.Analysis of the data was performed in R (v3.4.4). Raw counts for the 311 cases were read using the DESeq2 package (v1.18.1), and only genes with transcripts per million reads 1 or more in at least 5% of the cases were retained 15,486 genes). Differential expression analysis was performed between cases harboring a PRAME deletion , PRAME heterozygous-deleted clones , and PRAME homozygous-deleted clones . For the HBL-1 cell line, RNA-Seq was performed for 2 conditions: PRAME WT clones , and PRAME homozygous-deleted clones . RNA-Seq was performed on an Illumina HiSeq 2500 (paired-end 75 bp reads). Reads were aligned to the GRCh37 reference using the STAR aligner (v2.5.3a_modified), which also generated per-gene count data with HTSeq-Count. Raw counts were read into R (v4.1.0), and only genes with a total count of 10 or more across all samples were retained . Differential expression was performed between all PRAME WT clones  and all PRAME-deleted clones (heterozygous and homozygous) with DESeq2 (v1.18.1). GSEA Preranked (v4.1.0) was used to examine specific pathways between the 2 types with the P value as the ranking feature. RNA-Seq data were deposited in NCBI’s Gene Expression Omnibus (GSE190403).PRAME heterozygous and homozygous deletions were introduced into 3 DLBCL cell lines, SU-DHL-4, Karpas-422, and HBL-1. For the SU-DHL-4 and Karpas-422 cell lines, RNA-Seq was performed for 3 conditions: PRAME WT clones  databases and in-house whole-genome or whole-exome sequencing data.Human DLBCL cell lines Karpas-422, SU-DHL-4, WSU-DLCL2, SU-DHL-10, SU-DHL-6, DB, DOHH-2, HT, and SU-DHL-8 were purchased from DSMZ. The TOLEDO cell line was purchased from ATCC. OCI-LY1 and OCI-LY3 were provided by the Louis M. Staudt lab ; HBL-1 and OCI-LY10 were provided by the Michael Gold lab . Karpas-422, SU-DHL-4, SU-DHL-10, SU-DHL-8, SU-DHL-6, and DB were cultured in RPMI 1640 (Thermo Fisher Scientific) supplemented with 20% FBS (Thermo Fisher Scientific). WSU-DLCL2, DOHH-2, OCI-LY1, HT, HBL-1, and TOLEDO were cultured in RPMI 1640 supplemented with 10% FBS. OCI-LY1 and OCI-LY10 were cultured in IMDM supplemented with 10% and 20% FBS, respectively. All cell lines were confirmed to be negative for mycoplasma using Venor GeM Mycoplasma Detection Kit, PCR-based . All cell lines were authenticated by STR profiling  as described (Human PRAME-KO cells (PRAME heterozygous or homozygous KO) were generated using the CRISPR/Cas9 (CRISPR-associated) system. Target sequences were found in exon 4 of human PRAME, transcript variant 202 (NCBI: NM_206956.3), using the CRISPOR design tool . To identify clones carrying mutations within PRAME, genomic DNA was prepared from expanded individual clones with Quick DNA mini-prep kit (Zymo Research), and the CRISPR target site was amplified for Sanger sequencing using primers . Colonies carrying monoallelic and/or biallelic changes resulting in frameshifting indels were further verified by cloning amplified CRISPR target sites into TOPO TA vectors (Invitrogen) followed by Sanger sequencing of each allele.WSU-DLCL2 cells were transduced by lenti-GFP-shEZH2 (OriGene TL304713), and GFP-positive cells were enriched using a FACSAria III cell sorter (BD Biosciences). Western blotting was performed using EZH2 , PRAME , H3K27me3 , and GAPDH  antibodies.+ cells were negatively selected using the naive CD4+ T cell isolation kit II by MACS according to the manufacturer’s instructions. CD4+ cells were activated using anti-CD3  and anti-CD28  mAb–precoated 96-well plates. Next, 2 × 105 T cells and 4 × 103 DLBCL cells (SU-DHL-4 or Karpas-422) were cocultured at a 50:1 ratio in AIM-V medium (Invitrogen) supplemented with HLA-DR/DP/DQ mAb  for MHC II blockade for 3 days. T cell proliferation was assessed using CellTrace violet dye (Invitrogen). Prior to coculturing, CD4+ cells (1 × 107 cells/mL) were stained with 5 μM CellTrace violet dye and incubated at 37°C for 15 minutes with gentle vortexing every 5 minutes. Flow cytometry was performed on the FACSSymphony flow cytometer (BD Biosciences) using the following Abs/stains: LIVE/DEAD Yellow Fixable dye 1:1000 (Invitrogen), CD4 BV786 clone SK3 1:200 (BD Biosciences), CD8 APC-H7 clone SK1 1:200 (BD Biosciences), and CD69 FITC clone FN50 1:50 (BioLegend).PBMCs from healthy human donors were obtained from Miltenyi Biotec, and naive CD4+ T cells as described . At least 3 replicates of T cell/well were counted and PRAME-peptide/nonpeptide number ratio was calculated.Monocyte-derived DCs were pulsed with individual PRAME peptides  and cultured with autologous PBMCs to active peptide-specific CD8escribed . PBMCs dDLBCL cell lines were grown in 24-well plates, and cell viability was determined using the trypan blue automatic method . DLBCL cell lines were exposed at 6 concentrations  of tazemetostat (EPZ-6438) for 4 days and analyzed for cell viability as before. After 4 days of treatment, cells were harvested and cell lysates were prepared using RIPA buffer (Thermo Fisher Scientific), and total proteins were quantified using a BCA protein assay kit (Thermo Fisher Scientific). NuPAGE Novex Bis-Tris gradient gel (4%–12%) (Invitrogen) was used for protein loading (25 μg each sample) and transferred to a 0.45 μm nitrocellulose membrane (Bio-Rad) by semidry transfer. Blots were stained with anti-PRAME , anti-H3K27me3 , and anti-EZH2  following the manufacturer’s instructions. GAPDH  was included as an internal loading control. HRP-conjugated anti-rabbit or anti-mouse IgG (Promega) was used to visualize labeled proteins. ECL system (GE Life Sciences) on a Chemidoc digital imager (Bio-Rad) was used for protein detection. For the Pr20 mAb binding assay, DLBCL cell lines were incubated with Pr20 mAb or its isotype control human IgG1 mAb (3 μg/mL) for 30 minutes on ice and washed in PBS. Analysis was done by flow cytometry on a Beckman Dickinson Fortessa and analyzed with FlowJo 9.8.1 and FlowJo 10 software.PRAME promoter region were designed according to the ENCODE transcription binding site as previously described  in DLBCL cells  according to the manufacturer’s protocol. Primers for the escribed . Real-tiPLA was performed as previously described , 53. Bri2, 0.5% NP-40) and nuclei were collected on ice. CHAPS buffer  was used for nuclear extraction, and the lysate was incubated with 2 μg of PRAME  antibodies overnight at 4°C on a rocker. Protein A/G beads (Thermo Fisher Scientific) were added (20 μL), and samples were rotated for 1 hour at 4°C. Beads were collected by centrifugation and extensively washed with CHAPS lysis buffer. Proteins were eluted into SDS gel loading buffer by heating at 65°C. Proteins were separated using NuPAGE 4% to 12% gels (Thermo Fisher Scientific), transferred to PVDF membranes, and stained as described in EZH2i treatment assay, Western blotting, and Pr20 binding assay.DLBCL cells  were lysed using lysis buffer fl/+ mice were generated as described in Béguelin et al. (Ezh2(Y641F)fl/+ Cγ1-Cre mice to engineer BCL6 constitutive expression and mutant EZH2 activity in GCB cells. The bone marrow of these mice was next transplanted into lethally irradiated recipient C57BL/6J mice from The Jackson Laboratory (1 × 106 cells per recipient). Animals were immunized with sheep red blood cells every 3 weeks to ensure continuous formation of GCs and were observed for spleen growth. Three months after the bone marrow transplant, mice were treated with vehicle (0.5% sodium carboxymethyl cellulose + 0.1% Tween 80) or EZH2i EPZ011989 at 250 mg/kg (bid) by oral gavage administration for 28 consecutive days. Three mice per group were euthanized at 3 time points: right after treatment and 3 months and 4 months after treatment withdrawal. Spleen, submandibular lymph nodes, lung, liver, and kidney were collected and processed for formalin fixing and paraffin embedding.n et al. . IμBcl6 n et al. . We crost test. A P value less than 0.05 was considered statistically significant in all experiments.Statistical analyses were performed using 1-way ANOVA with Bonferroni’s correction for multiple comparisons or unpaired 2-tailed Human studies were reviewed and approved by the University of British Columbia British Columbia Cancer Research Ethics Board (H14-02304: Biomarkers in lymphoid cancer and H17-01829: PRAME deletions in DLBCL), in accordance with the Declaration of Helsinki. We obtained written informed consent from the patients, or informed consent was waived for the samples used in this retrospective study. All animal experiments were approved by the Research Animal Resource Center of the Weill Cornell Medical College (IACUC 2011-031: Molecular and epigenetic mechanisms of lymphoma genesis).Ezh2(Y641F)fl/+ Cγ1-Cre mice model treated with EZH2i and contributed to writing the manuscript. SPS, PAH, RDG, JMC, and DWS provided guidance and novel tools of the project and contributed to writing the manuscript. CS supervised and designed the study and wrote the manuscript.KT designed and performed experiments, analyzed the results, and wrote the manuscript. LCC, DE, AT, SH, and EV performed experiments, analyzed the results, and wrote the manuscript. SBN, TA, ET, MYL, SSH, MB, BW, AT, AWZ, AB, DK, GD, MK, SR, and SS performed experiments and analyzed the results. TD, SSM, CMB, and DAS performed the Pr20 binding assay. JSN, GDA, and BHN supervised and analyzed ELISPOT assays. AMM and WB generated the


Educating parents about the newborn screening (NBS) process is critical in ensuring that families are aware of their child’s NBS, which could contribute to better outcomes for the baby and experiences for the family. Successful education efforts result in expecting parents understanding the importance of NBS, feeling comfortable with the NBS process, and being aware of their choices after NBS is complete. Educating parents prenatally is challenging for many NBS programs for a variety of reasons. The COVID-19 pandemic added additional barriers to NBS programs’ ability to educate parents prenatally about NBS. By initiating a department-wide partnership among other programs with a similar target audience, Michigan’s NBS Program was able to host a virtual baby fair. Since the inaugural event, Michigan’s NBS Program has hosted seven virtual fairs with 15 participating programs. A total of 692 participants registered for the baby fair and received a resource packet, over 157 participants joined one of the live presentations, and 211 have viewed the YouTube videos of recorded fairs. Virtual baby fairs are a cost-effective and convenient approach to education that could be implemented in any NBS program to educate parents prenatally about NBS.  Newborn screening (NBS) is an important public health program. Michigan’s public health code requires all newborns to be screened for rare but serious disorders (MCL 333.5431). The Michigan NBS panel currently includes 58 disorders. Infants with these disorders benefit greatly from early treatment, as they appear healthy at birth but can become ill in a very short time. While screening is mandated for all newborns in Michigan, educating parents about the NBS process is critical to ensure that families are aware of their child’s NBS, which could contribute to better outcomes for the baby and experiences for the family. Successful education efforts result in expecting parents understanding the importance of NBS, feeling comfortable with the NBS process, and being aware of their choices after NBS is complete. International studies have found that parents generally have limited awareness of NBS ,2. ParenSince awareness of NBS improves parental experiences, inadequate education about NBS could compound inequities faced by families of medically underserved areas (MUA). A survey conducted by Expecting Health and RTI International showed that families living within MUAs are less likely to receive NBS education prenatally and are less aware of NBS when compared to families living outside of MUAs .The success of any NBS parental education initiative depends on several factors, including knowing when to start providing education about NBS to parents. Michigan’s NBS Program conducted a phone-based survey of parents whose child was diagnosed through NBS, and several parents commented they would have preferred to receive more information about NBS before their child’s birth . AdditioObstetrician-gynecologists and other obstetric care providers should make resources about newborn screening available to patients during pregnancy. Information can be disseminated through informational brochures and electronic sources and through review or discussion at some time during prenatal care. Integrating education about newborn screening into prenatal care allows parents to be prepared for having their child undergo screening as well as for receiving newborn screening test results.”  [“The prenatal timeframe may be the most optimal time for NBS education because it is a time when parents are most eager to learn how they can help their baby, and they have the time to absorb the information ,8.In addition to general NBS education, Michigan educates parents about the Michigan BioTrust for Health program, which oversees the storage and use of blood spots that remain after the NBS process is complete. While all blood spots go into storage after screening, parents can choose whether they want their baby’s residual dried blood spots to be available for use for de-identified medical research benefitting public health. Families are asked to document their consent decision about the research use of blood spots around the time that blood spots are collected for NBS. Like NBS education, a study on parental self-reported experiences of BioTrust education also found that parents preferred to receive BioTrust information prenatally . ParentsThe importance of educating parents prenatally about NBS is a well-established and supported concept among the NBS community . HoweverBecause not all pregnant persons receive the full standard of prenatal care, NBS and BioTrust staff need to take a multi-pronged approach to prenatal education and cannot rely on prenatal care providers alone. All hospitals and birthing attendants in Michigan are instructed by the MDHHS to provide parents with NBS and BioTrust education while in their care, including brochures, infographics, and/or a video with information about the programs. Additionally, Michigan maintains program websites with educational resources for parents including information about newborn screening, a timeline of when disorders were added to the Michigan NBS panel, disorder fact sheets, and information about DBS storage and uses through the BioTrust. In addition to the information families should receive in the hospital, NBS and BioTrust staff also attend in-person events. From 2016 to 2020, NBS staff attended 16 baby fairs in 8 of Michigan’s 83 counties to ensure that the most up to date and comprehensive educational materials were being distributed to families a. These The COVID-19 pandemic added more barriers to NBS programs’ ability to educate parents prenatally about NBS. As restrictions on in-person gatherings in Michigan were put in place, traditional ways of educating parents, including in-person baby fairs, birth education classes, and hospital tours, were cancelled. The NBS program needed to creatively think of a way to safely educate parents, while following guidelines in place due to the pandemic. In partnership with other areas of the Michigan Department of Health and Human Services (MDHHS), the Michigan NBS Program hosted a virtual baby fair. The virtual baby fair was an opportunity for MDHHS programs, including NBS and the BioTrust for Health, to share important resources and information to new and expecting parents from the comfort and safety of their home. By partnering with other programs, the Michigan NBS Program was able to promote the event to a larger audience and potentially attract more participants who might not otherwise seek out education about NBS. The virtual baby fair allowed the Michigan NBS Program to provide education to new parents across the state from subject matter experts, to ensure optimal consistency and currency in messaging. Staff from the Michigan NBS Program reached out to programs within the MDHHS with a similar target audience. From there, a team of representatives from each interested program formed a planning committee. Fifteen programs from the MDHHS participated in the inaugural baby fair, including NBS, BioTrust for Health, Infant Safe Sleep, Women, Infants, & Children (WIC), Immunizations, Early Hearing Detection and Intervention, Birth Defects Education and Outreach, State Breastfeeding Promotion, Maternal Infant Health Program, Childhood Lead Poisoning Prevention Program, Vital Records, Unintentional Injury Prevention, Michigan Home Visiting Initiative, Children’s Special Healthcare Services, and Eat Safe Fish. One event coordinator was identified to lead the efforts of the planning committee. The group met twice a month using a virtual platform, with meetings beginning four months prior to the baby fairs, to discuss logistics and promotion of the event. During the first round of virtual baby fairs, two date options were offered for participants: a weeknight and a weekend morning. A “save the date” with registration information was created to provide the public with the information they would need to sign up for the free event. Free promotion of the event was accomplished through MDHHS social media pages, emails to existing listservs, faxes to prenatal care provider offices, and WIC text message notifications. Registration was completed utilizing online survey software. Each registrant was asked to select which fair they planned to attend, provide contact information, and indicate their county of residence.The planning committee created a packet of information and resources, called the “MDHHS Virtual Baby Fair Resource Packet,” to supplement the information provided at the live events. Each program created a one-page flyer with a summary of the program, what they have to offer parents, educational materials, and contact information. Programs were instructed to include a snapshot of the most important messages they wanted parents to hear about their program to match information that may have been distributed to someone walking up to an informational table at an in-person baby fair. The packet, and any additional program resources, were sent electronically or mailed to registrants, depending on their preference. Everyone who registered for the event received this packet prior to the live event.An email reminder about the event was sent one week before and the day of each live event. On the day of the MDHHS Virtual Baby Fair, participants joined the event through a video teleconferencing software program, Microsoft Teams. The live baby fair presentation was meant to mimic an interaction someone might have at a traditional in-person baby fair vendor booth. Each program had five minutes to present, followed by two minutes for participants to ask questions. In total, the presentations lasted 1 h and 15 min. Questions were asked in the chat box or emailed if they preferred to ask anonymously. The live event was recorded and made into a YouTube video, a link to this video is listed in the After the event, each registrant received an email with the post-event survey and the YouTube link of the recorded event. The post-event survey included questions on how the registrant heard about the baby fair, the ease of using the video teleconferencing software platform, the usefulness of the information presented at the live event and provided in the resource packet, the length of the baby fair, and if other topics should be covered. Feedback on these post-event surveys was used to gain insight on how future events might be improved. In total, 378 people registered for the baby fair and received the Baby Fair Resource Packet. Of those who registered, 64 registrants (17%) requested a hard copy resource packet via mail. Approximately 70 participants joined one of the two live presentations .Since the first round of fairs, MDHHS has hosted five additional virtual baby fairs. These events were expanded to include additional MDHHS programs and were lengthened to 2 h. The format of the live event, the resource packet, and the promotion of the fairs did not change. To date, 692 new and expecting parents registered for one of the baby fairs and received the Baby Fair Resource Packet. Approximately 15% of all registrants requested a hard copy resource packet. Approximately 157 participants joined one of the live presentations, and 211 have viewed the YouTube videos of recorded fairs.As part of the resource packet, the NBS program flyer included a description of what NBS is, how many babies are found to have a disorder detected by NBS, a reminder that the parent does not need to request that NBS be performed, what they can expect from the newborn screen, and stressed the importance of identifying a primary care provider for their baby before delivery. The NBS program also included a QR code which linked to a NBS informational video for those who prefer information to be read to them. This video is an animated road map of the newborn screening process, explaining the three parts to the newborn screen, testing of the blood spot at the state laboratory, and that parents should receive the results of their baby’s NBS from their baby’s primary care provider. A flyer was also included for the Michigan BioTrust for Health program to provide families with information about how blood spots are used after screening is conducted and the options they have regarding usage of those spots. The goal of providing this information about the BioTrust to families was to prepare them for the consent decision they will be asked to make shortly after screening takes place at the hospital. Contact information for each program was listed, and links to the NBS and BioTrust websites were provided so participants could access additional resources. The NBS and BioTrust programs’ live presentations included an overview of what NBS is, how many babies are diagnosed with a NBS condition in Michigan, an overview of the three components of NBS, with pictures showing a newborn having the hearing screen and pulse oximetry screens conducted and the blood spot screen collected. Additional information included what parents can expect at each step of the NBS process , parents’ choices regarding the research use of leftover blood spots, and an overview of program brochures and infographics. Those educational materials were distributed to each registrant in the resource packet so that they could reference them after the baby fair.The virtual baby fairs have attracted registrants from 44 of Michigan’s 83 counties b. Of thoA post-event survey was sent to all registrants. Of the 692 registrants, 40 completed the survey (6%). All 40 respondents found the baby fair to be helpful to them as new or expecting parents, with 40% finding it somewhat helpful and 60% finding it very helpful. Among all respondents, 88% reported it was easy to connect to the live baby fair presentation using a video conferencing platform. For baby fairs with a length of 1 h and 15 min, 64% of respondents felt that the time was perfect, while 36% would attend a longer presentation where more information was presented. For baby fairs with a length of 2 h, 100% of respondents thought the length of time was perfect. No respondent reported that the length of the baby fair was too long. Most respondents, 91%, found the baby fair resource packet helpful, while 9% said it was not.As a result of the COVID-19 pandemic, the Michigan NBS Program developed a virtual education initiative that could be useful for all NBS programs as a tool to educate parents prenatally about NBS despite limited resources. The virtual baby fair allowed the Michigan NBS Program to distribute important information about NBS to parents and provided parents access to a plethora of health and safety information and a platform to ask questions. By providing a written resource, a live presentation, and a recording of the presentation on YouTube, parents had the opportunity to learn at their own pace using their preferred platform.A benefit to the virtual format was that expecting parents from a wide geographic range attended. More counties were represented than Michigan’s NBS Program had been able to reach with previous in-person baby fairs. From 2016–2020, Michigan NBS staff attended 16 in-person baby fairs in 8 counties a. The viThe baby fair was planned, organized, and conducted with little financial support. The NBS program covered the cost of postage for mailing the requested printed resources (under $100). In-person baby fairs typically cost about $1000 on average when including staff time and vendor fees. One staff member from the NBS program staff led coordination of the event, and one additional staff member assisted with logistics. The time commitment was most significant for the coordinator of the event, with most of the work being completed in the month prior to the event. This time was able to be incorporated into the time typically allotted for in-person education events. Additional staff time required of other participating program staff was minimal, with most of the work for them involving the assembly of the program page in the resource packet, attending the live presentation, and promotion of the event. The time commitment for all staff decreased with each event as the framework created for the inaugural event was used for subsequent baby fairs. The Michigan NBS Program experienced a few limitations with the baby fairs. To date, the Virtual Baby Fair has only been presented in English, which creates barriers for people who do not speak English. For all seven fairs, a low percent of registrants (23%) attended the live presentations. Additionally, the low response rate on the post-event surveys (6%) could limit the planning committee’s ability to implement changes that would make the event experience better, as the responses only represent a small percent of total registrants and attendees. Finally, the participating programs were limited to a five-minute presentation, which is a small amount of time to fit all the information that might be useful for parents to know about their program.Despite these limitations, the planning committee learned ways to improve the event with each fair. After the first round of baby fairs, the planning committee increased the length of the event to two hours and added more programs. Feedback from post-event surveys indicated that families thought this amount of time was appropriate. The committee also identified additional areas for improvement for future fairs. For the next round of virtual fairs, the committee will test the use of an event platform for registration that sends calendar reminders and instant emails to registrants following registration to confirm their registration. This could help increase the percent of registrants that attend the live presentation. Making the baby fair and resource packet and presentation available in more languages would make the baby fair accessible to a wider audience. This could potentially be accomplished by reaching out to local translation support organizations that might be willing to volunteer their time to help make it accessible to those who could benefit from the presentation in a different language. Additionally, to increase the attendance, the planning committee could consider applying for grant funding to support advertisement of the event on a larger scale and promoting the event to more prenatal care providers to distribute to parents.A future step for the Michigan NBS Program is to assess the impact of the virtual baby fair on parent knowledge of NBS. This could be done by surveying registrants with a pre- and post-event survey. The survey could allow the Michigan NBS Program to determine whether the baby fair is an effective way to deliver information to improve parental understanding of NBS. Questions could be included on the survey to help understand what programs were most helpful to expecting parents and encouraged their attendance.The virtual baby fair allowed the NBS program to educate more parents across Michigan than attending in-person events had in the past, at almost no additional cost to the program. This is especially important for smaller programs with limited staff and budget for education. The virtual format also allowed for expecting parents from a wide geographic to range to attend. The NBS program could have led this initiative alone and only educated parents exclusively about NBS. By taking a team approach to the baby fair and partnering with other programs, the event was promoted to a larger audience and potentially attracted more parents that might not otherwise seek out NBS education. Moving forward, the Michigan NBS Program plans to host virtual baby fairs twice a year to give every new and expecting parent the chance to receive information they need to prepare for their baby’s newborn screen.


Cxcl12, Dll4, and Psmb11 enables the cortex to support T lineage commitment and the generation and selection of CD4+CD8+ thymocytes. Although the importance of cTECs in T cell development is well defined, mechanisms that shape the cTEC compartment and regulate its functional specialization are unclear. Using a Cxcl12DsRed reporter mouse model, we show that changes in Cxcl12 expression reveal a developmentally regulated program of cTEC heterogeneity. Although cTECs are uniformly Cxcl12DsRed+ during neonatal stages, progression through postnatal life triggers the appearance of Cxcl12DsRed− cTECs that continue to reside in the cortex alongside their Cxcl12DsRed+ counterparts. This appearance of Cxcl12DsRed− cTECs is controlled by maturation of CD4−CD8−, but not CD4+CD8+, thymocytes, demonstrating that stage-specific thymocyte cross-talk controls cTEC heterogeneity. Importantly, although fate-mapping experiments show both Cxcl12DsRed+ and Cxcl12DsRed− cTECs share a common Foxn1+ cell origin, RNA sequencing analysis shows Cxcl12DsRed− cTECs no longer express Foxn1, which results in loss of the FOXN1-dependent cTEC gene signature and may explain the reduced capacity of Cxcl12DsRed− cTECs for thymocyte interactions. In summary, our study shows that shaping of the cTEC compartment during the life course occurs via stage-specific thymocyte cross-talk, which drives loss of Foxn1 expression and its key target genes, which may then determine the functional competence of the thymic cortex.In the thymus, cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells support αβT cell development from lymphoid progenitors. For cTECs, expression of a specialized gene signature that includes   Foxn1.cTECs undergo progressive changes during postnatal life caused by loss of Foxn1− cTECs remain within the cortex but lack cTEC signature genes.Foxn1− cTECs show an impaired ability to influence developing thymocytes. Importantly, many studies have shown the key roles that thymic stromal cells play in controlling thymocyte development (+UEA1+Ly51− medullary TECs (mTECs) are key in controlling T cell tolerance induction through the induction of both negative selection and Foxp3+ T cell development -expressing cTECs, which induce Notch signaling and direct progenitors toward a T cell fate (−CD8− double-negative (DN) stages, including CD44+CD25− DN1, CD44+CD25+ DN2, and CD44−CD25+ DN3, where they rearrange the Tcrb gene and express TCRβ protein as part of the cell-surface pre-TCR complex. Importantly, selection of TCRβ-expressing DN3 cells is also controlled by cTEC products, with CXCL12 and DLL4 acting in concert with the pre-TCR to generate large cohorts of preselection CD4+CD8+αβTCRlow thymocytes (+CD8+ thymocytes that results in the generation of single-positive (SP) CD4+ and CD8+ thymocytes. In this study, the unique ability of cTECs to support positive selection is at least in part attributed to their specialized Ag-processing capabilities –bound self-peptides that result in the effective positive selection of CD8+ thymocytes  analysis confirming their differential Cxcl12 gene expression. Interestingly, examination of cTEC heterogeneity across the life course revealed a developmentally regulated program where cTECs were uniformly Cxcl12DsRed+ at neonatal stages, with Cxcl12DsRed− cTECs appearing 1 wk after birth and persisting into adulthood. Importantly, whereas fate-mapping experiments show Cxcl12DsRed+ and Cxcl12DsRed− cTECs both derive from FOXN1+ cells, RNA sequencing (RNA-seq) analysis showed these populations to be transcriptionally distinct. Unlike Cxcl12DsRed+ cTECs, Cxcl12DsRed− cTECs lacked Foxn1 expression, and this was accompanied by a change in the gene expression profiles of FOXN1 targets, including Cxcl12 itself, as well as Psmbl11, and the Notch ligand Dll4. Furthermore, the emergence of Cxcl12DsRed− cTECs was impaired in Rag2−/−, but not Tcra−/−, mice, and Cxcl12DsRed− cTECs were impaired in their ability to form successful cellular interactions with thymocytes when compared with their Cxcl12DsRed+ counterparts. Taken together, our study identifies a developmentally regulated program of cTEC heterogeneity, where signals arising from the maturation of immature DN3 thymocytes cause transcriptional changes in the cTEC population that result in loss of Foxn1 expression and transcripts of its downstream targets. This then creates epithelial heterogeneity in the thymic cortex that may influence functionality within the cTEC compartment.Despite this importance of cTECs for thymus function, our understanding of the mechanisms that control their development remains incomplete. To address cTEC development and heterogeneity, we examined Ly51e Cxcl12 . We founCxcl12DsRed knockin (stock no. 022458) (Tcrα−/− (stock no. 002116 (Rag2−/−(stock no. 008449) (Foxn1Cre (stock no. 018448) (Tcra−/− and Rag2−/− mice were heterozygous littermate controls. RANKVenus BAC transgenic mice were generated as described previously (The following mice on a C57BL/6 background were purchased from The Jackson Laboratory and used at 10 wk of age unless otherwise stated:  022458) , which w 008449) , Foxn1Cr 018448) , and Ros 018448) . Controleviously . Husband− cells were enriched by the depletion of CD45+ cells using anti-CD45 beads and LS columns (Miltenyi Biotec). The following Abs were used for TEC analysis: anti-CD45 clone 30-F11 (eBioscience), anti-EpCAM1 clone G8.8 (eBioscience), anti-Ly51 clone 6C3 (BioLegend), anti–MHC II clone M5/114.15.2 (eBioscience), anti-CD80 clone 16-10A1 (BioLegend), CD104 clone 346-11A (BioLegend), and anti–MHC I 28-14-8. Biotinylated UEA-1 (Vector laboratories) was detected using streptavidin PECy7 (eBioscience). Cells were analyzed using a LSR Fortessa (Becton Dickinson) with data analysis carried out using FlowJo v10 (Becton Dickinson). For cell sorting, TEC subsets were identified using the earlier Abs and isolated using a FACSAria Fusion 1 cell sorter (Becton Dickinson). The sorting strategy for the different TEC subsets was as follows: Cxcl12DsRed+ cTEC, CD45−EpCAM1+UEA1−Ly51+CXCL12DsRed+; CXCL12DsRed− cTEC, CD45−EpCAM1+UEA1−Ly51+CXCL12DsRed−; mTEClo, CD45−EpCAM1+UEA1+Ly51−CD80−MHC II−; mTEChi, CD45−EpCAM1+UEA+Ly51−CD80+MHC II+; CD104+ mTEClo, CD45−EpCAM1+UEA1+Ly51−CD80−MHC II−CD104+; and CD104− mTEClo, CD45−EpCAM1+UEA1+Ly51−CD80−MHC II−CD104−.For TEC analysis, single-cell suspensions were generated by digesting thymic lobes with collagenase Dispase  and DNase 1 . CD45Foxn1Cre/Rosa26YFP/Cxcl12 mice was isolated and fixed in 2% paraformaldehyde  for 2 h, then overnight in 15% sucrose (Sigma). Thymic lobes were frozen on dry ice and sectioned at 7 μm within 24 h of freezing. eYFP protein in sections from Foxn1Cre/Rosa26YFP/Cxcl12DsRed was amplified using rabbit anti-GFP (ThermoFisher) and donkey anti-rabbit 488 (ThermoFisher). Sections were counterstained with DAPI (Sigma) and mounted using Prolong Diamond (ThermoFisher). Sections were imaged using Zeiss Zen 880 microscope (Zeiss) and analysis using Zeiss Zen Black (Zeiss).Thymus tissue from Actb (β-actin) (Qiagen) and indicated genes of interest (Sigma-Merck). Data shown are typical of at least two independently sorted sample sets; histograms represent the mean (± SEM) of replicate reactions. Primer sequences used were: Foxn1, forward 5′-CAAATTCTGCAGGGGTCAGA-3′ and reverse 5′-TGGGGTGCAATCCTCTGATA-3′; Cxcl12, forward 5′-GCTCTGCATCAGTGACGGTA-3′ and reverse 5′-TGTCTGTTGTTGTTCTTCAGC-3′; Psmb11, forward 5′-ATCGCTGCGGCTGATACTC-3′ and reverse 5′-GCAGGACATCATAGCTGCCAA-3′; Prss16, forward 5′-GTATTTCTGCACATAGGAGGCG-3′ and reverse 5′-TGTTCTAGGCTTATCACCAGGG-3′; Cd83, forward 5′-AGGGCCTATTCCCTGACGAT-3′ and reverse 5′-CTTCCTTGGGGCATCCTGTC-3′; Dll4, forward 5′-GAAGCGCGATGACCACTTCG-3′ and reverse 5′-TGGACGGCAGATGCACTCAT-3′; Ly75, forward 5′-GCTCAGGTAATGATCCATTCACC-3′ and reverse 5′-TTAGTTCCGCTACAGTCCTGG-3′; Ctsl, forward 5′-ATCAAACCTTTAGTGCAGAGTGG-3′ and reverse 5′-CTGTATTCCCCGTTGTGTAGC-3′; Epcam1, forward 5′-TTGCTCCAAACTGGCGTCTAA-3′ and reverse 5′-GCAGTCGGGGTCGTACA-3′; Aire, forward 5′-TGCATAGCATCCTGGACGGCTTCC-3′ and reverse 5′-CCTGGGCTGGAGACGCTCTTTGAG-3′; Trpm5, forward 5′-CCAGCATAAGCGACAACATCT-3′ and reverse 5′-GAGCATACAGTAGTTGGCCTG-3′; Ccl21a, forward 5′-ATCCCGGCAATCCTGTTCTC-3′ and reverse 5′-GGGGCTTTGTTTCCCTGGG-3′; and Actb (β-actin), QuantiTect Mm Actb 1SG Primer Assay .Real-time PCR was performed as described previously  on a CorFoxn1 high-confidence genes . Gene ontology analysis was performed using clusterProfiler (RNA samples were extracted using the Qiagen RNeasy kit. Libraries were prepared using the SMARTer Ultra Low Input RNA Kit for Sequencing as per the manufacturer’s instructions and sequenced on an Illumina NovaSeq platform. Reads were trimmed for adapter contamination using Trimmomatic (version 0.36) and aligned to the mm10 mouse genome using STAR (version 2.7.3a) , 31. Reace genes  was asseProfiler .−EpCAM1+ TECs were FACS sorted from 10-wk-old Cxcl12DsRed mice and neonatal day 2 wild-type (WT) mice and labeled with CFSE according to the manufacturer’s instructions (ThermoFisher). A single-cell suspension of WT adult thymocytes was labeled with CellTrace Violet according to the manufacturer’s instructions (ThermoFisher), and the two cell types were mixed at a 5:1 ratio (thymocytes:TEC). The mixed suspension was then centrifuged, the supernatant removed, and the cell pellet vortexed and incubated at 37°C for 20 min, a time point that enables successful conjugate formation between WT TECs and thymocytes  mice were uniformly 12DsRed+ . Althoug of life , with thn period , 1D. ColCxcl12DsRed+ and Cxcl12DsRed− cTECs. In this study, Cxcl12DsRed+ and Cxcl12DsRed− subsets of total CD45−EpCAM1+UEA1−Ly51+ cTECs were FACS sorted from 10-wk-old adult Cxcl12DsRed reporter mice, with experiments performed in triplicate to produce three independent biological replicates for each subset. This approach identified 946 genes differentially expressed between DsRed+ and DsRed− cTECs , Notch signaling (Dll4), and Ag processing/presentation . By contrast, Cxcl12DsRed+ and Cxcl12DsRed− cTEC subsets showed no reduction in levels of Epcam1 mRNA . Fo. FoCxcl1d− cTECs , as wellam1 mRNA . Importaker Ly51 . qPCR an subsets . Moreoveof mTECs . These fd− cTECs .Cxcl12DsRed− cTECs in relation to their Cxcl12DsRed+ counterparts, we searched for genes that were differentially expressed between the two subsets  that are important in the regulation of cortical T cell development. Despite this change in the cTEC-specific mRNA signature, Cxcl12DsRed− cTECs continue to reside within cortical areas alongside their Cxcl12DsRed+ counterparts, where they contribute to the reticular epithelial network of the adult thymic cortex.The presence of e mapped , 5B, index areas . Use of + mTECs . In this article, it is important to note that β5t-deficient mice are still able to positively select some SP8+ thymocytes (Psmb11 can to contribute to SP8 generation in normal mice. Alternatively, adult Foxn1− cTECs that lack Psmb11 may be incapable of positive selection because of other functional defects, such as a failure to interact with CD4+CD8+ thymocytes. Although it is interesting to note that Cxcl12DsRed− cTECs express significantly lower levels of MHC I relative to their Cxcl12DsRed+ counterparts, and form fewer cell–cell conjugates with thymocytes, further studies are required to examine the functional properties of the cTEC subsets described in this article. Relevant to this, our attempts to compare the functional abilities of FACS-sorted Cxcl12DsRed+ and Cxcl12DsRed− cTECs from adult mice in reaggregate thymus organ cultures were unsuccessful. In this study, intact three-dimensional structures consistently failed to form when using TECs isolated from adult mice, which is in contrast with the efficient generation of intact reaggregate thymus organ culture from embryonic TECs (The presence within the adult thymic cortex of cTECs that no longer express key genes regulating specific stages of thymocyte development raises multiple interesting scenarios. For example, it may be relevant to understanding progressive changes in thymus function under homeostatic conditions. In this study, because both eckpoint , absenceymocytes , a findinic TECs , 49. Thenic TECs , which mCxcl12DsRed− cTECs may also play a role in physically supporting the epithelial scaffold within the thymus cortex, a possibility raised recently in the context of the presence of FOXN1− TECs in thymus (Cxcl12DsRed− cTECs are interspersed in the cortex alongside Cxcl12DsRed+ cells. A final possibility is that alongside loss of Foxn1-mediated functional properties, Cxcl12DsRed− cTECs acquire new functional features that are important in adult thymus cortex organization and/or function. Again, further examination requires approaches to directly assess the functional properties of defined cTEC subsets.Beyond directly influencing specific stages of thymocyte development, n thymus . Such a +UEA1− cTEC compartment undergoes developmentally regulated changes in its cellular makeup that are driven by interactions with the maturation of immature CD4−CD8− thymocytes. We identify the emergence of a cTEC subset that retains its Ly51+UEA1− phenotype and positioning within the cortex but has ceased to express FOXN1, resulting in the lack of expression of key FOXN1 target genes that define the functional properties of cTECs. These findings demonstrate the emerging complexity of the thymic cortex and will aid in future studies that examine the role of this intrathymic site in thymocyte development.In summary, we show that the Ly51Data Supplement


Structured curricula are demanded to improve training programs of future urologists. This study aimed to evaluate the acceptance of the newly implemented residency rotation program at the University Hospital Frankfurt. Primary endpoint was resident's satisfaction with the current residency rotation program. Secondary endpoint was the fulfilment of the objectives and expectations by residents.A standardized 15-item, online-based survey was sent to every urologic resident of the University Hospital Frankfurt, completing their rotation between August 2020 and August 2022. In addition to baseline characteristics, training and working conditions were assessed. Descriptive statistics were applied.In total 15 rotations of the Residency Rotation Program at the University Hospital Frankfurt were evaluated, including urologic practice (5/15), Intermediate Care Unit (4/15), urooncology (4/15) and clinical exchange to St. Gallen (2/15). Overall, the majority were very (67%) or rather satisfied (2%) with their rotation. Of the pre-rotation defined objectives, 71% were fulfilled, 18% partially fulfilled and 8% not fulfilled. With respect to the expectations, 67% were fulfilled, 19% partly fulfilled and 4% were not fulfilled. All residents would recommend their respective rotations.Our results demonstrate that the residency rotation program at the University Hospital Frankfurt enjoys a high level of acceptance as well as a positive impact on urologic training. Satisfaction with the completed rotation was convincing, most of the expectations and objectives for the respective rotation could be fulfilled. These results help to ensure the quality of urologic curricula and to improve the structure of training programs for future urologists. Residency is probably the most formative time of a physician's career. Results of the current German national urology postgraduate training survey showed: more than 50% of respondents were dissatisfied with their training facility, although about 80% were satisfied with their choice of urology as their specialty. Reasons given for dissatisfaction included a lack of quality in training . To imprIn response to this demand, the training curriculum in urology  was published on the initiative of the German Society of Urology (DGU), the German Society of Residents in Urology (GeSRU) and the Professional Association of German Urologists (BvDU) . MoreoveThis study aimed to evaluate the acceptance and satisfaction of residents according to the newly implemented Residency Rotation Program at the University Hospital Frankfurt. The rate of achieved pre-defined expectations and objectives for each rotation by the residents is uncertain. We hypothesized a pronounced accordance between pre-defined and achieved expectations, as well as pre-defined objectives by the residents. Therefore, a questionnaire-based survey was performed.Kantonsspital St. Gallen in Switzerland since 03/2021. An additional research fellowship was offered for residents interested in clinical research based on a well-established cooperation with the University of Montréal Health Center in Montreal, Canada , as well as a former collaboration with Harvard Medical School, Brigham and Women's Hospital in Boston, USA .Starting in 2018, the Department of Urology at the University Hospital Frankfurt was one of the first hospitals in Germany to develop and implement a comprehensive and mandatory residency rotation program with specific goals . This roMain outcome-based learning objectives for each rotation were defined based on German training regulations [via SurveyMonkey®  and was tested for usability and technical functionality prior to circulation.To evaluate the residency rotation program at the University Hospital Frankfurt, a standardized, non-validated 15-item, online-based questionnaire was designed due to the Checklist for Reporting Results of Internet E-Surveys . The quevia mail to every urologic resident of the University Hospital Frankfurt, completing their rotation  between August 2020 and August 2022. Due to the anonymity of the survey, no ethics vote is required.The link to the questionnaire was sent Descriptive statistics included frequencies and proportions. Baseline characteristics were expressed as percentages or as mean values ± standard deviation (SD).To enhance the quality and transparence of research, our report and manuscript design follows the “Defined Criteria To Report INovations in Education” (DoCTRINE) Guidelines .Kantonsspital St. Gallen in Switzerland. The baseline characteristics of the participants are shown in In total, 15 rotations of the residency rotation program at the University Hospital Frankfurt were evaluated. Overall, 33% of the participating residents completed a rotation to a urologic practice, 27% to a surgical Intermediate Care Unit (IMC) and 27% the urooncologic rotation. Furthermore, 13% took part in the clinical exchange program to Overall, the majority were very (67%) or rather satisfied (27%) with their rotation, and only one person was partly satisfied (6%). Of the participating residents, 53% were very satisfied and 47% were rather satisfied with the acquisition of theoretical skills. In comparison, 33% were very satisfied, 53% rather satisfied, 7% partly satisfied and 7% rather dissatisfied with the acquisition of practical skills. Evaluating working conditions, 40% were very satisfied, 53% rather satisfied and 7% partly satisfied.Evaluating preparedness for the rotation through medical studies and the previous training period, 20% felt very good, 27% rather good, 33% partly good, and 20% rather poor prepared. Of the participating residents, 74% were satisfied with the organization of the upcoming scheduled rotation . In retrospect, the majority (93%) found the timing of their rotation to be good, 7% partly good. Considering the induction phase of the rotation, 67% were satisfied, and 33% were partly satisfied. Finally, 93% were satisfied with the supervision by their respective attending.All participating residents had an introductory and a final discussion at the beginning or the end of their rotation. An interim discussion took place for 40% of the respondents . During Of the 38 pre-rotation defined objectives, 71% were fulfilled, 18% were partially fulfilled and 8% were not fulfilled . In retrThis study aimed to evaluate the acceptance and satisfaction of residents according to the newly implemented Residency Rotation Program at the University Hospital Frankfurt. Moreover, we hypothesized a pronounced accordance between pre-defined and achieved expectations, as well as pre-defined and achieved objectives by the residents. Therefore, we performed a questionnaire-based survey and made several important observations.Kantonsspital St. Gallen enjoyed a high popularity (both 100%). These results confirm that interdisciplinary rotations and the gain of new experience and knowledge in other clinal filed within and outside the specialty of urology are crucial for a broad formation.First, we reported a high overall satisfaction within each rotation of the newly implemented residency rotation program at the University Hospital Frankfurt (94%). Specifically, the IMC rotation and the clinical exchange rotation to the Second, we also reported a high level of at least partly fulfilled pre-defined expectations (88%), as well as pre-defined objectives 89%). These results could explain the high level of. Setting and revisiting specific goals can be essential for achieving academic and career objectives and thereby increasing job satisfaction %. These .Third, the high level of satisfaction also reflected in a high rate of recommending each rotation to colleagues (100%). Comparable results for the implantation of structured rotations could also be shown for anaesthesiology residents . FurtherFourth, 93% of the respondents were satisfied with the supervision by their respective attending. Moreover, all participants reported an introductory and a final discussion. Specifically, these discussions were crucial to evaluate the accordance of pre-defined and achieved expectations, as well as pre-defined and achieved objectives. A close working relationship with the supervising attending might contribute to a high level of job satisfaction. Mentor accessibility and frequent interaction are known as key factors for a satisfying mentor-mentee relationship which is essential for the success of postgraduate training curricula . Since wKantonsspital St. Gallen. Unfortunately educational and surgical curricula remain variable across residency programs . Residents doing their rotation to the urologic practice and the IMC were generally in their first or second year of postgraduate training and had an average of 9 months of urological experience at the beginning of each rotation. A mandatory rotation program, including 6 to 12 months at the home department and 6-month rotations to urologic practice and IMC, may help recent graduates learn the basics of urology, develop routines and deal with clinical emergencies. Additionally, predefined milestones, periodical evaluations, and regular feedback could support the learning process . In compprograms . Therefoprograms .Taken together, we reported a high level of overall satisfaction and a high rate of accordance between pre-defined and achieved expectations, as well as pre-defined and achieved objectives. Moreover, a high level of satisfaction with the assigned supervisor was reported. These results confirm the acceptance of our rotation program, as well as high quality regarding the design and realization of each rotation. They help to identify importanct topics that may be improved or even further developed in existing training programs.Even with its strengths, the current study is not devoid of limitations. First and foremost are the limitations inherent to the single institutional nature and the limited sample size. Moreover, these observations need to be validated in a larger and multi-institutional cohort. Second, despite the anonymity of the survey participants may have given better ratings due to the departmental evaluation. Third, outcomes described in the current study were based on a subjective evaluation of each participant. It is noteworthy to mention that this survey represents a pilot study based on resident-reported oucomes on local level. In a second step, a national evaluation of the WECU program is already planned. Conducting a future study, a structured competence-orientated exam could be implemented before and after each rotation for objective progress assessment. Finally, the impact of such rotation programs on learning curves of urologic residents could be tested.The residency rotation program at the University Hospital Frankfurt enjoys a high level of acceptance. Satisfaction with the completed rotation and the training and working conditions was excellent. In addition, for the majority of participating residents timing of the respective rotation was appropriate. Introductory and final discussions with the supervisor took place regularly. Most of the expectations and objectives for each rotation could be fulfilled at least in part so that a positive impact on urologic training can be assumed.Evaluations serve to ensure structure and process quality. Moreover, our findings may help develop urologic training curricula and improve existing training programs' concepts.


Popliteal artery aneurysms are the most common peripheral artery aneurysm, accounting for ≥70% of peripheral aneurysms.A). The upper longitudinal incision can be extended over the great saphenous vein, and the inferior incision can be extended medially over the small saphenous vein if these vessels have been targeted as conduits. An interposition bypass using either a prosthetic or saphenous vein graft has demonstrated similar patency rates and major adverse limb events in this position and improved patency compared with a medial bypass in some studies, although the data is somewhat lacking.,In our video, we demonstrate the treatment of a 72-year-old man with an asymptomatic 3.8-cm popliteal aneurysm using an open posterior approach and prosthetic conduit. In brief, the posterior approach was performed with the patient in the prone position via an S-shaped incision from the medial side of the thigh extending to the lateral calf with the horizontal component over the flexor crease (In conclusion, the posterior approach to popliteal artery aneurysms is a durable repair option for patients with pathology confined to the popliteal fossa. The procedure is well tolerated and complements endovascular and open medial approaches to this disease in properly selected patients.


ARID1A in endometriosis-related OC, SMARCA4, and SMARCB1 in hypercalcemic type small cell ovarian carcinoma (SCCOHT), ACTL6A, CHRAC1, RSF1 amplification in high-grade serous OC. Here we review the available literature on CRCs’ involvement in OC to improve our understanding of its development and investigate CRCs as possible biomarkers and treatment targets for OC.Ovarian cancer (OC) is the fifth leading cause of women’s death from cancers. The high mortality rate is attributed to the late presence of the disease and the lack of modern diagnostic tools, including molecular biomarkers. Moreover, OC is a highly heterogeneous disease, which contributes to early treatment failure. Thus, exploring OC molecular mechanisms could significantly enhance our understanding of the disease and provide new treatment options. Chromatin remodeling complexes (CRCs) are ATP-dependent molecular machines responsible for chromatin reorganization and involved in many DNA-related processes, including transcriptional regulation, replication, and reparation. Dysregulation of chromatin remodeling machinery may be related to cancer development and chemoresistance in OC. Some forms of OC and other gynecologic diseases have been associated with mutations in specific CRC genes. Most notably,  Ovarian cancer (OC) is the third most common gynecologic malignancy and the second deadliest oncogynecologic disease in the world . It is eOne of the culprits of OC chemoresistance and early therapy failure is the heterogeneity of the disease. Histologically, OCs are subdivided into epithelial and non-epithelial tumors. The non-epithelial OC only comprises 10% of OC cases. These are malignant germ cell tumors  that are usually present in preadolescent women, and sex cord-stromal tumors  that are more common and typically affect postmenopausal women . Due to TP53 mutations and chromosomal instability, these tumors arise from the intraepithelial carcinoma in the fallopian tube. However, other origins, such as solid pseudoendometrioid transition tumors or borderline or low-grade serous OC, are possible. All other epithelial tumors  are considered less of a concern due to their slow-growing nature. Although borderline, mucinous, and seromucinous tumor origin is unclear, the endometroid and clear cell tumors are thought to originate from endometriosis and are similar to endometrial cancers [PIK3CA, PTEN, KRAS, BRAF, CTNNB1, and ARID1A genes are frequent amongst type I epithelial OCs and are considered potential OC biomarkers [Epithelial type of OC is further subdivided into five major types according to histologic differences: the endometrioid, clear cell, mucinous, low-grade, and high-grade serous OCs . Accordi cancers . Albeit omarkers .BRCA1 and BRCA2 germline mutations [TP53 mutations due to patients with the Li-Fraumeni syndrome [Regardless of type, a third of OC cases are related to homologous recombination gene insufficiency . About 2utations ,12. The syndrome . The secInside every human cell’s nucleus fits the genome comprising three billion DNA base pairs, almost two meters in length. This is achieved by winding the DNA around histone protein complexes forming nucleosomes that are the building blocks of the chromatin nucleoprotein structure. Each nucleosome has an octamer core made of H2A, H2B, H3, and H4 histone protein pairs and fits 147 bp of DNA. The nucleosomes are connected with linker DNA of 10–60 bp in length . The disThere are four conserved families of ATP-dependent chromatin remodelers in mammals: chromodomain helicase DNA binding (CHD), imitation switch (ISWI), inositol requiring 80 (INO80), and switch/sucrose non-fermenting (SWI/SNF) . The mulTraditionally, SWI/SNF chromatin remodelers are associated with increasing chromatin accessibility, INO80 mainly acts as a chromatin repressor, and ISWI and CHD are most often described as histone chaperones in chromatin assembly . HoweverSWI/SNF was the first CRC discovered and, thus, is the most well-studied. The complex gets its SWI/SNF (mating Type SWItch/Sucrose Non-Fermentable) name from the yeast where it was first discovered . The BrgSMARCA4) or BRM (SMARCA2) catalytic subunits. The different SWI/SNF complexes are distinguished by complex-specific accessory subunits , polybromo-associated BAF (PBAF), and recently characterized non-canonical, GLTSCR1/L-associated BAFs (ncBAF or GBAF) . Each cosubunits . cBAF noSWI/SNF complexes are well known for their essential functions in many pathways related to cancerogenesis. Most notably, loss of SWI/SNF or its subunits is known to impair DNA damage repair, cell cycle regulation, critical signaling pathways, such as PI3K/AKT/mTOR, and MYC regulation (described in detail below).SMARCA1) or SNF2H (SMARCA5) ATPases that, together with regulatory domains, form up to 16 CRCs with various functions: ACF, CHARC, RSF perform nucleosome spacing, WICH is primarily involved in replication by incorporating histones into newly synthesized DNA, NoRC perform transcriptional repression, NURF and CERF are responsible for transcriptional activation [ISWI (Imitation switch) is a highly conserved family of chromatin remodelers. ISWI contains SWI2/SNF2 family ATPase domain, a part of a broader superfamily of DEAD/H-helicases, a specific HAND-SANT-SLIDE DNA, and histone-tail binding domain. The human ISWI has either SNF2L  family consists of nine (CHD1-9) proteins. The CHD family shares a highly conservative helicase-ATPase domain from the SWItch2/SNF2 superfamily of ATPases and two tandem chromatin organization modifier domains (chromodomains), that read histone modifications. In particular, CHD1 binds to the H3H4me mark and CHD5 to H3K27me3 . CHD proCHD3 and CHD4 proteins are also included in the nucleosome remodeling and histone deacetylase complex (Mi-2/NuRD) that also contains histone deacetylases (HDAC1 and HDAC2), and other proteins  that bind to methylated DNA or histone tails . GeneralCHD5, with the discovery that its locus 1p36.31 is often deleted in glioma and other tumors [The first CHD gene linked with human cancer was r tumors . Curiousr tumors . CHD5 exr tumors . CHD5 rer tumors . MoreoveInositol requiring 80 (INO80) family, named after inositol-responsive gene expression regulation , is the uring HR .Just like other CRCs, INO80 also governs transcription. INO80 can activate mRNA transcription by −1 and +1 nucleosome sliding at transcription start sites where it is recruited by RNAPII and transcription elongation complex PAF1, however, at heterochromatin sites INO80 silences transcription by removing the H2A.Z mark, preventing H3K79 methylation and RNAPII eviction and degradation at replication fork–transcription complex collision sites .ARID1A  is the most frequently altered. ARID1A mutations are particularly prevalent in gynecologic cancers (found in 10–60% of ovarian and endometrioid carcinoma cases) [ARID1A mutation rates are found in ovarian clear cell carcinoma (OCCC) and endometroid ovarian carcinoma (ARID1A’s use as a gynecologic cancer biomarker.About 25% of human cancers harbor mutations in at least one of 29 genes encoding SWI/SNF proteins . Among ta cases)  and pre-a cases) . The higarcinoma  suggestiNone of the previous research found any sufficient correlation between ARID1A protein expression loss and clinical features such as age, FIGO grade, and disease survival . In a suARID1A alterations are loss-of-function mutations such as large deletions, frameshift, or nonsense mutations that lead to the largest BAF complex subunit loss and inactivation of the SWI/SNF complex. However, ARID1A mutations are not sufficient to promote tumorigenesis alone. Tumors with ARID1A loss often harbor mutations of the PI3K/AKT pathway, such as inactivating PTEN or activating PIK3CA mutations. In mice models, separate ARID1A or PI3K/AKT pathway mutations promoted ovarian hyperplasia, while concomitant inactivation of ARID1A together with PIK3CA activating mutations induced tumors, similar to ovarian clear cell carcinoma (OCCC) [Usually, a (OCCC) .ARID1A downregulation due to inactivating mutations has many downstream effects through epigenetic mark displacement, deregulating gene expression, and reduced protein interactions, primarily affecting DNA damage repair and signaling pathways .TP53 [ARID1A and TP53 mutations are mutually exclusive in gynecologic malignancies, however, p53 is indirectly regulated by ARID1A. ARID1A is an HDAC6 deacetylase transcriptional repressor. Thus, the loss of ARID1A leads to HDAC6 reactivation, which causes p53 lysine 120 residue deacetylation and apoptosis inhibition [SWI/SNF complex regulates active H3K27ac histone mark at enhancer and promoter regions through interaction with p300/CBR histone acetyltransferase . The aceTP53 . ARID1A hibition .ARID1A-deficient cells, topoisomerase IIARID1A silencing increases telomerase activity, adding to the survival of cancer cells [ARID1A’s deficiency has also been linked with telomere impairment. In cription . Moreovecription . Howevercription . ARID1A er cells .ARID1A’s mutational status in OC cells is also associated with reactive oxygen species (ROS) formation. ARID1A knockdown in OC cell lines increased intracellular ROS levels, as well as increased oxidative stress marker 8-hydrixyguanosine levels in OCCC patients with low ARID1A expression. Moreover, ARID1A-mutated cell lines were sensitive to ROS inductor elesclomol [SLC7A11 (cysteine transporter required by GSH) expression making them vulnerable to GSH and glutamate-cysteine ligase synthetase catalytic subunit (GCLC) inhibition and possible cancer treatment through increased ROS formation [esclomol . Anotherormation .ARID1A insufficiency leads to increased reliance on double-stranded DNA break (DSB) repair mechanisms [Telomere defects and DNA damage caused by chanisms . Inactivchanisms . Most nochanisms . BAF facchanisms  as well chanisms . Moreovechanisms . The losARID1A mutations often co-occur with PI3K/AKT pathway gene KRAS, PIK3CA, and PTEN alterations [ANXA1 (AKT activator) upregulation and PIK3IP1 (PI3K inhibitor) expression downregulation. In particular, PIK3IP1 is regulated by ARID1A, suppressing EZH2 methyltransferase that inhibits PIK3IP1 expression through the H3K27me3 epigenetic mark [ARID1A mutated tumors [In ovarian and endometrial cancers, erations . Additiotic mark . Therefod tumors .Besides its role in SWI/SNF, ARID members have also been reported to co-precipitate with members of E3 ubiquitin ligase. Specifically, ARID1B is linked with elongin C (EloC) through BC box motif and with the addition of cullin 2 and ROC1 form E3 ubiquitin ligase that targets H2B histone explicitly at lysine 120 for monoubiquitination. ARID1B is a paralog that shares most of its sequence with ARID1A. Thus, a similar E3 ubiquitin ligase complex may be formed with ARID1A too. Mutation and depletion of the ARID domain result in decreased ubiquitination of H2B, similar to VHL mutations, causing decreased ubiquitination of HIF1 (ccRCC) . The two (ccRCC) .ARID1A-mutated OC treatment.To sum up, ARID1A is highly involved in many pathways related to cancerogenesis , and itsARID1A mutations in cancers arising from endometriosis  makes SWI/SNF an attractive option for targeted therapy approaches. Although ARID1A has a mutually exclusive homolog ARID1B, and both ARID1A and ARID1B are often co-mutated in human cancers [ARID1A mutant cancers [The frequent loss of function  cancers , the cre cancers . A more  cancers .ARID1A mutant cancers may be more susceptible to ATR blockade [ARID1A mutational status may be a good predictor of immunotherapy outcomes: ARID1A deficient OC mouse model showed higher mutational burden, more tumor-infiltrating lymphocytes, and elevated levels of programmed cell death-ligand 1 (PD-1), providing an opportunity for immunotherapy [ARID1A tumors [ARID1A deficient OCCC cell lines, dasatinib, a tyrosine kinase inhibitor, was identified as a potential drug target. ARID1A mutated cell lines were sensitive to dasatinib treatment because of impaired cell cycle and YES1 (dasatinib target) deregulation [Due to ARID1A’s involvement in DDR responses, blockade  and PARPblockade . Clinicablockade . Althougblockade , ARID1A blockade . Howeverotherapy . A studyA tumors . In anotgulation .ARID1A is the most frequently mutated SWI/SNF subunit coding gene, other SWI/SNF members, albeit less often, are also altered in OC. In particular, up to 90% of a rare form of OC, small cell ovarian carcinoma, hypercalcemic type (SCCOHT), a rhabdoid-like tumor has germline or somatic mutations of SWI/SNF ATPase domain BRG1 coding gene SMARCA4 [SMARCA4 mutations cause hyperactivation of PRC2 repressive complex) [Although complex) , but it complex) . Moreovemote DSB .SMARCA4 in up to 5%, SMARCA1 at 2%, SMARCA2 at 1%, and SMARCC1 at 2% [SMARCA2 mutations are rare, most SCCOHT cases present with dual inactivation of both BGR1 and BRM, though, unlike SMARCA4, SMARCA2 is silenced epigenetically [SMARCB1  mutations instead of SMARCA4 [SMARCB1 mutations are also found in other undifferentiated OC cases and myoepithelium-like tumors of the vulvar region [MYC regulation: MYC is a transcriptional modulator of SMARCB1. However, ARID1A regulates MYC through binding with the MYC promoter region, and BRG1 (coded by SMARCA4) controls MYC partner MAX in a similar manner [Meanwhile, other forms of OC, such as undifferentiated uterine or ovarian carcinoma and OCCC, are found to harbor alterations in other SMARC genes . Most noC1 at 2% . Althougetically . Similar SMARCA4 . Moreover region . Interesr manner .Curiously, in HGSOC, SMARC protein SMARCC1 was shown to be regulated on the protein level through methylation by coactivator-associated arginine methyltransferase 1 (CARM1/PRTM4), which is overexpressed in HGSOC. The methylation causes BAF complex eviction from certain loci, including promoters of c-Myc pathway genes, thus altering gene expression .ACTL6A and ncBAF subunit gene BRD9, are overexpressed in most cancers, including OC. The amplification of these genes co-occurs with notable oncogene TP63 amplification, suggesting an oncogenic mechanism of OC [ACTL6A amplification has also been associated with follicle-stimulating hormone regulation through PIK3/AKT pathway. ACTL6A increase leads to enhanced glycolysis due to increased expression of phosphoglycerate kinase 1 (PGK1) [Although the loss of function mutations in SWI/SNF complex encoding genes occur in more than 20% of human cancers, some amplifications of genes coding for SWI/SNF subunits are also found. Most notably, a scaffolding protein, coded by sm of OC . However1 (PGK1) . Moreove1 (PGK1) .ARID1A, SMARC genes coding ATPase and regulatory subunits are frequently altered as well. An investigation is needed on whether these genes could serve as novel biomarkers for OC.Overall, SWI/SNF gene alterations, especially inactivating mutations, are frequent events in OC development. Besides SMARCA1) mutations have been linked with OCCC [SMARCA1 deletion has been associated with increased apoptosis through caspase activator Apaf-1 expression upregulation [Similarly, to SWI/SNF, ISWI ATPase domains are also encoded by SMARC genes that frequent alterations in OC. Most notably, ISWI ATPase SNF2L , forms either RSF-1 or RSF-5 CRC [SMARCA5) is overexpressed in OC tissues together with its binding partner RSF1 which is also often upregulated in various tumors, including OC [TP53, the DNA damage caused by RSF-5 upregulation goes unsolved [CFLAR, XIAP, BCL2, BCL2L1) and inflammation (PTGS2) gene expression adding to paclitaxel resistance in ovarian cells [Depending on which ATPase it binds, ISWI protein remodeling and spacing factor 1  are known oncogenic drivers regulating proliferation and survival in many cancer types [CHRAC1 expression was found to be increased due to miR-512-3p downregulation and was related to enhanced proliferation, invasion, migration, and apoptosis [In addition to er types . In cisppoptosis . RBBP4 ipoptosis .CHD5 gene are detected in OC, CHD5 is also downregulated by increased promoter methylation in OC patient samples [CHD5 promoter was methylated the most often, although the OC cell line (MDAH2774) showed no increase in methylation [CHD5 polymorphism (rs9434741) has also been associated with endometriosis, an OC precursor [CHD5 increased mutation and methylation, CHD5 mRNA expression was found to be downregulated by at least 2-fold in 41% (32/72) of invasive epithelial ovarian carcinomas in comparison with 12 controls and correlated with shorter disease-free survival times [CHD5 is the main CHD family protein linked with cancer . Althoug samples . In one hylation . CHD5 porecursor , howeveral times .CHD5, CHD CRCs are also found to be dysregulated in ovarian and other cancers and have a role in chemoresistance. CHD8 overexpression has been linked with poor survival in both HGOSC and endometrial cancer cases [Besides er cases . CHD8 bithway OC . Similarthway OC . Other Nthway OC . Meanwhitastasis .ACT6LA, shared with SWI/SNF family remodelers. ACT6LA amplification affects multiple CRCs and causes platinum resistance in OC [PART1, which targets miR-512-3p and causes CHRAC1 (another CRC gene) upregulation and cisplatin-resistant OC cell proliferation and migration [miR-381, which is downregulated in OC tissues [Although several INO80 subunits are overexpressed in melanoma , cervicace in OC . Howeverce in OC . Yin yance in OC  in OC ceigration . In OC p tissues . In anot tissues .RUVBL1, single nucleotide polymorphisms rs1057156 (A>G) and rs149652370 (A>G) were significantly associated with poor overall survival. The rs1057156 (A>G) variant was found to lower miR-4294 binding affinity causing increased RUVBL1 expression in OC cells [In 495 Chinese epithelial OC patient study analyzing INO80 module gene OC cells . In OC pOC cells .TP53, BRCA1/2, MYC or RAS, CRCs and their involvement in cancer pathways are known only for a few decades [ARID1A and other genes in OC and other cancers, providing ample ground for possible therapeutic options [Overall, at least 18 different CRC subunits are affected in OC. While SWI/SNF genes are mostly affected by inactivating mutations, amplification and epigenetic changes also affect CRC coding genes . These a decades , thus, a options . HoweverRecently identified numerous mutations and amplifications in chromatin remodeling genes open a possibility for new therapeutic approaches. Of note, FDA approval of EZH2 inhibitor tazemetostat therapy for follicular lymphoma shows a promising step in epigenetic therapy application, and increases hope that the same inhibitor could be used for SWI/SNF deficient HGSOC, OCCC, endometrial OC, or SCCOHT patient treatment . Phase I


Study findings support that both program-related  and non-program-related  factors are important to applicants when making ROL decisions. Applicants who matched with their top choice program initially experienced emotional benefits, but these emotional differences did not persist into PGY-1, where all matched applicants had similar levels of emotion and life satisfaction. The emotional cost and benefits of matching with programs of different ROL positions  were most apparent shortly after matching but in the long-term, a stronger predictor of PGY-1 emotions was perceived person-program alignment. Person-program alignment  also predicted burnout in the first few months of a residency program. These findings show that, when applicants are making ranking decisions, they undervalue factors that predict stress and burnout during residency.In the medical residency match process, applicants’ ranking decisions are influenced by multiple factors related to training, geography, and lifestyle expectations. Ranking decisions directly impact match results, with implications for emotional outcomes such as happiness and stress. The present study explored the decision factors considered most important by applicants when creating rank order lists (ROLs), and how match outcomes and program factors predicted happiness, enthusiasm, stress, and life satisfaction. Senior medical students ( Every March on Match Day, senior medical students find out where they will complete their residency training, the culmination of a months-long application process facilitated by the National Resident Matching Program (NRMP). Match outcomes are impacted by metrics of applicant qualifications , as well as ranking behaviors .The Match’s underlying algorithm works optimally when applicants create their rank order lists (ROLs) based on their personal preferences . HoweverOur investigation aims to provide further insight into the decision-making process students engage in as they attempt to identify their preferred residency programs, and the relative importance they give to ranking factors. Many studies on ROL decisions focus on a particular specialty area  or analyA second aim of this investigation was to identify factors that actually were important to emotional responses as students learned the outcome of Match Day, as well as several months after beginning their residency program. We assessed respondents at three timepoints: shortly before Match Day, shortly after Match Day, and in the fourth month of their residency program.Senior medical students at a large public university in California were contacted to participate in the study with approval from the IRB. Several weeks before Match Day, students were invited by a classmate in student leadership to participate in a study that involved three online surveys. Students who elected to participate received $25 for completing the first survey, and $50 each for the second and third surveys. Two cohorts of students (the graduating classes of 2016 and 2017) were recruited for the study, and data collection ran from March 2016 to October 2017. This study was part of a larger investigation about emotional forecasting ,11, and not at all) to 9 (most extreme possible). The selected states – happiness, excitement, and life satisfaction – were chosen because of their relevance to well-being [not important) to 9 (extremely important).The first survey was sent out 2 weeks before Match Day, after students had submitted their ROLs, and completed by a week before Match Day. In the survey, participants rated their level of general happiness, excitement, stress, and life satisfaction from 1  to 9 (perfectly suited to my needs). These priority items . They also reported their current perception of the alignment between their priorities and those of the program on a scale from 1 (shown in  were pulI enjoy my work. I have no symptoms of burnout) to 5 (I feel completely burned out and often wonder if I can go on. I am at the point where I may need some changes or may need to seek some sort of help). Single-item measures of burnout have been validated against longer scales such as the Maslach Burnout Inventory and are recommended for increasing response rates among physicians specifically [The third, final survey was administered in mid-October, selected because it was several months into post-graduate year one (PGY-1). Applicants again rated their responses of happiness, enthusiasm, stress, and life satisfaction using the same scale as at Time 1. They also rated the person-program alignment using the same scale as at Time 2. Participants also reported the level of burnout they were currently experiencing from 1  responses to an open ended prompt about the top five factors considered and these were examined as the percentage of participants who gave each category of response, and 2) ratings of the importance of each factor on a continuous Likert-type scale, which were summarized as mean rated importance and standard deviations. The impact of match outcome  on responses was analyzed using an Analysis of Variance (ANOVA), which permitted the examination of whether responses on continuous Likert-type scales differed among the categories of the rank of match. Because ANOVAs are omnibus tests, independent contrasts were conducted to compare each category included in the ANOVA. Whether responses related to the perceived alignment between the person and the program was also examined, using linear regression analyses to examine the degree to which continuous Likert-type ratings of person-program alignment predicted responses to the match outcome that were also rated on continuous Likert-type scales.n = 182 who completed Time 1 out of 203 eligible participants, 89.6% response rate; 179 responded to Time 2; 167 responded to Time 3) included 86 males (47.5%) and 95 females (52.5%); one participant did not report gender. The average age of participants was 28 years (range 25–36). Most participants (174) matched with a residency program through the NRMP, representing 72 different residency locations throughout the nation. Two matched into specialty areas that do not use the NRMP, two did not match into any program, and one participant was matched with a residency program through the Supplemental Offer and Acceptance Program after Match Day. Of matched participants (n = 174), 94 matched with the program they ranked first, 33 with the second ranked program, 16 with the third ranked program, and 31 with the program ranked fourth or lower.The sample , and inter-rater agreement was high (agreeing on 97.6% of classifications). The top five decision factors are described in The second source of information about ranking considerations came from ratings of the importance of specific factors when making ROL decisions at Time 1 see . Of the F representing the omnibus statistic associated with the ANOVA and p representing the statistical significance associated with the F statistic. These analyses revealed that the difference in happiness among groups was significant, F = 22.7, p < 0.001. Those who matched with their top choice program expressed the highest levels of happiness compared to those who matched with programs they ranked second, t(126) = 2.1, p = 0.04, third, t(109) = 6.2, p < 0.001, or fourth or lower, t(126) = 7.3, p < 0.001. This pattern held for excitement, F = 14.4, p < 0.001, and life satisfaction, F = 12.8, p < 0.001. Stress, a negative emotion, F = 3.8, p = 0.01, had the reverse pattern.The repeated measurement nature of the study permitted analyses of the relationship between ROL outcomes (of matching with a top choice or lower) and predicted responses such as happiness, enthusiasm, stress, and life satisfaction. Only two respondents did not match with any program, and this group was too small to analyze. To investigate the emotional impact of different match outcomes, Analyses of Variance (ANOVA) compared responses at Time 2 (shortly after Match Day) among applicants who matched into a program they ranked first, second, third, or fourth or lower. The rank of the match was treated as categorical in analyses because there were qualitative differences between ranks that were not equivalent intervals and because the category ‘4th or lower’ captured multiple possible outcomes. Means and standard deviations are shown in F = 0.5, p = 0.7, excitement, F = 0.3, p = 0.9, stress, F = 0.8, p = 0.5, or life satisfaction, F = 0.2, p = 0.9.At Time 3, however, several months into PGY-1, participants reported similar levels of responses regardless of the ROL position of their matched program see . An ANOVWe also analyzed the impact of perceived person-program alignment on responses shortly after Match Day (Time 2), shown in We also analyzed the impact of perceived person-program alignment on responses several months into residency training (Time 3), shown in R2 = 0.2, F = 5.0, p < 0.001. As with other responses, burnout was not predicted by perceived person-program alignment on the reputation of the program , location ; relationship factors , training quality , or finance factors . Perceived alignment on post-residency opportunities predicted greater burnout , potentially due to stress related to doing well in the program when opportunities were greater. Perceived alignment on program culture predicted less burnout in the first few months of starting a residency program , accounting for approximately 30% of experienced burnout.A similar regression analysis was conducted on reported burnout at Time 3, months into their new residency program, The medical residency match process is becoming more competitive, with fewer applicants matching with their top choice program every year. In 2021, only 46.4% of U.S. allopathic seniors matched with their top ranked program [Perceived person-program alignment with the program culture of the residency program, however, did predict responses, immediately after Match Day as well as months after the start of residency. Other factors that residents reported relying on to make their rank order decisions did not predict responses after Match Day or during residency, including geographic location, training quality, relationship factors, and financial factors. After they began their residency programs, the perception that the program culture aligned with their needs predicted greater happiness and enthusiasm, and less stress and burnout. Cognitive research supports that social interactions are uniquely emotional and memorable sources of information, and have an outsize impact on decisions , which mIn recent years, there has been growing concern about medical resident burnout, which affects medical trainees at higher rates than other US physicians ,24 and iProgram culture factors that impact person-program alignment, such as feelings about the program, morale and rapport, life balance, caseloads and call schedules, should be considered by both medical students and residency program leaders throughout the application process. It would be beneficial for programs to discuss information related to program values and expectations with students and encourage consideration of these factors during interviews through interview questions or advising sessions prior to interviews. This has the potential to improve the quality of decisions and reduce stress and burnout among residents.Limitations of this study include that it used a single-school sample, had a relatively small sample size, the sample of unmatched applicants was too small for analysis, some match ranks were relatively small, and it only assessed resident burnout at one timepoint. The percentage of students in our sample who matched with their top choice program (54.0%) was slightly above the national rate for U.S. allopathic seniors in 2017, 48.4%.Future research should examine the link between perceived program priority alignment and emotions throughout residency.This study expands on existing research on decision factors in the residency match process and demonstrates that person-program alignment on program culture is important for emotional responses and burnout among residents. Although the initial emotional costs or benefits of matching with a certain program fade relatively shortly after Match Day, program culture alignment with respect to factors like call schedule and patient caseload predicted greater happiness, enthusiasm and life satisfaction, as well as lower stress and burnout in the first few months of residency. Our study provides insight into the processes involved in rank order decision-making, as well as the importance of program priorities factors for optimizing well-being and minimizing burnout during residency.


There is a need for improved communication in assisted living communities (ALCs) for staff and family caregivers of persons with dementia. This study is a cluster randomized trial in 10 ALCs  of a PC education (PCEAL) program for dementia care providers facilitated by hospice nurse educators. Baseline, 3- and 6-month data was collected for documentation of advance care planning (ACP) discussions. The PCEAL program had a moderate effect on ACP discussions at the 3-month follow in the treatment (N=17) compared to the control group (N= 6) . The findings indicate that the PCEAL program potentially improves ACP discussions with family members of persons with dementia. We will discuss recommendations for tailoring the PCEAL program to include other direct and administrative staff and to elicit the perspective of families about quality of communication.