Guidelines for Manufacturing and Application of Organoids: Liver.

Recent amendments to regulatory frameworks have placed a greater emphasis on the utilization of in vitro testing platforms for preclinical drug evaluations and toxicity assessments. This requires advanced tissue models capable of accurately replicating liver functions for drug efficacy and toxicity predictions. Liver organoids, derived from human cell sources, offer promise as a reliable platform for drug evaluation. However, there is a lack of standardized quality evaluation methods, which hinders their regulatory acceptance. This paper proposes comprehensive quality standards tailored for liver organoids, addressing cell source validation, organoid generation, and functional assessment. These guidelines aim to enhance reproducibility and accuracy in toxicity testing, thereby accelerating the adoption of organoids as a reliable alternative or complementary tool to animal testing in drug development. The quality standards include criteria for size, cellular composition, gene expression, and functional assays, thus ensuring a robust hepatotoxicity testing platform.

Guidelines for Packaging, Transport, and Storage of Source Cells for Organoids.

This paper presents guidelines for the systematic management of packaging, storage, transportation, and traceability of source cells used for organoid research. Given the important role of source cells in organoid studies, it is important to ensure the preservation of their quality and integrity throughout transportation and distribution processes. The proposed guidelines, therefore, call for a cohesive strategy through these stages to minimize the risks of contamination, deterioration, and loss-threats that significantly compromise the safety, efficacy, and efficiency of source cells. Central to these guidelines is the quality control measures that include roles and responsibilities across the entire supply chain, with recommendations specific to packaging materials, transportation facilities, and storage management. Furthermore, the need for an integrated management system is emphasized, spanning from source cell collection to the final application. This system is crucial for maintaining the traceability and accountability of source cells, facilitating the sharing, distribution, and utilization on a global scale, and supporting to advance organoid research and development.

Guidelines for Manufacturing and Application of Organoids: Skin.

To address the limitations of animal testing, scientific research is increasingly focused on developing alternative testing methods. These alternative tests utilize cells or tissues derived from animals or humans for in vitro testing, as well as artificial tissues and organoids. In western countries, animal testing for cosmetics has been banned, leading to the adoption of artificial skin for toxicity evaluation, such as skin corrosion and irritation assessments. Standard guidelines for skin organoid technology becomes necessary to ensure consistent data and evaluation in replacing animal testing with in vitro methods. These guidelines encompass aspects such as cell sourcing, culture techniques, quality requirements and assessment, storage and preservation, and organoid-based assays.

Virtual fitness buddy ecosystem: a mixed reality precision health physical activity intervention for children.

6-11-year-old children provide a critical window for physical activity (PA) interventions. The Virtual Fitness Buddy ecosystem is a precision health PA intervention for children integrating mixed reality technology to connect people and devices. A cluster randomized, controlled trial was conducted across 19 afterschool sites over two 6-month cohorts to test its efficacy in increasing PA and decreasing sedentary behavior. In the treatment group, a custom virtual dog via a mixed reality kiosk helped children set PA goals while sharing progress with parents to receive feedback and support. Children in the control group set PA goals using a computer without support from the virtual dog or parents. 303 children had 8+ hours of PA data on at least one day of each of the 3 intervention time intervals. Conversion of sedentary time was primarily to light-intensity PA and was strongest for children with low baseline moderate-to-vigorous PA than children above 45 min of baseline moderate-to-vigorous PA. Findings suggest that the VFB ecosystem can promote sustainable PA in children and may be rapidly diffused for widespread public health impact.

Guidelines for Manufacturing and Application of Organoids: Lung.

The objective of standard guideline for utilization of human lung organoids is to provide the basic guidelines required for the manufacture, culture, and quality control of the lung organoids for use in non-clinical efficacy and inhalation toxicity assessments of the respiratory system. As a first step towards the utilization of human lung organoids, the current guideline provides basic, minimal standards that can promote development of alternative testing methods, and can be referenced not only for research, clinical, or commercial uses, but also by experts and researchers at regulatory institutions when assessing safety and efficacy.

Guidelines for Manufacturing and Application of Organoids: Heart.

Cardiac organoids have emerged as invaluable tools for assessing the impact of diverse substances on heart function. This report introduces guidelines for general requirements for manufacturing cardiac organoids and conducting cardiac organoid-based assays, encompassing protocols, analytical methodologies, and ethical considerations. In the quest to employ recently developed three-dimensional cardiac organoid models as substitutes for animal testing, it becomes imperative to establish robust criteria for evaluating organoid quality and conducting toxicity assessments. This guideline addresses this need, catering to regulatory requirements, and describes common standards for organoid quality and toxicity assessment methodologies, commensurate with current technological capabilities. While acknowledging the dynamic nature of technological progress and the potential for future comparative studies, this guideline serves as a foundational framework. It offers a comprehensive approach to standardized cardiac organoid testing, ensuring scientific rigor, reproducibility, and ethical integrity in investigations of cardiotoxicity, particularly through the utilization of human pluripotent stem cell-derived cardiac organoids.

Guidelines for Manufacturing and Application of Organoids: Brain.

This study offers a comprehensive overview of brain organoids for researchers. It combines expert opinions with technical summaries on organoid definitions, characteristics, culture methods, and quality control. This approach aims to enhance the utilization of brain organoids in research. Brain organoids, as three-dimensional human cell models mimicking the nervous system, hold immense promise for studying the human brain. They offer advantages over traditional methods, replicating anatomical structures, physiological features, and complex neuronal networks. Additionally, brain organoids can model nervous system development and interactions between cell types and the microenvironment. By providing a foundation for utilizing the most human-relevant tissue models, this work empowers researchers to overcome limitations of two-dimensional cultures and conduct advanced disease modeling research.

Essential Guidelines for Manufacturing and Application of Organoids.

An organoid is a self-organized three-dimensional structure derived from stem cells that mimics the structure, cell composition, and functional characteristics of specific organs and tissues and is used for evaluating the safety and effectiveness of drugs and the toxicity of industrial chemicals. Organoid technology is a new methodology that could replace testing on animals testing and accelerate development of precision and regenerative medicine. However, large variations in production can occur between laboratories with low reproducibility of the production process and no internationally agreed standards for quality evaluation factors at endpoints. To overcome these barriers that hinder the regulatory acceptance and commercialization of organoids, Korea established the Organoid Standards Initiative in September 2023 with various stakeholders, including industry, academia, regulatory agencies, and standard development experts, through public and private partnerships. This developed general guidelines for organoid manufacturing and quality evaluation and for quality evaluation guidelines for organoid-specific manufacturing for the liver, intestines, and heart through extensive evidence analysis and consensus among experts. This report is based on the common standard guideline v1.0, which is a general organoid manufacturing and quality evaluation to promote the practical use of organoids. This guideline does not focus on specific organoids or specific contexts of use but provides guidance to organoid makers and users on materials, procedures, and essential quality assessment methods at end points that are essential for organoid production applicable at the current technology level.

Guidelines for Manufacturing and Application of Organoids: Kidney.

Recent advancements in organoid technology have led to a vigorous movement towards utilizing it as a substitute for animal experiments. Organoid technology offers versatile applications, particularly in toxicity testing of pharmaceuticals or chemical substances. However, for the practical use in toxicity testing, minimal guidance is required to ensure reliability and relevance. This paper aims to provide minimal guidelines for practical uses of kidney organoids derived from human pluripotent stem cells as a toxicity evaluation model in vitro.

Pseudomonas aeruginosa N-3-Oxododecanoyl Homoserine Lactone Disrupts Endothelial Integrity by Activating the Angiopoietin-Tie System.

The activation of the angiopoietin (Angpt)-Tie system is linked to endothelial dysfunction during sepsis. Bacterial quorum-sensing molecules function as pathogen-associated molecular patterns. However, their impact on the endothelium and the Angpt-Tie system remains unclear. Therefore, this study investigated whether treatment with N-3-oxododecanoyl homoserine lactone (3OC12-HSL), a quorum-sensing molecule derived from Pseudomonas aeruginosa, impaired endothelial function in human umbilical vein endothelial cells. 3OC12-HSL treatment impaired tube formation even at sublethal concentrations, and immunocytochemistry analysis revealed that it seemed to reduce vascular endothelial-cadherin expression at the cell-cell interface. Upon assessing the mRNA expression patterns of genes associated with the Angpt-Tie axis, the expressions of Angpt2, Forkhead box protein O1, Tie1, and vascular endothelial growth factor 2 were found to be upregulated in the 3OC12-HSL-treated cells. Moreover, western blot analysis revealed that 3OC12-HSL treatment increased Angpt2 expression. A co-immunoprecipitation assay was conducted to assess the effect of 3OC12-HSL on the IQ motif containing GTPase activating protein 1 (IQGAP1) and Rac1 complex and the interaction between these proteins was consistently maintained regardless of 3OC12-HSL treatment. Next, recombinant human (rh)-Angpt1 was added to assess whether it modulated the effects of 3OC12-HSL treatment. rh-Angpt1 addition increased cellular viability, improved endothelial function, and reversed the overall patterns of mRNA and protein expression in endothelial cells treated with 3OC12-HSL. Additionally, it was related to the increased expression of phospho-Akt and the IQGAP1 and Rac1 complex. Collectively, our findings indicated that 3OC12-HSL from Pseudomonas aeruginosa can impair endothelial integrity via the activation of the Angpt-Tie axis, which appeared to be reversed by rh-Angpt1 treatment.

De-biased Disentanglement Learning for Pulmonary Embolism Survival Prediction on Multimodal Data.

Health disparities among marginalized populations with lower socioeconomic status significantly impact the fairness and effectiveness of healthcare delivery. The increasing integration of artificial intelligence (AI) into healthcare presents an opportunity to address these inequalities, provided that AI models are free from bias. This paper aims to address the bias challenges by population disparities within healthcare systems, existing in the presentation of and development of algorithms, leading to inequitable medical implementation for conditions such as pulmonary embolism (PE) prognosis. In this study, we explore the diversity of biases in healthcare systems, which highlights the need for a holistic framework to reduce bias by complementary aggregation. By leveraging de-biasing deep survival prediction models, we propose a framework that disentangles identifiable information from images, text reports, and clinical variables to mitigate potential biases within multimodal datasets. Our study offers several advantages over traditional clinical-based survival prediction methods, including richer survival-related characteristics and bias-complementary predicted results. By improving the robustness of survival analysis through this framework, we aim to benefit patients, clinicians, and researchers by improving fairness and accuracy in healthcare AI systems. The code is available at https://github.com/zzs95/fairPE-SA.

Dual-targeted nano-encapsulation of neonatal porcine islet-like cell clusters with triiodothyronine-loaded bifunctional polymersomes.

There is growing evidence that neonatal porcine islet-like cell clusters (NPCCs) isolated from piglets can be used to treat type 1 diabetes in humans. However, graft rejection is a common complication in humans owing to the prevalence of xenoantigens in porcine. Therefore, researchers have investigated various islet encapsulation techniques that could protect against these antigens. To this end, this study presents a robust nano-encapsulation method based on bifunctional polymersomes (PSomes), in which N-hydroxysuccinimide (NHS) and maleimide (Mal) groups conjugated to the PSomes terminal interact with the amine and thiol groups on the surface of NPCCs to induce dual targeting via two covalent bonds. The findings indicate that the ratio of NHS to Mal on PSomes is optimal for dual targeting. Moreover, triiodothyronine (T3) is known to promotes pancreatic islet maturation and differentiation of endocrine cells into beta cells. T3 encapsulated in PSomes is shown to increase the glucose sensitivity of NPCCs and enhance insulin secretion from NPCCs. Furthermore, improvements in the nano-encapsulation efficiency and insulin-secreting capability of NPCCs through dual targeting via dual-Psomes are demonstrated. In conclusion, the proposed nano-encapsulation technique could pave the way for significant advances in islet nano-encapsulation and the imprevement of NPCC immaturity via T3 release.

Rapid quantitative PCR equipment using photothermal conversion of Au nanoshell.

The emergence of infectious diseases worldwide necessitates rapid and precise diagnostics. Using gold nanoshells in the PCR mix, we harnessed their unique photothermal properties in the near-infrared regime to attain efficient heating, reaching ideal photothermal PCR cycle temperature profile. Our photothermal PCR method expedited DNA amplification while retaining its detection sensitivity. Combining photothermal quantitative PCR with real-time fluorometry and non-invasive temperature measurement, we could amplify the target DNA within just 25 min, with a minimum detectable DNA amount of 50 picograms. This innovation in photothermal qPCR, leveraging the photothermal properties of gold nanoshells, will pave the way for immediate point-of-care diagnostics of nucleic acid biomarkers.

Rapid Antibacterial Activity Assessment of Chimeric Lysins.

Various chimeric lysins have been developed as efficacious antibiotics against multidrug-resistant bacteria, but direct comparisons of their antibacterial activities have been difficult due to the preparation of multiple recombinant chimeric lysins. Previously, we reported an Escherichia coli cell-free expression method to better screen chimeric lysins against Staphylococcus aureus, but we still needed to increase the amounts of expressed proteins enough to be able to detect them non-isotopically for quantity comparisons. In this study, we improved the previous cell-free expression system by adding a previously reported artificial T7 terminator and reversing the different nucleotides between the T7 promoter and start codon to those of the T7 phage. The new method increased the expressed amount of chimeric lysins enough for us to detect them using Western blotting. Therefore, the qualitative comparison of activity between different chimeric lysins has become possible via the adjustment of the number of variables between samples without protein purification. We applied this method to select more active chimeric lysins derived from our previously reported chimeric lysin (ALS2). Finally, we compared the antibacterial activities of our selected chimeric lysins with reported chimeric lysins (ClyC and ClyO) and lysostaphin and determined the rank orders of antibacterial activities on different Staphylococcus aureus strains in our experimental conditions.

The Application of Large Language Models for Radiologic Decision Making.

BACKGROUND AND PURPOSE: Large language models (LLMs) have seen explosive growth, but their potential role in medical applications remains underexplored. Our study investigates the capability of LLMs to predict the most appropriate imaging study for specific clinical presentations in various subspecialty areas in radiology. METHODS AND MATERIALS: Chat Generative Pretrained Transformer (ChatGPT), by OpenAI and Glass AI by Glass Health were tested on 1,075 clinical scenarios from 11 ACR expert panels to determine the most appropriate imaging study, benchmarked against the ACR Appropriateness Criteria. Two responses per clinical presentation were generated and averaged for the final clinical presentation score. Clinical presentation scores for each topic area were averaged as its final score. The average of the topic scores within a panel determined the final score of each panel. LLM responses were on a scale of 0 to 3. Partial scores were given for nonspecific answers. Pearson correlation coefficient (R-value) was calculated for each panel to determine a context-specific performance. RESULTS: Glass AI scored significantly higher than ChatGPT (2.32 ± 0.67 versus 2.08 ± 0.74, P = .002). Both LLMs performed the best in the Polytrauma, Breast, and Vascular panels, and performed the worst in the Neurologic, Musculoskeletal, and Cardiac panels. Glass AI outperformed ChatGPT in 10 of 11 panels, except Obstetrics and Gynecology. Maximum agreement was in the Pediatrics, Neurologic, and Thoracic panels, and the most disagreement occurred in the Vascular, Breast, and Urologic panels. CONCLUSION: LLMs can be used to predict imaging studies, with Glass AI's superior performance indicating the benefits of extra medical-text training. This supports the potential of LLMs in radiologic decision making.

Is There a Difference in the Incidence of Depression between Radiation and Surgical Treatments in Patients with Prostate Cancer?

PURPOSE: Patients with cancer have a high risk of depression. However, a few studies have assessed differences in the incidence of depression among patients with prostate cancer (PC) based on whether they received radiotherapy (RTx) or surgical treatment. MATERIALS AND METHODS: We analyzed data from the National Health Insurance Sharing Service database regarding the entire Korean adult population with PC (n=210,924) between 2007 and 2017. The adjusted hazard ratios (HRs) of depression associated with treatment were estimated using propensity score-matched Cox proportional hazards models and Kaplan-Meier survival analyses. RESULTS: Our final cohort comprised 9,456 patients with PC; of which, 8,050 men underwent surgery. During a mean follow-up duration of 7.1 years, 503 (5.3%) patients were newly diagnosed with depression. A significant difference in the incidence of depression was noted between the RTx and surgery groups (RTx vs. surgery: 5.55% vs. 5.28%; p=0.011) in the unmatched cohort. In the matched cohort, older age (≥70 years, HR: 1.596, p<0.001) and poor Charlson comorbidity index scores (HR: 1.232, p=0.039) were correlated with the risk of depression. In addition, the adjusted HR for depression in the surgery group was 0.843 (p=0.221) compared with that in the RTx group. Kaplan-Meier analyses revealed that no significant difference in the cumulative probability of persistent depression was detected between the RTx and surgery groups in matched cohort (p=0.3386). CONCLUSIONS: In this nationwide population-based study, no significant differences in the risk of depression were observed between the surgical and RTx groups.

Effects of G and SH Truncation on the Replication, Virulence, and Immunogenicity of Avian Metapneumovirus.

Four mutants varying the length of the G and SH genes, including a G-truncated mutant (ΔG) and three G/SH-truncated mutants (ΔSH/G-1, ΔSH/G-2, and ΔSH/G-3), were generated via serially passaging the avian metapneumovirus strain SNU21004 into the cell lines Vero E6 and DF-1 and into embryonated chicken eggs. The mutant ΔG particles resembled parental virus particles except for the variance in the density of their surface projections. G and G/SH truncation significantly affected the viral replication in chickens' tracheal ring culture and in infected chickens but not in the Vero E6 cells. In experimentally infected chickens, mutant ΔG resulted in the restriction of viral replication and the attenuation of the virulence. The mutants ΔG and ΔSH/G-1 upregulated three interleukins (IL-6, IL-12, and IL-18) and three interferons (IFNα, IFNß, and IFNγ) in infected chickens. In addition, the expression levels of innate immunity-related genes such as Mda5, Rig-I, and Lgp2, in BALB/c mice were also upregulated when compared to the parental virus. Immunologically, the mutant ΔG induced a strong, delayed humoral immune response, while the mutant ΔSH/G-1 induced no humoral immune response. Our findings indicate the potential of the mutant ΔG but not the mutant ΔSH/G-1 as a live attenuated vaccine candidate.

Adjunctive effect of 470-nm and 630-nm light-emitting diode irradiation in experimental periodontitis treatment: a preclinical study.

PURPOSE: This study investigated the adjunctive effect of light-emitting diodes (LEDs) in the treatment of experimental periodontitis. METHODS: Experimental periodontitis was induced by placing ligatures around the mandibular second, third, and fourth premolars of 6 beagles for 3 months. After ligature removal, periodontitis progressed spontaneously for 2 months. The animals' hemimandibles were allocated among the following 3 groups: 1) no treatment (control), 2) scaling and root planing (SRP), and 3) SRP with LED irradiation at 470-nm and 630-nm wavelengths (SRP/LED). The probing pocket depth (PPD) and gingival recession (GR) were measured at baseline, 6 weeks, and 12 weeks. The clinical attachment level (CAL) was calculated. After 12 weeks, histological and histomorphometric assessments were performed. The distances from the gingival margin to the apical extent of the junctional epithelium (E) and to the connective tissue (CT) attachment were measured, as was the total length of soft tissue (ST). RESULTS: PPD and CAL increased at 12 weeks compared with baseline in the control group (6.31±0.43 mm to 6.93±0.50 mm, and 6.46±0.60 mm to 7.61±0.78 mm, respectively). PPD and CAL decreased at 12 weeks compared with baseline in the SRP group (6.01±0.59 to 4.81±0.65 mm, and 6.51±0.98 to 5.39±0.93 mm, respectively). PPD and CAL decreased at 12 weeks compared with baseline in the SRP/LED group (6.03±0.39 to 4.46±0.47 mm, and 6.11±0.47 to 4.78±0.57 mm, respectively). The E/ST and CT/ST ratios significantly differed among the 3 groups (P<0.05). The clinical parameters and histologic findings demonstrated that 470-nm and 630-nm wavelength LED irradiation accompanying SRP could improve treatment results. CONCLUSIONS: Within the study limitations, 470 nm and 630 nm wavelength LED irradiation might provide additional benefits for periodontitis treatment.

Cell Death Induced by the Combination of Ephedra sinica Extract and Radiation in HNSCC is Positively Related to BAX and p-MLKL Expression.

BACKGROUND: Numerous studies have proven the efficacy and safety of natural products, and are widely used as attractive cancer treatments. The investigation of effective natural products for improving cancer treatment is a promising strategy. Combination treatment with radiosensitizers and radiotherapy (RT) is considered necessary for therapeutic improvement in head and neck squamous cell carcinoma(HNSCC). OBJECTIVE: This study aims to investigate whether Ephedra sinica (ES) extract could induce selective cell death in cancer cells and serve as a radiosensitizer for HNSCC. METHODS: HNSCC cells were pretreated with ES extract before radiation, and the radiosensitizing activity was assessed using a colony formation assay. Radiation-induced cell death was evaluated using an annexinV-FITC assay. Western blotting was performed to confirm cell death-related gene expression, including apoptosis and necrosis markers. RESULTS: ES extract significantly inhibited HNSCC cell viability (FaDu and SNU1076), while having minimal effect on normal HaCaT cells. When HNSCC cells were irradiated with 2, 4, or 8 Gy and cultured with ES extract (25 µg/mL), they exhibited increased radiation sensitivity compared to non-treated cells. The combination of ES extract and radiation resulted in increased cell death compared to non-treated, ES-treated, or irradiated cells. The apoptosis marker BAX and necrosis marker p-MLKL expression levels were also elevated following the combination treatment. CONCLUSION: ES extract demonstrated significant cytotoxic potential in HNSCC cells without affecting normal cells. It enhanced the radiosensitivity of HNSCC cells by upregulating BAX and p-MLKL expression, leading to increased cell death. These results suggest ES extract exhibits a potential radiosensitizing capacity in HNSCC.

Tracing the Evolutionary Pathways of Serogroup O78 Avian Pathogenic Escherichia coli.

Avian pathogenic E. coli (APEC) causes severe economic losses in the poultry industry, and O78 serogroup APEC strains are prevalent in chickens. In this study, we aimed to understand the evolutionary pathways and relationships between O78 APEC and other E. coli strains. To trace these evolutionary pathways, we classified 3101 E. coli strains into 306 subgenotypes according to the numbers and types of single nucleotide polymorphisms (RST0 to RST63-1) relative to the consensus sequence (RST0) of the RNA polymerase beta subunit gene and performed network analysis. The E. coli strains showed four apparently different evolutionary pathways (I-1, I-2, I-3, and II). The thirty-two Korean O78 APEC strains tested in this study were classified into RST4-4 (45.2%), RST3-1 (32.3%), RST21-1 (12.9%), RST4-5 (3.2%), RST5-1 (3.2%), and RST12-6 (3.2%), and all RSTs except RST21-1 (I-2) may have evolved through the same evolutionary pathway (I-1). A comparative genomic study revealed the highest relatedness between O78 strains of the same RST in terms of genome sequence coverage/identity and the spacer sequences of CRISPRs. The early-appearing RST3-1 and RST4-4 prevalence among O78 APEC strains may reflect the early settlement of O78 E. coli in chickens, after which these bacteria accumulated virulence and antibiotic resistance genes to become APEC strains. The zoonotic risk of the conventional O78 APEC strains is low at present, but the appearance of genetically distinct and multiple virulence gene-bearing RST21-1 O78 APEC strains may alert us to a need to evaluate their virulence in chickens as well as their zoonotic risk.