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Background: Staphylococcus aureus causes bloodstream and skin infections in humans. The prevalence of multidrug-resistant S. aureus strains means new antibiotics are needed. A novel antimicrobial drug named SK-03-92, a synthetic aromatic organic stilbenoid compound, kills S. aureus cells within 30 min, but an increase in both biofilm formation and persister cells occurs. SK-03-92 treatment downregulates transcription of the biofilm regulating protein regulator (brpR) gene and biofilm regulating protein sensor (brpS) gene in S. aureus. BrpR/BrpS system may be a LytTR regulatory system tied to biofilm formation, creation of persister cells, and late-stage competence in S. aureus. The aim of this study was to determine what biofilm, late-stage competence, and persister-associated genes were regulated in a brpR mutant compared to wild-type strains. Methods: In this study, involvement of BrpR in regulating other genes was assessed by comparing transcriptional changes in a brpR mutant strain to the S. aureus parent strain via RNA sequencing (RNA-Seq). Bioinformatic analysis was then performed on the RNA-Seq data to assess what biochemical pathways might be involved. Results: From these analyses, 440 genes were identified that had significant differences in transcript abundance when comparing the brpR mutant to wild-type strains. Quantitative reverse transcription polymerase chain reaction analysis confirmed bacA, icd, metE, and pdhA transcript levels were lower, whereas alr and mraY were higher in the brpR mutant versus wild-type strain. Furthermore, an enzymatic assay targeting NADH production from the pyruvate dehydrogenase complex showed lower levels in the mutant compared to wild-type strain. Conclusions: Overall, the study demonstrated several biosynthetic pathways tied to biofilm formation and late-stage competency may be regulated by BrpR and some potential leads for the mechanism of action of the SK-03-92 drug were uncovered.

Background TP53 mutational status ( TP53 ms) is prognostically important in myeloid neoplasms. Immunohistochemistry (IHC) of p53 has been used as a surrogate for TP53 mutations, with faster results than next-generation sequencing (NGS) and cytogenetic analysis. Given the challenges posed by heterogeneous p53 IHC expression, this study aims to evaluate the interobserver interpretation of p53 stain as a surrogate marker for TP53m . Methods Fifty-three bone marrow (BM) specimens previously diagnosed as myeloid neoplasms or nonneoplastic BM specimens with available concurrent TP53ms were evaluated. p53 IHC staining was performed on all 53 core biopsy or clot sections. TP53 mutation was found in 24 cases ( TP53 m). Twenty-nine specimens were TP53 wild-type and served as the control group. Six hematopathologists (HPs) blinded to the diagnosis and TP53 m independently scored the p53 staining pattern of the biopsies/clot sections for overall staining percentage (OSP) and staining intensity (dark staining percentage as a percentage of total cells (DCP)). Results The intraclass correlation was excellent for OSP (0.927) and good for DCP (0.848). For OSP, a cutoff of 30% resulted in sensitivity of 83.3%, specificity of 89.7%, PPV of 87.0%, and NPV of 86.7%. For DCP, a cutoff of 10% achieved sensitivity of 87.5%, specificity of 96.6%, PPV of 95.4%, and NPV of 90.3%. Additionally, DCP significantly correlated with the VAF of TP53 m (R = 0.469, p = 0.028). Conclusion High inter-rater reliability for OSP and DCP supports integrating p53 IHC into bone marrow evaluations for rapid TP53 m assessment. With superior specificity and predictive values, DCP proves to be a more reliable diagnostic marker for TP53 mutations. While this approach shows promise for myeloid neoplasms, further validation is needed to overcome challenges and expand its clinical utility for tailored treatment strategies.

ABSTRACT Background While vaccination is a key strategy to mitigate the severity of coronavirus disease 2019 (COVID‐19) in children, the risk factors for symptomatic, medically attended (hereon referred to as “symptomatic”) COVID‐19 and the impact of vaccination are not well defined among pediatric solid organ transplant recipients (PSOTR). Methods PSOTR aged six months through 21 years at 11 centers in the Pediatric Infectious Diseases Transplant Network (PIDTRAN) were analyzed retrospectively for symptomatic COVID‐19 between July 2021 and May 2023. Using a Cox proportional hazards model, risk factors associated with COVID‐19‐associated hospitalization, in‐hospital oxygen use, mechanical ventilation, and the impact of COVID‐19 vaccination were analyzed. Results Of 1370 PSOTR, 410 (29%) had symptomatic COVID‐19 infection. Among those with symptomatic infection, 115 (28%) were hospitalized, 35 required in‐hospital oxygen, and three required mechanical ventilation. In the study, 45% were unvaccinated against COVID‐19. Compared to unvaccinated PSOTR, incompletely vaccinated PSOTR had 56% lower hazard (95% CI 0.26, 0.72, p = 0.001) of COVID‐19‐associated hospitalization whereas completely vaccinated did not have a statistically significant difference in hazard of hospitalization. Receipt of at least one dose of the bivalent vaccine was also associated with 92.5% lower hazard (95% CI 0.010, 0.55, p = 0.011) of hospitalization. Risk factors for hospitalization included chronic kidney disease, underlying heart disease, neurodevelopmental delay, and lymphocytopenia. Conclusions Most PSOTR with symptomatic COVID‐19 did not require hospitalization or respiratory support. Several comorbidities and lymphocytopenia are associated with severe COVID‐19 in PSOTR. COVID‐19 vaccination, mostly, protected PSOTR against COVID‐19‐associated hospitalization, but vaccine uptake among PSOTR was low. image

Abstract Although the human genome encodes a vast repertoire of noncoding RNAs that regulate gene expression, the noncoding genome remains underexplored due to technical challenges. Specifically, during transcriptomic sequencing data alignment, the overlap between noncoding and coding loci can create ambiguous read alignments that are subsequently discarded from downstream analysis. For this reason, most of the noncoding genome is excluded from standard genomic annotations used for sequencing alignment. To address this challenge and enable concurrent profiling of the coding and noncoding transcriptome, we systematically integrated standard coding (GENCODE) and noncoding (LncBook) genome annotations, preserving coding gene annotations and removing overlapping noncoding regions. The resulting integrated genome annotation expanded the number of annotated noncoding genes from 40,785 to 138,296 while preserving all coding genes and reducing ambiguous read assignment. To evaluate the utility of our integrated genome annotation for uncovering novel, biologically relevant noncoding RNAs (ncRNAs), we realigned CD138-positive bulk RNA-seq (N = 942) and CD138-negative single-cell RNA-seq (N = 478) data from the MMRF CoMMpass study, generating a comprehensive coding-noncoding atlas of the myeloma bone marrow microenvironment with noncoding genes representing 51% of highly variable genes and displaying significant cell type specificity. Tumor expression profiling based on this integrated profiling identified 15 clusters, including two enriched for amp(1q21) or t(4;14) and associated with shorter progression-free survival (PFS). Differential expression and systematic filtering yielded 19 candidate high-risk ncRNAs, including previously uncharacterized ENSG00000310209 , which was associated with poor PFS (HR = 1.141, P = 0.0025), increased IRF4 activity, Wnt pathway activation, CCL5 signaling, and the accumulation of anergic-like CD8+ T cells. These findings establish integrated coding-noncoding analysis as a strategic approach for discovering functional ncRNAs from transcriptomic sequencing data.

Photodynamic priming (PDP), a fallout of photodynamic therapy, transiently modulates the tumor microenvironment (TME), enhances therapeutic susceptibility, and promotes immunogenic cell death through the release of damage-associated molecular patterns (DAMPs). Pancreatic ductal adenocarcinoma (PDAC) remains non-responsive to current therapies with a desmoplastic and immunosuppressive TME that limits drug delivery and blunts responses to immune checkpoint blockade. We investigated whether PDP could enhance anti-PD1 therapy responses in PDAC using patient-derived organoids (PDOs). PDOs were treated with Visudyne and red light (25, 75, and 100 J/cm²), followed by assessment of cytotoxicity, DAMP expression (HSP60, calreticulin, HMGB1), and transcriptomic changes. Monocyte-derived dendritic cells (mDCs) from healthy donors were cocultured with PDP-treated (25 J/cm 2 ) PDOs, then with matched naïve T cells. mDC and T cell activation markers were analyzed. Pembrolizumab (anti-PD1) was added to PDO-mDC-T cell cocultures to evaluate combined effects on PDO viability and T cell activation. PDP induced dose-dependent cytotoxicity and upregulated DAMPs. Gene profiling in PDOs showed increased IFN-γ , TNF-α , and CXCL12 , with reduced PD-L1 , TGF-β1 , and FOXP1 expression. mDCs exposed to PDP-treated PDOs upregulated CD40, CD86, and MHC-II, driving activation of CD4 + and CD8 + T cells, evidenced by elevated PD1 expression. Addition of pembrolizumab further decreased PDO viability and amplified effector cytokines ( IFN-γ , TNF-α , FASLG , granzyme B, IL-2 , IL-21 ). PDP was associated with modulation of the PDAC TME toward a more immunogenic phenotype by enhancing tumor immunogenicity, activating dendritic cells and T cells, and potentiating PD1 blockade. These findings provide mechanistic support for further preclinical and clinical evaluation of PDP combined with checkpoint inhibition in PDAC.

Healthcare Systems Science (HSS) is an important part of medical education because it helps physicians understand how healthcare systems work and how to improve them. However, it is not commonly included in graduate medical training. This manuscript describes the creation and use of a radiation oncology (RO) HSS curriculum that was built with medical school faculty and adapted to the needs of residents in our residency program. The curriculum included sixteen sessions over ten months and covered six domains: person centered care, population centered care, team-based care, high value care, leadership, and health policy economics and technology. Each session included a short lecture and a case discussion on topics such as financial toxicity, telemedicine, workflow improvement, burnout, health equity, and billing. The curriculum was used successfully with regular participation from residents and faculty, showing that it can fit into an existing residency schedule. Residents completed pre- and post-intervention surveys assessing understanding, consideration, confidence, and satisfaction related to HSS domains using 5-point Likert-scale items. Among residents with paired survey data, scores for understanding of HSS, incorporation of HSS into care planning, confidence applying HSS principles, and satisfaction with HSS integration all improved following curriculum completion. This experience shows that adding HSS to RO residency training is practical and useful, and it may also be a helpful model for other residency programs.

Abstract Background Adults with congenital heart disease (ACHD) represent a rapidly expanding population with evolving mortality patterns. Despite improved survival, excess mortality persists. Objective To evaluate the incidence, causes, and predictors of mortality in a contemporary ACHD cohort. Methods We performed a retrospective cohort study of adults (≥18 years) first evaluated at Mayo Clinic from 2002–2023. Baseline clinical, imaging, and electrocardiographic data were analyzed. Vital status was determined using institutional records and the Accurint national mortality database. Kaplan-Meier analysis and Cox proportional hazard models were used to evaluate mortality and identify independent predictors of mortality Results A total of 7,678 ACHD patients were included, with median age of 36.8 years and median follow-up of 11.4 years. During 78,768 patient-years of follow-up, 1,116 patients died (median age at death 57.2 years), corresponding to an annual mortality rate of 1.4%. The cumulative rate of all-cause mortality at 5, 10, 15, and 20 years was 6.9%, 12.0%, 19.0%, and 26.2%, respectively. When stratified by CHD complexity, the annual death rate in patients with severe CHD (2.4%/year) was twice that of patients with moderate or mild CHD (both 1.2%/year). Older age and ACHD subtypes, specifically, cyanotic heart disease (HR 3.9, 95% CI 2.9–5.3) and Fontan physiology (HR 3.2, 95% CI 2.3–4.4), were strongly associated with increased mortality. Additional independent predictors included male sex, ventricular dysfunction, advanced NYHA class, prior heart failure hospitalization, hypertension, smoking, coronary artery disease, renal dysfunction, and abnormal hemoglobin levels. Cardiovascular causes accounted for 57.7% of deaths with known etiology, predominantly heart failure (48.9%) and sudden cardiac death (21.9%), while non-cardiovascular causes were driven mainly by infection and malignancy. Conclusions In this large contemporary ACHD cohort, mortality was driven by ventricular dysfunction, heart failure, and systemic end-organ involvement in addition to the underlying congenital anatomy. Both cardiovascular and non-cardiovascular causes contributed significantly to mortality. These findings underscore the need for comprehensive multidisciplinary ACHD care focused on early recognition of cardiac functional decline, management of acquired comorbidities, and end-organ dysfunction.

Background/Objectives: Retained airway secretions are a common complication of mechanical ventilation and a major contributor to ventilator-associated pneumonia. Airway Pressure Release Ventilation following a Time-Controlled Adaptive Ventilation protocol (APRV-TCAV) has been anecdotally observed to improve secretion clearance, but the mechanism is not well established. Because TCAV sets the release time (TLow) from each patient’s own expiratory flow decay, we hypothesized that the resulting expiratory-dominant flow profile would generate a net antegrade force on secretions. This bench study compared secretion movement between APRV-TCAV and conventional Volume-Control Continuous Mandatory Ventilation (VC-CMV), with and without an in-line oscillatory device. Methods: A critical care ventilator was connected to a 3 L test lung via a 7 mm endotracheal tube (ETT). Simulated mucus (1% guar gum) was instilled into the ETT, and secretion movement was measured under VC-CMV (positive end-expiratory pressure (PEEP) 5, 10, 15 cmH2O) and APRV-TCAV (PHigh 20, 25, 30 cmH2O; PLow 0 cmH2O). Each condition was repeated with an in-line oscillatory device. Results: All VC-CMV settings produced retrograde secretion movement toward the test lung; all APRV-TCAV settings produced antegrade movement. The largest antegrade movement was observed at the widest PHigh-to-PLow differential. Adding the oscillatory device raised peak expiratory flow above peak inspiratory flow in every condition, yet abolished antegrade movement under APRV-TCAV. Conclusions: In this bench study, APRV-TCAV reversed the direction of secretion movement within the ETT, with larger antegrade movement at wider PHigh-to-PLow differentials. This suggests a potential mechanism by which the ventilator flow profile itself may influence secretion movement. The directional effect depended on the intact release-phase waveform and was abolished by in-line oscillatory therapy despite preserved peak flow asymmetry.

PURPOSE Hematology care delivery is changing rapidly for patients, care teams, and health care institutions. This study simultaneously evaluated utilization data for synchronous/real-time patient appointments, asynchronous/time-delayed patient communications, and provider preferences in a large academic hematology practice to inform sustainable care delivery and workflows in the digital era. METHODS We conducted a retrospective analysis of aggregated adult hematology appointment and patient communication data at Mayo Clinic (Rochester, MN) from January 2023 to January 2025. Data were stratified by appointment modality, patient characteristics, and electronic health record (EHR) message volumes. Additionally, a voluntary, anonymous survey assessed hematology provider preferences regarding appointment modalities, in-basket burdens, and billing practices. RESULTS Among 83,713 completed appointments, 80.8% were in-person, 12.9% video, and 6.4% telephone. Patients age 80 years and older and rural cohorts used virtual modalities at rates comparable with younger and metropolitan cohorts. Asynchronous communication volume was considerable, with 155,036 patient-initiated telephone calls generating a digital cascade of 407,578 EHR messages (a 1:2.6 ratio) and 49,798 patient-initiated advice request in-basket messages generating 343,030 additional EHR messages (a 1:6.9 ratio). Providers (n = 53) strongly preferred in-person appointments for new consults (92.5%) and active therapy management (90.6%) but favored virtual appointments for surveillance (52.8%). Although 86.8% of providers reported spending >30 minutes daily on in-basket management, 94.3% reported not billing for this time. CONCLUSION Telehealth is a substantial component of modern hematology practice, used across all age groups. However, digital cascading and proliferation of asynchronous communications present an invisible and uncaptured workload. Optimizing care delivery could include implementation of a stratified hybrid model, prioritizing in-person appointments for high-acuity needs and virtual appointments for low-acuity needs, alongside standardized workflows to mitigate burdens of the digital era.