[TSR2 overexpression inhibits proliferation and invasion of gastric cancer cells by downregulating the PI3K/AKT signaling pathway].

OBJECTIVE: To investigate the expression of TSR2 in gastric cancer and explore its correlation with progression of gastric cancer and the possible mechanism. METHODS: We retrospectively analyzed TSR2 expression in clinical specimens from 105 gastric cancer patients and the impact of TSR2 expression level on disease progression and 5-year postoperative survival of the patients. GO and KEGG enrichment analyses were used to predict the biological functions and mechanisms of TSR2. In gastric cancer MGC-803 cells with lentivirus-mediated TSR2 overexpression or knockdown, the changes in cell proliferation, invasion, and migration were assessed with CCK-8 and Transwell assays, and the expressions of p-PI3K and p-AKT were detected using Western blotting. RESULTS: TSR2 expression was significantly lower in gastric cancer tissues than in the adjacent tissues with significant correlations with CEA level, CA19-9 level, and T and N staging (P < 0.05). A low TSR2 expression, CEA≥5 µg/L, CA19-9≥37 kU/L, T3-T4 stages, and N2-N3 staged were identified as independent risk factors affecting 5-year survival rate of the patients following radical surgery (P < 0.05), and a high TSR2 expression was associated with a higher 5-year survival rate of the patients (P < 0.001). Bioinformatics analysis suggested the functional involvement of TSR2 with the PI3K/AKT signaling pathway. MGC-803 cells overexpressing TSR2 showed significantly lowered proliferation, migration, and invasion capacities (P < 0.05), while TSR2 knockdown produced the opposite effects (P < 0.05). Western blotting showed that TSR2 overexpression reduced the phosphorylation of PI3K and AKT, and TSR2 knockdown caused the opposite changes in MGC-803 cells (P < 0.05). CONCLUSION: TSR2 is lowly expressed in gastric cancer tissues to adversely affect the patients' prognosis, and its overexpression inhibits gastric cancer cell proliferation, invasion, and migration possibly by downregulating the PI3K/AKT pathway.

Mapping nationwide concentrations of sulfate and nitrate in ambient PM2.5 in South Korea using machine learning with ground observation data.

Particulate matter (PM) is a major air pollutant in Northeast Asia, with frequent high PM episodes. To investigate the nationwide spatial distribution maps of PM2.5 and secondary inorganic aerosols in South Korea, prediction models for mapping SO42- and NO3- concentrations in PM2.5 were developed using machine learning with ground-based observation data. Specifically, the random forest algorithm was used in this study to predict the SO42- and NO3- concentrations at 548 air quality monitoring stations located within the representative radii of eight intensive air quality monitoring stations. The average concentrations of PM2.5, SO42-, and NO3- across the entire nation were 17.2 ± 2.8, 3.0 ± 0.6, and 3.4 ± 1.2 µg/m3, respectively. The spatial distributions of SO42- and NO3- concentrations in 2021 revealed elevated concentrations in both the western and central regions of South Korea. This result suggests that SO42- concentrations were primarily influenced by industrial activities rather than vehicle emissions, whereas NO3- concentrations were more associated with vehicle emissions. During a high PM2.5 event (November 19-21, 2021), the concentration of SO42- was primarily influenced by SOX emissions from China, while the concentration of NO3- was affected by NOX emissions from both China and Korea. The methodology developed in this study can be used to explore the chemical characteristics of PM2.5 with high spatiotemporal resolution. It can also provide valuable insights for the nationwide mitigation of secondary PM2.5 pollution.

[Overexpression of BZW1 promotes invasion and metastasis of gastric cancer cells by regulating Wnt/ß-catenin signaling and promoting epithelial-mesenchymal transition].

OBJECTIVE: To investigate the expression level of basic leucine zipper and W2 domain-containing protein 1 (BZW1) in gastric cancer, its impact on patient prognosis and the underlying mechanisms. METHODS: TIMER, UALCAN and Kaplan-Meier Plotter databases were used for analyzing BZW1 expression level gastric cancer tissues and its correlation with tumor grade and stage and the patients' prognosis. We further analyzed BZW1 expressions, disease progression, and postoperative 5-year survival in 102 patients undergoing radical surgery for gastric cancer at our hospital between January, 2014 and December, 2016. Gastric cancer MGC803 cells were examined for changes in migration, invasion, and epithelial-mesenchymal transition (EMT) following lentivirus-mediated BZW1 overexpression or knockdown. RESULTS: The protein and mRNA expressions of BZW1 in gastric cancer tissues were 3.30 and 6.54 times of those in adjacent tissues, respectively (P < 0.01). BZW1 expression in gastric cancer tissues were positively correlated with peripheral blood CEA and CA199 levels (P < 0.01). A high BZW1 expression was an independent risk factor for 5-year survival of gastric cancer patients after radical surgery (P < 0.05, HR=2.070, 95%CI: 1.021-4.196). At the cut-off value of 3.61, BZW1 expression had a sensitivity of 75.56% and a specificity of 71.93% for predicting postoperative 5-year mortality (P < 0.01). In MGC803 cells, BZW1 overexpression obviously promoted cell migration and invasion (P < 0.05), enhanced cellular expressions of N-cadherin and vimentin (P < 0.05) and inhibited the expression of E-cadherin (P < 0.05). Enrichment analysis suggested the involvement of BZW1 in the Wnt/ß-catenin signaling pathway. Western blotting confirmed that BZW1 overexpression promoted while BZW1 knockdown inhibited the expressions of Wnt3a, ß-catenin and C-myc in MGC803 cells (P < 0.05). CONCLUSION: BZW1 is highly expressed in gastric cancer tissues to affect the patient prognosis possibly by activation the Wnt/ß-catenin signaling pathway to promote EMT of gastric cancer cells.

Evidence of the Role of Inflammation and the Hormonal Environment in the Pathogenesis of Adrenal Myelolipomas in Congenital Adrenal Hyperplasia.

Adrenal myelolipomas (AML) are composed of mature adipose and hematopoietic components. They represent approximately 3 percent of adrenal tumors and are commonly found in patients with congenital adrenal hyperplasia (CAH). CAH provides a unique environment to explore AML pathogenesis. We aimed to evaluate the role of the immune system and hormones that accumulate in poorly controlled CAH in the development of AML. When compared to normal adrenal tissue, CAH-affected adrenal tissue and myelolipomas showed an increased expression of inflammatory cells (CD68, IL2Rbeta), stem cells (CD117) B cells (IRF4), and adipogenic markers (aP2/FABP4, AdipoQ, PPARγ, Leptin, CideA), and immunostaining showed nodular lymphocytic accumulation. Immunohistochemistry staining revealed a higher density of inflammatory cells (CD20, CD3, CD68) in CAH compared to non-CAH myelolipomas. In vitro RNA-sequencing studies using NCI-H295R adrenocortical cells with exogenous exposure to ACTH, testosterone, and 17-hydroxyprogesterone hormones, showed the differential expression of genes involved in cell cycle progression, phosphorylation, and tumorigenesis. Migration of B-lymphocytes was initiated after the hormonal treatment of adrenocortical cells using the Boyden chamber chemotaxis assay, indicating a possible hormonal influence on triggering inflammation and the development of myelolipomas. These findings demonstrate the important role of inflammation and the hormonal milieu in the development of AML in CAH.

Validation of Korean Olfactory Questionnaire for Perioperative Olfactory Assessment in Endoscopic Endonasal Skull Base Surgery.

Objective: To analyze changes in olfactory function after endoscopic endonasal skull base surgery and compare performance of the olfactory questionnaire with those of conventional psychophysical tests. Methods: Patients were classified into 5 categories for olfactory function evaluation (normal, mild hyposmia, moderate hyposmia, severe hyposmia, and anosmia) based on a self-assessment. Patients also underwent the butanol threshold test (BTT), Cross-Cultural Smell Identification Test (CCSIT), and 11-item olfactory questionnaire. Subjects with normosmia preoperatively and who were followed up at least 6 months after surgery were analyzed. Receiver operating characteristic curves and confusion matrix analysis were performed for BTT, CCSIT, and olfactory questionnaire to compare their diagnostic abilities. The effects of age, preoperative olfaction, septal flap, tumor pathology, and tumor size on postoperative olfaction were evaluated using multivariate linear regression analysis. Results: Data from 108 patients were analyzed. Postoperative changes in the olfactory questionnaire were significantly associated with changes in the BTT and CCSIT. The area under the curve for postoperative self-olfactory function classification was highest for olfactory questionnaire (0.894), followed by BTT (0.767) and CCSIT (0.688). Patient age at the time of surgery and preoperative BTT score were significantly related to postoperative olfactory outcomes. Conclusion: The olfactory questionnaire correlated well with conventional psychosomatic olfactory function tests. In combination with clinical parameters and preoperative psychosomatic olfactory function tests, the olfactory questionnaire is suitable for assessing subjective olfactory function after endoscopic endonasal skull base surgery.

Effects of long-range transport on carboxylic acids, chlorinated VOCs, and oxidative potential in air pollution events.

In the context of air quality research, the collection and analysis of fine particulate matter (PM2.5, with a diameter less than 2.5 µm) and volatile organic compound (VOCs) play a pivotal role in understanding and addressing environmental issues across the Korean Peninsula. PM2.5 and VOCs were collected over 4-hr intervals from October 17 to November 26, 2021 during the 2021 Satellite Integrated Joint Monitoring of Air Quality campaign at Olympic Park in the Republic of Korea to understand the factors controlling air quality over the Seoul Metropolitan Area. Source apportionment was performed using the positive matrix factorization (PMF) model incorporating PM2.5 and VOCs. The factor identified by chlorinated VOCs as a major component was presumed to be due to transboundary influx and was referred to as the long-range transport factor. The long-range transport factor of PM2.5 was composed of NO3-, SO42-, NH4+, and di-carboxylic acids. Back trajectory analysis showed that the airflows originated from China and passed through the west coast of Korea to the Korean Peninsula. In the PMF results using PM2.5 and VOCs, long-range transport factors were identified in both analyses, and the high correlation observed between these factors confirms that they were transported from abroad. The dithiothreitol oxidation potential normalized to quinine showed the highest oxidation potential during the same period as the long-range transport factors increased. In conclusion, PM2.5 from external sources significantly contribute to elevated levels of dithiothreitol assay-oxidative potential (DTT-OP) in Korea. The toxic concentration, expressed as the mean ± standard deviation, was determined to be 0.29 ± 0.05 µM/m³, peaking at 0.39 µM/m³. This level is 1.8 times higher than that observed outside the event period. A notable increase in secondary pollutants was observed during these periods. These pollutants are known to enhance oxidative potential, thereby potentially impacting human health.

An Ontario Health (Cancer Care Ontario) Clinical Practice Guideline: Surveillance Strategies in Patients with Stage I, II, III or Resectable IV Melanoma Who Were Treated with Curative Intent.

AIMS: To make recommendations on managing the surveillance of patients with stage I, II, III or resectable IV melanoma who are clinically free of disease following treatment with curative intent. MATERIALS AND METHODS: This guideline was developed by Ontario Health's (Cancer Care Ontario's) Program in Evidence-Based Care and the Melanoma Disease Site Group (including seven medical oncologists, four surgical oncologists, three dermatologists, one radiation oncologist and one patient representative). The MEDLINE, EMBASE, Cochrane Library, PROSPERO databases and the main relevant guideline websites were searched. Internal and external reviews were conducted, with final approval by the Program in Evidence-Based Care and the Melanoma Disease Site Group. The Grading of Recommendations, Assessment, Development and Evaluation approach was followed, and the Modified Delphi method was used. RESULTS: Based on the current evidence (eight eligible original study papers and four relevant guidelines) and the clinical opinions of the authors of this guideline, the initial recommendations were made. To reach 75% agreement for each recommendation, the Melanoma Disease Site Group (16 members) voted twice and one recommendation was voted on three times. After a comprehensive internal and external review process (including national and international reviewers), 12 recommendations, three weak recommendations and six qualified statements were ultimately made. CONCLUSIONS: After a systematic review, a comprehensive internal and external review process and a consensus process, the current guideline has been created. The guideline authors believe that this guideline will help clinicians, patients and policymakers make well-informed healthcare decisions that will guide them in clinical melanoma surveillance and ultimately assist in improving patient outcomes.

Enhancement of denitrification by sulfur-based carrier in sequencing batch reactor (SBR) for advanced wastewater treatment.

This study was to enhance the nitrogen removal efficiency in the sequencing batch reactor (SBR) process by adding sulfur-based carriers. The nitrogen removal efficiency of the control group was compared with that of the experimental group through a two-series operation of SBR1 without carrier and SBR2 with the carrier under the condition of no external carbon source. A total nitrogen (T-N) removal efficiency of 6.6%, 72.6%, and 79.9% was observed in SBR1, SBR2 (5%), and (10%), respectively. The T-N removal efficiency was improved in the system with carriers, which showed an increase in the removal efficiency of approximately 91.7%. The results suggest that the inclusion of the carrier led to an elevation in the sulfur ratio, implying an augmented surface area for sulfur-based denitrifying microorganisms. Additionally, CaCO3 contributed essential alkalinity for sulfur denitrification, thereby preventing a decline in pH. Regardless of the carrier, the efficiency of organic matter removal surpassed 89%, indicating that the sulfur-based carrier did not adversely affect the biological reaction associated with organic matter. Therefore, autotrophic denitrification was successfully performed using a sulfur carrier in the SBR process without an external carbon source, improving the nitrogen removal efficiency.

Correlation between aggression at the veterinary clinic and problem behaviors at home for cats in the USA.

OBJECTIVES: The aim of the study was to determine whether cats that exhibit aggression during veterinary visits are more likely to have behavior problems at home. METHODS: An online, anonymous, cross-sectional survey was developed and distributed to residents in the USA who were aged over 18 years and who were the primary owners of at least one cat. The survey collected information about cat and household factors, and utilized a validated questionnaire instrument for obtaining behavioral information of pet cats. RESULTS: Aggression at the veterinary clinic was reported in 42.6% of the cats. The frequency of aggression exhibited at the veterinary clinic was lower in cats that lived in multi-cat households. Most cats did not receive medications intended to reduce fear, anxiety and/or pain before veterinary visits. Aggression at the veterinary clinic was positively associated with behavior problems at home, including stranger-directed aggression, owner-directed aggression, resistance to restraint, familiar cat aggression, dog-directed aggression, house-soiling, separation-related behaviors and scratching claws on inappropriate surfaces indoors. CONCLUSIONS AND RELEVANCE: Cats that exhibit aggression at the veterinary clinic are more likely to exhibit aggression and anxiety-related behaviors at home. Veterinarians should screen cats that exhibit aggression at the veterinary clinic for behavior problems at home to institute prompt diagnosis and treatment.

Dental implant as a potential risk factor for maxillary sinus fungus ball.

Fungus ball is the most common form of non-invasive fungal sinusitis, and maxillary sinus is the most commonly involved site. Maxillary sinus fungus ball (MFB) accounts for a considerable proportion of unilateral maxillary sinusitis. The prevalence of MFB has recently increased; however, its contributing factors are unclear. This study analyzed the association between MFB and dental implants. One hundred one patients who underwent unilateral maxillary sinus surgery were divided into two groups based on surgical biopsy results: unilateral bacterial sinusitis (UBS, n = 45) and MFB (n = 56). Stratified random sampling of 30 patients from each group was performed to adjust for age. The number of dental implants on maxillary teeth and degree of penetration into the maxillary sinus was radiologically evaluated. The number of patients with dental implants was greater (P = 0.085) and the number of implants was significantly higher (P = 0.031) in the MFB group. Dental implant can be a potential risk factor for MFB development. Therefore, dental implant surgeons should take caution in penetrating the maxillary sinus floor during implant insertion and otolaryngologists should consider the possibility of fungus ball when assessing patients with sinusitis who have dental implants.

TGF-ß and BMP signaling are associated with the transformation of glioblastoma to gliosarcoma and then osteosarcoma.

Background: Gliosarcoma, an isocitrate dehydrogenase wildtype (IDH-WT) variant of glioblastoma, is defined by clonal biphasic differentiation into gliomatous and sarcomatous components. While the transformation from a glioblastoma to gliosarcoma is uncommon, the subsequent transformation to osteosarcoma is rare but may provide additional insights into the biology of these typically distinct cancers. We observed a patient initially diagnosed with glioblastoma, that differentiated into gliosarcoma at recurrence, and further evolved to osteosarcoma at the second relapse. Our objective was to characterize the molecular mechanisms of tumor progression associated with this phenotypic transformation. Methods: Tumor samples were collected at all 3 stages of disease and RNA sequencing was performed to capture their transcriptomic profiles. Sequential clonal evolution was confirmed by the maintenance of an identical PTEN mutation throughout the tumor differentiation using the TSO500 gene panel. Publicly available datasets and the Nanostring nCounter technology were used to validate the results. Results: The glioblastoma tumor from this patient possessed mixed features of all 3 TCGA-defined transcriptomic subtypes of an IDH-WT glioblastoma and a proportion of osteosarcoma signatures were upregulated in the original tumor. Analysis showed that enhanced transforming growth factor-ß (TGF-ß) and bone morphogenic protein signaling was associated with tumor transformation. Regulatory network analysis revealed that TGF-ß family signaling committed the lineage tumor to osteogenesis by stimulating the expression of runt-related transcription factor 2 (RUNX2), a master regulator of bone formation. Conclusions: This unusual clinical case provided an opportunity to explore the modulators of longitudinal sarcomatous transformation, potentially uncovering markers indicating predisposition to this change and identification of novel therapeutic targets.

Cost-effectiveness of full and partial opioid agonists for opioid use disorder in outpatient settings: United States healthcare sector perspective.

INTRODUCTION: Studies show that medications for opioid use disorder (MOUD) reduce illicit opioid use, emergency healthcare services, opioid-related overdose, and death. However, few studies have investigated the long-term cost-effectiveness of MOUD in office-based opioid treatment (OBOT) and opioid treatment program (OTP) settings. We aimed to estimate the cost, utility, quality-adjusted life years gained (QALYs), and incremental cost-effectiveness ratios (ICERs) of three MOUD compared to each other and counseling without medication from a US healthcare sector perspective. METHODS: Our study developed a Markov model to conduct a cost-effectiveness analysis of counseling and three MOUD in the OBOT and OTP settings: sublingual buprenorphine/naloxone (BUPNX), buprenorphine extended-release (XR-BUP) injection, and oral methadone. The model included five health states representing combinations of receiving or off treatment while either using or not actively using illicit opioids, and death. The cycle length was one month; the time-horizon was ten years. The study obtained model inputs from systematic reviews of published literature and public data. A 3 % annual discount rate was applied to cost and utility calculation. The primary outcomes included total costs, life-years (LYs), QALYs, and ICERs. We also conducted a scenario analysis using a hypothetical OBOT outpatient setting with methadone. RESULTS: In the base-case OBOT setting, the total costs and QALYs, respectively, were counseling $22,848, 5.60; BUPNX $29,875, 5.82; and XR-BUP $63,936, 5.87. ICERs were $32,345/QALY (BUPNX vs. counseling) and $625,858/QALY (XR-BUP vs BUPNX). In the OTP setting, the total costs of counseling, methadone, BUPNX, and XR-BUP were $20,124, $27,000, $33,500, and $75,272, respectively. QALYs of methadone were 5.86. QALYs of counseling, BUPNX, and XR-BUP remained the same as in the OBOT setting. Incremental ICERs were $26,714/QALY (methadone vs counseling) and $3,337,623/QALY (XR-BUP vs methadone). BUPNX was dominated by methadone. In the scenario analysis, BUPNX was also dominated by methadone. CONCLUSIONS: Outpatient MOUD resulted in important gains in quality of life and life expectancy. In both OBOT and OTP settings, XR-BUP was not cost-effective. BUPNX was cost-effective in the OBOT setting, while it was dominated by methadone in the OTP setting. The cost-effectiveness of BUPNX and XR-BUP could be enhanced if the costs of these medications were reduced.

Characterization of Enlarged Tongues in Cloned Piglets.

Although the efficiency of cloning remains very low, this technique has become the most reliable way to produce transgenic pigs. However, the high rate of abnormal offspring such as an enlarged tongue lowers the cloning efficiency by reducing the early survivability of piglets. Thus, the present study was conducted to identify the characteristics of the enlarged tongue from cloned piglets by histologic and transcriptomic analysis. As a result, it was observed that the tissues from enlarged tongues (n = 3) showed isolated and broken muscle bundles with wide spaces while the tissues from normal tongues (n = 3) showed the tight connection of muscle bundles without space by histological analysis. Additionally, transmission electron microscopy results also showed the formation of isolated and broken muscle bundles in enlarged tongues. The transcriptome analysis showed a total of 197 upregulated and 139 downregulated genes with more than 2-fold changes in enlarged tongues. Moreover, there was clear evidence for the difference between groups in the muscle system process with high relation in the biological process by gene ontology analysis. The analysis of the Kyoto Encyclopedia of Gene and Genomes pathway of differentially expressed genes indicated that the pentose phosphate pathway, glycolysis/gluconeogenesis, and glucagon signaling pathway were also involved. Conclusively, our results could suggest that the abnormal glycolytic regulation may result in the formation of an enlarged tongue. These findings might have the potential to understand the underlying mechanisms, abnormal development, and disease diagnosis in cloned pigs.

Attitudes and practices toward feral cats of male and female dog or cat owners and non-owners in Seoul, South Korea.

The number of pet cats in South Korea has sharply increased since 2010. Problems have arisen with feral or stray cats, creating conflict among residents, to such an extent that the government provides some sites for people to offer feeding stations for the stray cats. This study investigated hypotheses on people's attitudes toward feral cats in Seoul, South Korea: (i) dog and cat owners would show more positive attitudes than non-owners toward feral cats; (ii) females would have more positive attitudes toward feral cats than males; (iii) the number of Seoul-provided feeding stations by district would be correlated with people's positive attitudes toward feral cats. Responses from 7,394 participants were used for the final analyses with 3,179 males, 3,607 females, and 599 others (includes "decline to state"). Cat owners reported more extremely positive attitudes toward feral cats than people who had no cats. Females more often had cats than males, and they were more extremely positive toward pet and feral cats than males, and strongly opposed to culling as a management strategy. The attitudes toward feral cats of people with only dogs were intermediate between people with cats and people without pets, more resembling those of people without pets. There was a correlation between the number of city-provided feeding stations and people's attitudes toward feral cats, but only in the areas with at least 40 feeder stations; having many city-provided feeding stations was associated with very negative attitudes to feral cats. Whether the very large number of feeding stations were provided in the two neighborhoods due to a previously excessive number of feral cats in those neighborhoods, vs. those feeding stations attracting or provisioning an ever-growing number of feral cats, is unknown. These results show sharp differences in attitudes between cat owners and non-owners, and between males and females. Results resemble findings in other studies, perhaps with more extreme differences between sub-groups. The study revealed that attitudes toward pet and feral cats in Seoul were complex and varied with pet ownership, with sex, and with neighborhood context.

Potential Application of Muscle Precursor Cells from Male Specific-Pathogen-Free (SPF) Chicken Embryos in In Vitro Agriculture.

This study examined the potential benefits of male specific-pathogen-free (SPF) White Leghorn embryos in cellular agriculture for sustainable and ethical poultry meat production-addressing traditional farming challenges, including disease outbreaks of Salmonella and Avian influenza. We isolated myogenic precursor cells (MPCs) from the thigh muscles (Musculus femoris) of 12.5-day-old embryos from 10 SPF White Leghorns that tested negative for Salmonella. We randomly selected MPCs from three males and three females, isolated them using a modified pre-plating (pp) method, and compared their in vitro development. After 1 h (pp1) and 2 h (pp2) of incubation, they were transferred to a new dish to remove fast-adhering cells and cultured (pp3). Isolated MPCs had a 69% positive reaction to Pax7. During proliferation, no differences were observed in PAX7, MYF5, or MYOD expression between the male and female MPCs. However, after five days of differentiation, the expression of late myogenic factors-MYOG and MYF6-significantly increased in all MPCs. Notably, MYOG expression was 1.9 times higher in female than in male MPCs. This impacted MYMK's expression pattern. Despite this, the myotube fusion index did not differ between the sexes. Muscle cells from male SPF-laying chicken embryos are promising for developing clean animal-cell-derived protein sources via resource recycling.

Comparative transcriptome analysis between long- and short-term survival after pig-to-monkey cardiac xenotransplantation reveals differential heart failure development.

Cardiac xenotransplantation is the potential treatment for end-stage heart failure, but the allogenic organ supply needs to catch up to clinical demand. Therefore, genetically-modified porcine heart xenotransplantation could be a potential alternative. So far, pig-to-monkey heart xenografts have been studied using multi-transgenic pigs, indicating various survival periods. However, functional mechanisms based on survival period-related gene expression are unclear. This study aimed to identify the differential mechanisms between pig-to-monkey post-xenotransplantation long- and short-term survivals. Heterotopic abdominal transplantation was performed using a donor CD46-expressing GTKO pig and a recipient cynomolgus monkey. RNA-seq was performed using samples from POD60 XH from monkey and NH from age-matched pigs, D35 and D95. Gene-annotated DEGs for POD60 XH were compared with those for POD9 XH (Park et al. 2021). DEGs were identified by comparing gene expression levels in POD60 XH versus either D35 or D95 NH. 1,804 and 1,655 DEGs were identified in POD60 XH versus D35 NH and POD60 XH versus D95 NH, respectively. Overlapped 1,148 DEGs were annotated and compared with 1,348 DEGs for POD9 XH. Transcriptomic features for heart failure and inhibition of T cell activation were observed in both long (POD60)- and short (POD9)-term survived monkeys. Only short-term survived monkey showed heart remodeling and regeneration features, while long-term survived monkey indicated multi-organ failure by neural and hormonal signaling as well as suppression of B cell activation. Our results reveal differential heart failure development and survival at the transcriptome level and suggest candidate genes for specific signals to control adverse cardiac xenotransplantation effects.

Insensitive Effects of Inflammatory Cytokines on the Reference Genes of Synovial Fluid Resident-Mesenchymal Stem Cells Derived from Rheumatoid Arthritis Patients.

Mesenchymal stem cells derived from rheumatoid arthritis patients (RA-MSCs) provide an understanding of a variety of cellular and immunological responses within the inflammatory milieu. Sustained exposure of MSCs to inflammatory cytokines is likely to exert an influence on genetic variations, including reference genes (RGs). The sensitive effect of cytokines on the reference genes of RA-SF-MSCs may be a variation factor affecting patient-derived MSCs as well as the accuracy and reliability of data. Here, we comparatively evaluated the stability levels of nine RG candidates, namely GAPDH, ACTB, B2M, EEF1A1, TBP, RPLP0, PPIA, YWHAZ, and HPRT1, to find the most stable ones. Alteration of the RG expression was evaluated in MSCs derived from the SF of healthy donors (H-SF-MSCs) and in RA-SF-MSCs using the geNorm and NormFinder software programs. The results showed that TBP, PPIA, and YWHAZ were the most stable RGs for the normalization of H-SF-MSCs and RA-SF-MSCs using RT-qPCR, whereas ACTB, the most commonly used RG, was less stable and performed poorly. Additionally, the sensitivity of RG expression upon exposure to proinflammatory cytokines (TNF-α and IL-1ß) was evaluated. RG stability was sensitive in the H-SF-MSCs exposed to TNF-α and IL-1ß but insensitive in the RA-SF-MSCs. Furthermore, the normalization of IDO expression using ACTB falsely diminished the magnitude of biological significance, which was further confirmed with a functional analysis and an IDO activity assay. In conclusion, the results suggest that TBP, PPIA, and YWHAZ can be used in SF-MSCs, regardless of their exposure to inflammatory cytokines.

Adipose Tissue-Derived Mesenchymal Stem Cells Extend the Lifespan and Enhance Liver Function in Hepatocyte Organoids.

In this study, we generated hepatocyte organoids (HOs) using frozen-thawed primary hepatocytes (PHs) within a three-dimensional (3D) Matrigel dome culture in a porcine model. Previously studied hepatocyte organoid analogs, spheroids, or hepatocyte aggregates created using PHs in 3D culture systems have limitations in their in vitro lifespans. By co-culturing adipose tissue-derived mesenchymal stem cells (A-MSCs) with HOs within a 3D Matrigel dome culture, we achieved a 3.5-fold increase in the in vitro lifespan and enhanced liver function compared to a conventional two-dimensional (2D) monolayer culture, i.e., more than twice that of the HO group cultured alone, reaching up to 126 d. Although PHs were used to generate HOs, we identified markers associated with cholangiocyte organoids such as cytokeratin 19 and epithelial cellular adhesion molecule (EPCAM). Co-culturing A-MSCs with HOs increased the secretion of albumin and urea and glucose consumption compared to HOs cultured alone. After more than 100 d, we observed the upregulation of tumor protein P53 (TP53)-P21 and downregulation of EPCAM, albumin (ALB), and cytochrome P450 family 3 subfamily A member 29 (CYP3A29). Therefore, HOs with function and longevity improved through co-culturing with A-MSCs can be used to create large-scale human hepatotoxicity testing models and precise livestock nutrition assessment tools.