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Abstract Nonsense mutations – the underlying cause of approximately 11% of all genetic diseases – prematurely terminate protein synthesis by mutating a sense codon to a premature stop or termination codon (PTC). An emerging therapeutic strategy to suppress nonsense defects is to engineer sense-codon decoding tRNAs to readthrough and restore translation at PTCs. However, the readthrough efficiency of the engineered suppressor tRNAs (sup-tRNAs) largely varies in a tissue- and sequence context-dependent manner and has not yet yielded optimal clinical efficacy for many nonsense mutations. Here, we systematically analyze the suppression efficacy at various pathogenic nonsense mutations. We discover that the translation velocity of the sequence upstream of PTCs modulates the sup-tRNA readthrough efficacy. The PTCs most refractory to suppression are embedded in a sequence context translated with an abrupt reversal of the translation speed leading to ribosomal collisions. Moreover, modeling translation velocity using Ribo-seq data can accurately predict the suppression efficacy at PTCs. These results reveal previously unknown molecular signatures contributing to genotype-phenotype relationships and treatment-response heterogeneity, and provide the framework for the development of personalized tRNA-based gene therapies.

Understanding the mechanisms that underlie de novo mutations (DNMs) can be essential for interpreting human evolution, including aspects such as rapidly diverging genes, conservation of non-coding regulatory elements, and somatic DNA adaptation, among others. DNM accumulation in Homo sapiens is often limited to evaluation of human trios or quads across a single generation. Moreover, human SNPs in exons, pseudogenes, or other non-coding elements can be ancient and difficult to date, including polymorphisms attributable to founder effects and identity by descent. In this report, we describe multigenerational evolution of a human coding locus devoid of natural selection, and delineate patterns and principles by which DNMs have accumulated over the past few thousand years. We apply a data set comprising cystic fibrosis transmembrane conductance regulator (CFTR) alleles from 2,393 individuals homozygous for the F508del defect. Additional polymorphism on the F508del background diversified subsequent to a single mutational event during recent human history. Because F508del CFTR is without function, SNPs observed on this haplotype are effectively attributable to factors that govern accumulating de novo mutations. We show profound enhancement of transition, synonymous, and positionally repetitive polymorphisms, indicating appearance of DNMs in a manner evolutionarily designed to protect protein coding DNA against mutational attrition while promoting diversity.

Forum papers are thought-provoking opinion pieces or essays founded in fact, sometimes containing speculation, on a civil engineering topic of general interest and relevance to the readership of the journal. The views expressed in this Forum article do not necessarily reflect the views of ASCE or the Editorial Board of the journal. [ABSTRACT FROM AUTHOR]

Abstract Objective Osteoarthritis (OA) is a degenerative joint disease caused by the breakdown of joint cartilage and adjacent bone. Joint injury, being overweight, differences in leg length, high levels of joint stress, abnormal joint or limb development, and inherited factors have been implicated in the etiology of OA. In addition to physical damage to the joint, a role for inflammatory processes has been identified as well. Small heterodimer partner-interacting leucine zipper protein (SMILE) regulates transcription and many cellular functions. Among the proteins activated by SMILE is the peroxisome proliferator-activated receptor (PPAR) γ, which mediates the activities of CD4 + T helper cells, including Th1, Th2, and Th17, as well as Treg cells. PPAR-γ binds to STAT3 to inhibit its transcription, thereby suppressing the expression of the NF-κB pathway, and in turn, the expression of the inflammatory cytokines interferon (IFN), interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, which are sub-signals of STAT3 and NF-κB. Methods OA was induced in control C57BL/6 mice and in C57BL/6-derived SMILE-overexpressing transgenic (SMILE Tg) mice. The protein expression levels in the joint and spleen tissues were analyzed by immunohistochemistry and immunofluorescence images. In addition, flow cytometry was performed for detecting changes of the changes of immune cells. Results Less cartilage damage and significantly reduced levels of OA biomarkers (MMP13, TIMP3 and MCP-1) were observed in SMILE Tg mice. Immunohistochemistry performed to identify the signaling pathway involved in the link between SMILE expression and OA revealed decreased levels of IL-1β, IL-6, TNF-α, and phosphorylated AMPK in synovial tissues as well as a significant decrease in phosphorylated STAT3 in both cartilage and synovium. Changes in systemic immune cells were investigated via flow cytometry to analyze splenocytes isolated from control and SMILE Tg mice. SMILE Tg mice had elevated proportions of CD4 + IL-4 + cells (Th2) and CD4 + CD25 + Foxp3 + cells (Treg) and a notable decrease in CD4 + IL-17 + cells (Th17). Conclusion Our results show that overexpressed SMILE attenuates the symptoms of OA, by increasing AMPK signaling and decreasing STAT3, thus reducing the levels of inflammatory immune cells.

The clearwing moth, Synanthedon bicingulata Staudinger, 1887 (Lepidoptera: Sesiidae), is a serious pest that infests cherry trees. This species, which is distributed in South Korea, was once regarded as S. hector found in Japan, but renamed as S. bicingulata based on morphology. Molecular data for this species are not available. Hence, we sequenced the 16,255-bp long complete mitochondrial genome (mitogenome) of S. bicingulata. Phylogenetic analyses in the Cossoidea superfamily supported monophyly of the Synanthedon genus and Synanthedonini tribe. This newly sequenced mitogenome of S. bicingulata will be useful for studies in a diverse field of evolutionary study.

Abstract Background Critical limb ischemia (CLI) is a condition characterized by insufficient blood flow to the lower limbs, resulting in severe ischemia and potentially leading to amputation. This study aims to identify novel vasculogenic precursor cells (VPCs) in human bone marrow and evaluate their efficacy in combination with bone marrow-derived mesenchymal stem cells (BM-MSCs) for the treatment of CLI. Methods Ex vivo cultured VPCs and BM-MSCs from bone marrow were characterized and their effects on neovascularization and long-term tissue regeneration were tested in a mouse CLI model. Results VPCs, expressing high levels of hepatocyte growth factor and c-MET, were identified from human bone marrow aspirates. These cells exhibited strong vasculogenic capacity in vitro but possessed a cellular phenotype distinct from those of previously reported endothelial precursor cells in circulation or cord blood. They also expressed most surface markers of BM-MSCs and demonstrated multipotent differentiation ability. Screening of 376 surface markers revealed that VPCs uniquely display CD141 (thrombomodulin). CD141+VPCs are present in BM aspirates as a rare population and can be expanded ex vivo with a population doubling time of approximately 20 h, generating an elaborate vascular network even under angiogenic factor-deficient conditions and recruiting BM-MSCs to the network as pericyte-like cells. Intramuscular transplantation of a combination of human CD141+VPCs and BM-MSCs at a ratio of 2:1 resulted in limb salvage, blood flow recovery, and regeneration of large vessels in the femoral artery-removed CLI model, with an efficacy superior to that of singular transplantation. Importantly, large arteries and arterioles in dual cell transplantation expressed human CD31 in the intima and human α-smooth muscle actin in media layer at 4 and 12 weeks, likely indicating their lineage commitment to endothelial cells and vascular smooth muscle, respectively, in vivo. Conclusion Dual-cell therapy using BM-derived CD141+ VPCs and BM-MSCs holds potential for further development in clinical trials to treat peripheral artery disease and diabetic ulcers.

Abstract We consider multipartite quantum state discrimination and provide a specific relation between the properties of entanglement witness and quantum nonlocality inherent in the confidence of measurement. We first provide the definition of the confidence of measurement as well as its useful properties for various types of multipartite measurement. We show that globally-maximum confidence that cannot be achieved by local operations and classical communication strongly depends on the existence of a certain type of entanglement witness. We also provide conditions for an upper bound on maximum of locally-achievable confidence. Finally, we establish a method in terms of entanglement witness to construct quantum state ensembles with nonlocal maximum confidence.

Disseminated tuberculosis (TB), resulting from the hematogenous spread of tubercle bacilli, typically affects immunocompromised individuals, such as those infected with the human immunodeficiency virus. However, risk factors in immunocompetent populations are not well understood. Here, we report a rare case of disseminated TB with CD4+ T-cell depletion in a previously healthy 35-year-old man. The patient presented with a 2-month history of intermittent gross hematuria, dysuria, loose stools, and weight loss. His medical history was unremarkable except for a herpes zoster infection 4 years prior to presentation. Laboratory tests revealed microscopic hematuria and pyuria; however, the urine culture was negative. Urine specimens tested positive for TB-polymerase chain reaction. Abdominal computed tomography revealed a focal filling defect in the left kidney, segmental wall thickening of the terminal ileum, and multiple enlarged lymph nodes with central necrosis. Chest computed tomography revealed active pulmonary TB. Colonoscopy confirmed intestinal TB in the terminal ileum and ileocecal valve, with positive TB-polymerase chain reaction results from sputum and ileal ulcer tissue. The patient was diagnosed with disseminated TB and was treated with standard anti-TB drugs. Although the human immunodeficiency virus test results were negative, the patient’s CD4+ T-cell count was significantly low (278/μL). Follow-up tests after 1 month showed negative TB cultures; however, the patient’s CD4+ T-cell depletion persisted, with counts remaining low after 1 year. This case highlights the rare occurrence of disseminated TB in immunocompetent individuals with CD4+ T-cell depletion and emphasizes the importance of CD4+ T-cell assessment in healthy patients presenting with disseminated TB.

Abstract Background The mitochondria are essential organelles not only providing cellular energy in the form of ATP, but also regulating the inflammatory response and the cell death program. Mitochondrial dysfunction has been associated with various human diseases, including metabolic syndromes as well as inflammatory and neurodegenerative diseases. Acute respiratory distress syndrome (ARDS) is an acute pulmonary disorder characterized by uncontrolled alveolar inflammation, apoptotic lung epithelial/endothelial cells, and pulmonary edema. Despite the high mortality of ARDS, an effective pharmacotherapy to treat this disease has not been established yet. Therefore, identifying a novel targeted therapy for ARDS is important. Recently, exogenous mitochondrial transplantation was reported to be beneficial for treating mitochondrial dysfunction. The current study aimed to investigate the therapeutic effect of mitochondrial transplantation on ARDS in vitro and in vivo. Methods Mitochondria were isolated from human stem cells. For in vitro efficacy of mitochondrial transplantation on the inflammation and cell death, murine alveolar macrophages MH-S and human pulmonary microvascular endothelial cells HPMECs were exposed to LPS, respectively. The ARDS mice model established by a single intratracheal instillation of LPS was used for in vivo efficacy of intravenously treated mitochondria. Results Our results showed that the mitochondria isolated from human stem cells exhibited an anti-inflammatory effect against alveolar macrophages and an anti-apoptotic effect against the alveolar epithelial cells. Furthermore, intravenous mitochondrial treatment was associated with the attenuation of lung injury in the LPS-induced ARDS mice. Conclusion Dual effects of mitochondria on anti-inflammation and anti-apoptosis support the potential of mitochondrial transplantation as a novel therapeutic strategy for ARDS.

Objective This study aimed to evaluate the prevalence of urinary incontinence (UI) and its associated risk factors among pregnant Korean women, as UI significantly impacts their quality of life. Methods A cross-sectional study involving singleton pregnant women was conducted between April and December 2023. Data were collected using a questionnaire assessing demographic information and UI symptoms. The International Consultation on Incontinence Questionnaire-UI short form was used to diagnose UI. Results A total of 824 pregnant women from three centers participated, with an overall prenatal UI prevalence of 40.2% (331/824). Stress UI was most common (77.1%), followed by mixed UI (16.9%), and urgency UI (6.0%). Risk factors for UI included prior delivery mode, specifically vaginal delivery (adjusted odds ratio [aOR], 5.61; 95% confidence interval [CI], 1.40–22.50; P=0.015) and combined vaginal and cesarean delivery (aOR, 23.14; 95% CI, 1.77–302.74; P=0.017). Additionally, second trimester (aOR, 1.99; 95% CI, 1.19–3.32; P=0.009) and third trimester (aOR, 4.44; 95% CI, 2.65–7.40; P

Objective: Female urethral diverticulum (UD), an evagination of the urethral mucosa into the surrounding connective tissue, is extremely rare in pregnancy. No clear guidelines on the optimal management of UD have been established, except for a common conservative approach. Here, we discuss how to manage UD with pregnancy. Case report: A 39-year-old gravida 4, para 0, abortion 3 (G4P0A3) woman at 34+0 gestational weeks (GW) visited our outpatient department with a 6-cm septate vaginal mass. Transvaginal ultrasound sonography (TVUS) revealed a 5.5 x 4.9-cm multicystic mass, which was confirmed as UD with pelvic MRI. She was admitted because of preterm labor. A cesarean section was performed at 36+5 GW due to a previous myomectomy, and a healthy male baby was born. UD was still observed in the patient two months after delivery. Periurethral diverticulectomy was performed, and pathological analysis revealed UD with chronic inflammation and edema. Conclusion: Previous reports and our case report show that UD can develop during pregnancy and that pelvic MRI is suitable for its accurate diagnosis. Vaginal delivery is possible in pregnant women with the small size of the UD. UD aspiration can permit vaginal delivery in a few cases; however, pus can occur at the aspirated site after the operation. If UD is still observed after delivery, urethral diverticulectomy is recommended.

Abstract Acinetobacter baumannii (AB) has emerged as a major pathogen in vulnerable and severely ill patients. It remains unclear whether early mortality (EM) due to AB bacteremia is because of worse clinical characteristics of the infected patients or the virulence of the pathogen. In this study, we aimed to investigate the effect of AB virulence on EM due to bacteremia. This retrospective study included 138 patients with AB bacteremia (age: ≥ 18 years) who were admitted to a tertiary care teaching hospital in South Korea between 2015 and 2019. EM was defined as death occurring within 7 days of bacteremia onset. The AB clinical isolates obtained from the patients’ blood cultures were injected into 15 Galleria mellonella larvae each, which were incubated for 5 days. Clinical isolates were classified into high- and low-virulence groups based on the number of dead larvae. Patients’ clinical data were combined and subjected to multivariate Cox regression analyses to identify the risk factors for EM. In total, 48/138 (34.8%) patients died within 7 days of bacteremia onset. The Pitt bacteremia score was the only risk factor associated with EM. In conclusion, AB virulence had no independent effect on EM in patients with AB bacteremia.

Purpose As introduced, multimodal pain management bundle for ileostomy reversal may be considered to reduce postoperative pain and hospital stay. The aim of this study was to evaluate clinical efficacy of perioperative multimodal pain bundle for ileostomy. Methods Medical records of patients who underwent ileostomy reversal after rectal cancer surgery from April 2017 to March 2020 were analyzed. Sixty-seven patients received multimodal pain bundle protocol with ileostomy reversal (group A) and 41 patients underwent closure of ileostomy with conventional pain management (group B). Results Baseline characteristics, including age, sex, body mass index, American Society of Anesthesiologists classification, diabetes mellitus, and smoking history, were not significantly different between the groups. The pain score on postoperative day 1 was significant lower in group A (visual analog scale, 2.6±1.3 vs. 3.2±1.2; P=0.013). Overall consumption of opioid in group A was significant less than group B (9.7±9.5 vs. 21.2±8.8, P

Jung-Hee Jang,1,&ast; Hyeong Joon Jun,2,&ast; ChaYoung Lee,3,&ast; Eunjin Sohn,4 Ojin Kwon,1 Dong-Hoon Kang,3 Muhammad Umar,2,5 In Chul Jung,3 Soo-Jin Jeong4 1Korean Medicine Science Research Division, Korea Institute of Oriental Medicine, Daejeon, Republic of Korea; 2Korean Medicine Data Division, Korea Institute of Oriental Medicine, Daejeon, Republic of Korea; 3Department of Oriental Neuropsychiatry, College of Korean Medicine, Daejeon University, Daejeon, Republic of Korea; 4Korean Medicine Convergence Research Division, Korea Institute of Oriental Medicine, Daejeon, Republic of Korea; 5Korean Convergence Medical Science, University of Science and Technology, Daejeon, Republic of Korea&ast;These authors contributed equally to this workCorrespondence: Soo-Jin Jeong, Korean Medicine Convergence Research Division, Korea Institute of Oriental Medicine, Daejeon, 34054, Republic of Korea, Tel +82-42-868-9651, Email sjijeong@kiom.re.kr In Chul Jung, Department of Oriental Neuropsychiatry, College of Korean Medicine, Daejeon University, Daejeon, 34520, Republic of Korea, Tel +82-42-470-9000, Email npjeong@dju.krPurpose: We aimed to investigate the efficacy of a combined herbal formula and electroacupuncture (EA) for mild cognitive impairment (MCI), a neurodegenerative disease leading to dementia, and its underlying mechanisms of action.Patients and Methods: This was a prospective open-label observational pilot study at Daejeon Korean Medicine Hospital of Daejeon University in South Korea from March 2022 to March 2023. We included six Korean patients (50% male) aged ≥ 45 years and < 85 years with MCI, a clinical dementia rating score of 0.5, and a Montreal Cognitive Assessment-Korea (MoCA-K) score ≤ 22. The exclusion criterion was impaired cognitive function. Patients received combined therapy, including a herbal formula and EA, for 12– 24 weeks. We prescribed the herbal formulas Gamiguibi-tang, Yukmijihwang-tang, and Banhasasim-tang to the patients for at least 70% of the treatment period, in combination with EA. Moreover, we investigated changes in cognitive and cognition-related symptoms and cytokine expression in the blood following combined traditional medicine therapy. At baseline and after 12 and 24 weeks, we administered the MoCA-K and cognitive-related questionnaires. We analyzed network pharmacology to reflect the herbal formula intervention mechanism comprehensively.Results: The median score [interquartile range] of MoCA-K at baseline was 19.5 [16.0, 22.0], which improved significantly (24.5 [24.0, 26.0], p < 0.01) over 24 weeks following combined therapy. We obtained no significant conclusion regarding cytokine changes due to the small sample size. In network pharmacology, we analyzed the brain, head, heart, peripheral nerves, peripheral nervous system, and pancreas as the enriched organs from the common targets of the three herbal formulas.Conclusion: Combined herbal medicine and EA improved cognitive function in patients with MCI. We assume the underlying mechanism of herbal formulas to be antioxidative and anti-inflammatory changes in cytokine expression. Combined traditional medicine has potential therapeutic application in preventing MCI progression to dementia.Plain Language Summary: This was a single-centered study focusing on the therapeutic effect of combined herbal medicine and electroacupuncture in patients with mild cognitive impairment, including a small number of participants, a relatively long treatment intervention of 12 weeks, and a follow-up assessment of 24 weeks. The intervention was a combination of a herbal formula and electroacupuncture treatment customized for each participant. The blood cytokine analyses of the participants were compared with the network analysis of the predicted target organs and pathways for the herbal formulas administered. Because each participant was not given the exact same intervention, we were unable to identify the specific treatment that produced the predicted effect. The observational study design of the study limited the ability to accurately assess causation between intervention and outcome. However, combined traditional medicine has potential therapeutic application in preventing mild cognitive impairment progression to dementia.Keywords: network pharmacology, inflammation, combination treatment, dementia

This study is the first to sequence the complete mitochondrial genome (mitogenome) of Perforatus perforatus Bruguière, 1789 (Balanomorpha: Balanidae). The 15,536-bp long P. perforatus mitogenome contained a typical set of animal mitochondrial genes, along with one control region. The P. perforatus mitogenome had an inverted gene block (trnP-ND4L-ND4-trnH-ND5-trnF) between trnS(gct) and trnT. This inverted gene block had been detected six species in three subfamilies of the Balanidae family (Balaninae, Acastinae and Megabalaninae), but our results show that it is also present in Concavinae, in which P. perforatus is included. The phylogenetic tree based on the concatenated sequences of the 13 protein-coding genes and two rRNA genes showed that P. perforatus is closely associated with Acasta sulcate and Balanus trigonus within Balanidae.

Abstract Hepatocellular carcinoma frequently recurs after surgery, necessitating personalized clinical approaches based on tumor avatar models. However, location-dependent oxygen concentrations resulting from the dual hepatic vascular supply drive the inherent heterogeneity of the tumor microenvironment, which presents challenges in developing an avatar model. In this study, tissue samples from 12 patients with hepatocellular carcinoma are cultured directly on a chip and separated based on preference of oxygen concentration. Establishing a dual gradient system with drug perfusion perpendicular to the oxygen gradient enables the simultaneous separation of cells and evaluation of drug responsiveness. The results are further cross-validated by implanting the chips into mice at various oxygen levels using a patient-derived xenograft model. Hepatocellular carcinoma cells exposed to hypoxia exhibit invasive and recurrent characteristics that mirror clinical outcomes. This chip provides valuable insights into treatment prognosis by identifying the dominant hepatocellular carcinoma type in each patient, potentially guiding personalized therapeutic interventions.

BackgroundMolecular screening using next-generation sequencing (NGS) in the pathologic evaluation of lung cancer is considered the standard in clinical practice; hence, we evaluated the diagnostic yields of various sampling methods for NGS.MethodsNGS data from patients with lung cancer at the Pusan National University Hospital (Busan, South Korea), admitted October, 2020–April, 2023, was obtained. The sampling methods by which NGS data was obtained were divided into surgical and nonsurgical. Surgical methods included thoracoscopic surgery, surgical biopsy from the metastatic site, and lymph node excisional biopsy, whereas nonsurgical methods included bronchoscopy procedures and medical thoracoscopic biopsy.ResultsIn total, we obtained 319 patients’ NGS data:150 (47.0%) and 169 (53.0%) was obtained using surgical and nonsurgical methods, respectively. The overall diagnostic yield of NGS analysis was 97.5% for all samples. There were no significant differences in the success rates of deoxyribonucleic acid sequencing between surgical and nonsurgical sampling methods (98.0% vs. 96.4%, p = 0.313). On the other hand, the success rate of ribonucleic acid (RNA) sequencing was significantly lower in the surgical method group (78.0% vs. 92.3%; p < 0.001). Multivariate analysis showed that surgical sampling significantly correlated with RNA sequencing failure (Odd Ratio 4.128, 95% Confidence Interval 1.681–10.133, p = 0.002).ConclusionsSmall samples obtained using nonsurgical procedures are suitable for NGS analysis in clinical practice. However, surgical sampling showed a relatively lower success rate for RNA sequencing than nonsurgical sampling. This information may help in the development of protocols to reduce RNA degradation during the surgical process.

Summary: Background: Interstitial lung disease (ILD) poses a serious threat in patients with rheumatoid arthritis (RA). However, the impact of cornerstone drugs, including methotrexate (MTX) and TNF inhibitor, on RA-associated ILD (RA-ILD) remains controversial. Methods: Using an SKG mouse model and single-cell transcriptomics, we investigated the effects of MTX and TNF blockade on ILD. Findings: Our study revealed that MTX exacerbates pulmonary inflammation by promoting immune cell infiltration, Th17 activation, and fibrosis. In contrast, TNF inhibitor ameliorates these features and inhibits ILD progression. Analysis of data from a human RA-ILD cohort revealed that patients with ILD progression had persistently higher systemic inflammation than those without progression, particularly among the subgroup undergoing MTX treatment. Interpretation: These findings highlight the need for personalized therapeutic approaches in RA-ILD, given the divergent outcomes of MTX and TNF inhibitor. Funding: This work was funded by GENINUS Inc., and the National Research Foundation of Korea, and Seoul National University Hospital.

Purpose: To investigate the relationship between urine cytology results after overnight continuous saline irrigation (OCSI) following transurethral resection of bladder tumor (TURBT) and bladder tumor recurrence in non-muscle invasive bladder cancer (NMIBC). Materials and Methods: A retrospective study was conducted on patients diagnosed with NMIBC between 2016 and 2020 after undergoing TURBT at our hospital. All patients received OCSI following TURBT and had urine cytology test at postoperative 1 day. Urine cytology was classified into three groups: Negative, low-grade urothelial neoplasm (LGUN)+atypical urothelial cells (AUC), and suspicious for high-grade urothelial carcinoma (SHGUC)+high-grade urothelial carcinoma (HGUC). Recurrence-free survival (RFS) in each group was compared using the Kaplan–Meier method. Univariable and multivariable Cox regression analyses were performed to evaluate independent prognostic factors. Results: A total of 172 patients were included in this study. Based on urine cytology group (after OCSI), RFS did not reach the median value in the Negative group. In the LGUN+AUC group, the median RFS was 615.00 days. In the SHGUC+HGUC group, the median RFS was 377.00 days. In survival analysis, the Negative group had a longer RFS than the SHGUC+HGUC group (p=0.013). However, Cox regression analysis showed that SHGUC+HGUC was not an independent prognostic factor for recurrence. Conclusions: Urine cytology results after OCSI following TURBT in NMIBC were associated with bladder tumor recurrence. Specifically, SHGUC or HGUC in urine cytology after OCSI showed earlier recurrence than negative cases. However, further research is needed to accurately determine whether it is an independent prognostic factor.

Abstract Heart disease involves irreversible myocardial injury that leads to high morbidity and mortality rates. Numerous cell-based cardiac in vitro models have been proposed as complementary approaches to non-clinical animal research. However, most of these approaches struggle to accurately replicate adult human heart conditions, such as myocardial infarction and ventricular remodeling pathology. The intricate interplay between various cell types within the adult heart, including cardiomyocytes, fibroblasts, and endothelial cells, contributes to the complexity of most heart diseases. Consequently, the mechanisms behind heart disease induction cannot be attributed to a single-cell type. Thus, the use of multi-cellular models becomes essential for creating clinically relevant in vitro cell models. This study focuses on generating self-organizing heart organoids (HOs) using human-induced pluripotent stem cells (hiPSCs). These organoids consist of cardiomyocytes, fibroblasts, and endothelial cells, mimicking the cellular composition of the human heart. The multi-cellular composition of HOs was confirmed through various techniques, including immunohistochemistry, flow cytometry, q-PCR, and single-cell RNA sequencing. Subsequently, HOs were subjected to hypoxia-induced ischemia and ischemia-reperfusion (IR) injuries within controlled culture conditions. The resulting phenotypes resembled those of acute myocardial infarction (AMI), characterized by cardiac cell death, biomarker secretion, functional deficits, alterations in calcium ion handling, and changes in beating properties. Additionally, the HOs subjected to IR efficiently exhibited cardiac fibrosis, displaying collagen deposition, disrupted calcium ion handling, and electrophysiological anomalies that emulate heart disease. These findings hold significant implications for the advancement of in vivo-like 3D heart and disease modeling. These disease models present a promising alternative to animal experimentation for studying cardiac diseases, and they also serve as a platform for drug screening to identify potential therapeutic targets.