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Three-dimensional cell spheroids show promise for the reconstruction of native tissues. Herein, we report a sophisticated, uniform, and highly reproducible spheroid culture system for tissue reconstruction. A mesh-integrated culture system was designed to precisely control the uniformity and reproducibility of spheroid formation. Furthermore, we synthesized hexanoyl glycol chitosan, a material with ultralow cell adhesion properties, to further improve spheroid formation efficiency and biological function. Our results demonstrate improved biological function in various types of cells and ability to generate spheroids with complex structures composed of multiple cell types. In conclusion, our spheroid culture system offers a highly effective and widely applicable approach to generating customized spheroids with desired structural and biological features for a variety of biomedical applications.
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Técnicas de Cultura de Células , Quitosana , Medicina Regenerativa , Esferoides Celulares , Esferoides Celulares/citologia , Quitosana/química , Humanos , Técnicas de Cultura de Células/métodos , Engenharia Tecidual/métodos , AnimaisRESUMO
BACKGROUND: S-588410, a cancer peptide vaccine (CPV), comprises five HLA-A*24:02-restricted peptides from five cancer-testis antigens. In a phase 2 study, S-588410 was well-tolerated and exhibited antitumor efficacy in patients with urothelial cancer. Therefore, we aimed to evaluate the efficacy, immune response, and safety of S-588410 in patients with completely resected esophageal squamous cell carcinoma (ESCC). METHODS: This phase 3 study involved patients with HLA-A*24:02-positive and lymph node metastasis-positive ESCC who received neoadjuvant therapy followed by curative resection. After randomization, patients were administered S-588410 and placebo (both emulsified with Montanide™ ISA 51VG) subcutaneously. The primary endpoint was relapse-free survival (RFS). The secondary endpoints were overall survival (OS), cytotoxic T-lymphocyte (CTL) induction, and safety. Statistical significance was tested using the one-sided weighted log-rank test with the Fleming-Harrington class of weights. RESULTS: A total of 276 patients were randomized (N = 138/group). The median RFS was 84.3 and 84.1 weeks in the S-588410 and placebo groups, respectively (P = 0.8156), whereas the median OS was 236.3 weeks and not reached, respectively (P = 0.6533). CTL induction was observed in 132/134 (98.5%) patients who received S-588410 within 12 weeks. Injection site reactions (137/140 patients [97.9%]) were the most frequent treatment-emergent adverse events in the S-588410 group. Prolonged survival was observed in S-588410-treated patients with upper thoracic ESCC, grade 3 injection-site reactions, or high CTL intensity. CONCLUSIONS: S-588410 induced immune response and had acceptable safety but failed to reach the primary endpoint. A high CTL induction rate and intensity may be critical for prolonging survival during future CPV development.
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INTRODUCTION: This study is a retrospective case series to compare the accuracy of the Barrett toric calculator using predicted posterior corneal astigmatism (PCA) and PCA measurements using swept-source optical coherence tomography (SS-OCT) and a Scheimpflug camera. This evaluation was conducted across different types of anterior and posterior astigmatism. METHODS: A total of 146 eyes from 146 patients implanted with toric intraocular lenses were included. Mean absolute prediction error, standard deviation of prediction error, and the percentage of eyes with prediction errors within ±0.50 diopters (D) were calculated using vector analysis. Biometric measurements were conducted using the IOLMaster 700 and Pentacam HR. A subgroup analysis was conducted based on the orientation of both anterior and posterior corneal astigmatism. RESULTS: The Barrett toric calculator with predicted PCA yielded the best results, with 78.1% having a prediction error ≤ 0.50 D, which was a significantly higher percentage than the Barrett formula with the two versions of measured PCA (P < 0.05). In the subgroup with a horizontally steep meridian PCA using the IOLMaster 700, the Barrett formula with predicted PCA yielded the best results, with 78.3% of cases having a prediction error of less than 0.5 D. This percentage was significantly higher than the other two measured PCA subgroups (P < 0.05). CONCLUSION: The Barrett toric formula with predicted PCA demonstrated a statistically significantly higher proportion of cases with a prediction error ≤ 0.5 D compared to the two measured PCA formulas (from the IOLMaster 700 or Pentacam). This trend persisted even when the posterior corneal astigmatism was horizontally steep.
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BACKGROUND: Myocardial infarction (MI), a representative form of ischemic heart disease, remains a huge burden worldwide. This study aimed to explore whether extracellular vesicles (EVs) secreted from hyaluronic acid (HA)-primed induced mesenchymal stem cells (HA-iMSC-EVs) could enhance the cardiac repair after MI. RESULTS: HA-iMSC-EVs showed typical characteristics for EVs such as morphology, size, and marker proteins expression. Compared with iMSC-EVs, HA-iMSC-EVs showed enhanced tube formation and survival against oxidative stress in endothelial cells, while reduced reactive oxygen species (ROS) generation in cardiomyocytes. In THP-1 macrophages, both types of EVs markedly reduced the expression of pro-inflammatory signaling players, whereas HA-iMSC-EVs were more potent in augmenting anti-inflammatory markers. A significant decrease of inflammasome proteins was observed in HA-iMSC-EV-treated THP-1. Further, phospho-SMAD2 as well as fibrosis markers in TGF-ß1-stimulated cardiomyocytes were reduced in HA-iMSC-EVs treatment. Proteomic data showed that HA-iMSC-EVs were enriched with multiple pathways including immunity, extracellular matrix organization, angiogenesis, and cell cycle. The localization of HA-iMSC-EVs in myocardium was confirmed after delivery by either intravenous or intramyocardial route, with the latter increased intensity. Echocardiography revealed that intramyocardial HA-iMSC-EVs injections improved cardiac function and reduced adverse cardiac remodeling and necrotic size in MI heart. Histologically, MI hearts receiving HA-iMSC-EVs had increased capillary density and viable myocardium, while showed reduced fibrosis. CONCLUSIONS: Our results suggest that HA-iMSC-EVs improve cardiac function by augmenting vessel growth, while reducing ROS generation, inflammation, and fibrosis in MI heart.
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Células-Tronco Mesenquimais , Infarto do Miocárdio , Humanos , Ácido Hialurônico/farmacologia , Células Endoteliais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteômica , Infarto do Miocárdio/terapia , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Células-Tronco Mesenquimais/metabolismo , FibroseRESUMO
Out of practicality, ambient air rather than oxygen is preferred as a fuel in electrochemical systems, but CO2 and H2O present in air cause severe irreversible reactions, such as the formation of carbonates and hydroxides, which typically degrades performance. Herein, we report on a Na-air battery enabled by a reversible carbonate reaction (Na2CO3·xH2O, x = 0 or 1) in Nasicon solid electrolyte (Na3Zr2Si2PO12) that delivers a much higher discharge potential of 3.4 V than other metal-air batteries resulting in high energy density and achieves > 86 % energy efficiency at 0.1 mA cm-2 over 100 cycles. This cell design takes advantage of moisture in ambient air to form an in-situ catholyte via the deliquescent property of NaOH. As a result, not only reversible electrochemical reaction of Na2CO3·xH2O is activated but also its kinetics is facilitated. Our results demonstrate the reversible use of free ambient air as a fuel, enabled by the reversible electrochemical reaction of carbonates with a solid electrolyte.
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A halotolerant consortium between microalgae and methanotrophic bacteria could effectively remediate in situ CH4 and CO2, particularly using saline wastewater sources. Herein, Methylomicrobium alcaliphilum 20Z was demonstrated to form a mutualistic association with Chlorella sp. HS2 at a salinity level above 3.0%. Co-culture significantly enhanced the growth of both microbes, independent of initial inoculum ratios. Additionally, increased methane provision in enclosed serum bottles led to saturated methane removal. Subsequent analyses suggested nearly an order of magnitude increase in the amount of carbon sequestered in biomass in methane-fed co-cultures, conditions that also maintained a suitable cultural pH suitable for methanotrophic growth. Collectively, these results suggest a robust metabolic coupling between the two microbes and the influence of the factors other than gaseous exchange on the assembled consortium. Therefore, multi-faceted investigations are needed to harness the significant methane removal potential of the identified halotolerant consortium under conditions relevant to real-world operation scenarios.
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Chlorella , Methylococcaceae , Metano/metabolismo , Chlorella/metabolismo , Methylococcaceae/metabolismo , Bactérias/metabolismoRESUMO
Crystalline systems consisting of small-molecule building blocks have emerged as promising materials with diverse applications. It is of great importance to characterize not only their static structures but also the conversion of their structures in response to external stimuli. Femtosecond time-resolved crystallography has the potential to probe the real-time dynamics of structural transitions, but, thus far, this has not been realized for chemical reactions in non-biological crystals. In this study, we applied time-resolved serial femtosecond crystallography (TR-SFX), a powerful technique for visualizing protein structural dynamics, to a metal-organic framework, consisting of Fe porphyrins and hexazirconium nodes, and elucidated its structural dynamics. The time-resolved electron density maps derived from the TR-SFX data unveil trifurcating structural pathways: coherent oscillatory movements of Zr and Fe atoms, a transient structure with the Fe porphyrins and Zr6 nodes undergoing doming and disordering movements, respectively, and a vibrationally hot structure with isotropic structural disorder. These findings demonstrate the feasibility of using TR-SFX to study chemical systems.
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Engineered human cardiac tissues have been utilized for various biomedical applications, including drug testing, disease modeling, and regenerative medicine. However, the applications of cardiac tissues derived from human pluripotent stem cells are often limited due to their immaturity and lack of functionality. Therefore, in this study, we establish a perfusable culture system based on in vivo-like heart microenvironments to improve human cardiac tissue fabrication. The integrated culture platform of a microfluidic chip and a three-dimensional heart extracellular matrix enhances human cardiac tissue development and their structural and functional maturation. These tissues are comprised of cardiovascular lineage cells, including cardiomyocytes and cardiac fibroblasts derived from human induced pluripotent stem cells, as well as vascular endothelial cells. The resultant macroscale human cardiac tissues exhibit improved efficacy in drug testing (small molecules with various levels of arrhythmia risk), disease modeling (Long QT Syndrome and cardiac fibrosis), and regenerative therapy (myocardial infarction treatment). Therefore, our culture system can serve as a highly effective tissue-engineering platform to provide human cardiac tissues for versatile biomedical applications.
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Células Endoteliais , Células-Tronco Pluripotentes Induzidas , Humanos , Diferenciação Celular , Miócitos Cardíacos , Engenharia Tecidual/métodosRESUMO
The combination of X-ray free-electron lasers (XFELs) with serial femtosecond crystallography represents cutting-edge technology in structural biology, allowing the study of enzyme reactions and dynamics in real time through the generation of `molecular movies'. This technology combines short and precise high-energy X-ray exposure to a stream of protein microcrystals. Here, the XFEL structure of carbonic anhydrase II, a ubiquitous enzyme responsible for the interconversion of CO2 and bicarbonate, is reported, and is compared with previously reported NMR and synchrotron X-ray and neutron single-crystal structures.
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Anidrase Carbônica II , Anidrase Carbônica II/química , Cristalografia por Raios X , Proteínas/química , Síncrotrons , Raios X , HumanosRESUMO
Background: In previous studies, several asthma phenotypes were identified using clinical and demographic parameters. Transcriptional phenotypes were mainly identified using sputum and bronchial cells. Objective: We aimed to investigate asthma phenotypes via clustering analysis using clinical variables and compare the transcription levels among clusters using gene expression profiling of the blood. Methods: Clustering analysis was performed using 6 parameters: age of asthma onset, body mass index, pack-years of smoking, forced expiratory volume in 1 s (FEV1), FEV1/forced vital capacity, and blood eosinophil counts. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples and RNA was extracted from selected PBMCs. Transcriptional profiles were generated (Illumina NovaSeq 6000) and analyzed using the reference genome and gene annotation files (hg19.refGene.gft). Pathway enrichment analysis was conducted using GO, KEGG, and REACTOME databases. Results: In total, 355 patients with asthma were included in the analysis, of whom 72 (20.3%) had severe asthma. Clustering of the 6 parameters revealed 4 distinct subtypes. Cluster 1 (n = 63) had lower predicted FEV1 % and higher pack-years of smoking and neutrophils in sputum. Cluster 2 (n = 43) had a higher proportion and number of eosinophils in sputum and blood, and severe airflow limitation. Cluster 3 (n = 110) consisted of younger subjects with atopic features. Cluster 4 (n = 139) included features of late-onset mild asthma. Differentially expressed genes between clusters 1 and 2 were related to inflammatory responses and cell activation. Th17 cell differentiation and interferon gamma-mediated signaling pathways were related to neutrophilic inflammation in asthma. Conclusion: Four clinical clusters were differentiated based on clinical parameters and blood eosinophils in adult patients with asthma form the Cohort for Reality and Evolution of Adult Asthma in Korea (COREA) cohort. Gene expression profiling and molecular pathways are novel means of classifying asthma phenotypes.
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Background: The purposes of this study were to determine the accuracy of our cup positioning method and to evaluate the dislocation rate after total hip arthroplasty (THA). Methods: After positioning the patient in the lateral decubitus position on the operation table, an anteroposterior view of the hip was taken. The pelvic pitch was measured on the X-ray. A positive pitch was defined as the caudal rotation of the upper hemipelvis. Our target abduction of the cup was 43°. We used the cup holder to guesstimate the cup abduction. In a preliminary study, we found that the weight of the cup holder increased the pelvic pitch by 5°. Thus, the target abduction of the cup holder was calculated by a formula: 43° - pelvic pitch - 5°. During the cup insertion, the cup holder was anteverted to the calculated target according to the concept of combined anteversion. We evaluated 478 THAs (429 patients), which were done with the use of the method. Results: The mean cup abduction was 43.9° (range, 32.0°-53.0°) and the mean error of cup abduction was 2.4° (standard deviation [SD], 2.0°; range, 0.0°-11.0°). The mean cup anteversion was 28.5° (range, 10.0°-42.0°) and the mean error of cup anteversion was 6.7° (SD, 5.2°; range, 0.0°-27.6°). Of all, 82.4% of the cups (394 / 478) were within the safe zone: 30°-50° abduction and 10°-35° anteversion. During 2- to 5-year follow-up, no hip dislocated. Conclusions: Our adjusting method according to the pelvic pitch can be a reliable option for optimizing the cup abduction in THA.
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Artroplastia de Quadril , Prótese de Quadril , Luxações Articulares , Humanos , Acetábulo/cirurgia , Artroplastia de Quadril/métodos , Radiografia , Movimento , Luxações Articulares/cirurgiaRESUMO
We present the investigation of 1,2,4,5-tetrazine derivatives as low-cost and synthetically modular organic electrode materials in rechargeable aqueous Zn-ion batteries (AZIBs). The substituents at the 3,6-positions of tetrazine were found to be critical for cycling stability. While heteroatom substituents (chloro, methoxy, and pyrazole) lead to the rapid decomposition of electrode materials in the electrolyte, the installation of phenyl groups enhances the cycling stability via π-π stacking. Spectroscopic characterization suggests a cooperative Zn2+ and H+ insertion mechanism. This unique cooperativity of Zn2+ and H+ leads to a steady discharge plateau in contrast to the undesirable sloping voltage profile typically observed in Zn-organic batteries.
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Hepatitis B virus (HBV), a leading cause of developing hepatocellular carcinoma affecting more than 290 million people worldwide, is an enveloped DNA virus specifically infecting hepatocytes. Myristoylated preS1 domain of the HBV large surface protein binds to the host receptor sodium-taurocholate cotransporting polypeptide (NTCP), a hepatocellular bile acid transporter, to initiate viral entry. Here, we report the cryogenic-electron microscopy structure of the myristoylated preS1 (residues 2-48) peptide bound to human NTCP. The unexpectedly folded N-terminal half of the peptide embeds deeply into the outward-facing tunnel of NTCP, whereas the C-terminal half formed extensive contacts on the extracellular surface. Our findings reveal an unprecedented induced-fit mechanism for establishing high-affinity virus-host attachment and provide a blueprint for the rational design of anti-HBV drugs targeting virus entry.
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Vírus da Hepatite B , Simportadores , Humanos , Vírus da Hepatite B/genética , Hepatócitos/metabolismo , Ligação Proteica , Ligação Viral , Peptídeos/metabolismo , Simportadores/metabolismo , Internalização do VírusRESUMO
OBJECTIVE: While depression associated with disability has been extensively studied, how depression could differ depending on whether the disability is congenital or acquired remains to be investigated. The objective of this study was to compare depression and its related factors among people with congenital and acquired physical disabilities. METHODS: We used the 2016 Panel Survey of Employment for the Disabled in Korea, a population-based survey for people with disability registered with the Korean government. Among 4577 participants, a total of 2128 participants with physical disability were analyzed using Chi-square test and binary logistic regression analysis. RESULTS: The prevalence of depression was 12.9% in those with congenital physical disability and 16.0% in those with acquired physical disability. Stress due to disability and family relationship satisfaction were associated with higher and lower odds of depression, respectively, in both disability groups. Discrimination due to disability, divorce, social participation, and subjective social status were significant predictors of depression only in people with acquired disability. CONCLUSIONS: Compared to those with congenital disability, individuals with acquired disability can be more susceptible to issues relating to social relationship, social standing and discrimination. Findings of this study suggest that acquired disability and adapting to changes associated with it can be a source of mental distress in addition to living with it. More efforts are needed to address discrimination, provide supportive social relationships, and provide supportive living conditions in order to reduce depression in persons living with disability, especially those with acquired disability.
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Exoskeleton robots are a promising solution to reduce musculoskeletal disorders (MSDs) in different work environments, but a specific usability scale for evaluating them is lacking. This study aimed to develop and verify a preliminary Exoskeleton Usability Questionnaire (EUQ) for the lower limb exoskeletons by creating a draft survey questionnaire from existing questions in prior studies. An experiment was conducted with 20 participants who performed a specific task while wearing three lower limb robots and provided subjective feedback using the developed questionnaire. Data were analysed using exploratory and confirmatory factor analysis (CFA), resulting in a usability evaluation questionnaire for exoskeleton robots clustered into four main factors: mobility, adjustability, handling and safety. This study's findings are expected to be useful in evaluating the usability of the lower limb exoskeletons in both general production sites and agricultural work, which can aid in reducing the prevalence of lower limb MSDs.Practitioner Summary: This study developed a preliminary subjective usability evaluation questionnaire for exoskeleton robots. The questionnaire is clustered into four main factors: mobility, adjustability, handling and safety. These findings provide a valuable tool for assessing exoskeleton usability, potentially reducing musculoskeletal disorders (MSDs) in various work environments.
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Hydroxycarboxylic acid receptors (HCAR1, HCAR2, and HCAR3) transduce Gi/o signaling upon biding to molecules such as lactic acid, butyric acid and 3-hydroxyoctanoic acid, which are associated with lipolytic and atherogenic activity, and neuroinflammation. Although many reports have elucidated the function of HCAR2 and its potential as a therapeutic target for treating not only dyslipidemia but also neuroimmune disorders such as multiple sclerosis and Parkinson's disease, the structural basis of ligand recognition and ligand-induced Gi-coupling remains unclear. Here we report three cryo-EM structures of the human HCAR2-Gi signaling complex, each bound with different ligands: niacin, acipimox or GSK256073. All three agonists are held in a deep pocket lined by residues that are not conserved in HCAR1 and HCAR3. A distinct hairpin loop at the HCAR2 N-terminus and extra-cellular loop 2 (ECL2) completely enclose the ligand. These structures also reveal the agonist-induced conformational changes propagated to the G-protein-coupling interface during activation. Collectively, the structures presented here are expected to help in the design of ligands specific for HCAR2, leading to new drugs for the treatment of various diseases such as dyslipidemia and inflammation.
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Receptores Acoplados a Proteínas G , Transdução de Sinais , Humanos , Ácidos Carboxílicos , Ligantes , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Cyanase plays a vital role in the detoxification of cyanate and supplies a continuous nitrogen source for soil microbes by converting cyanate to ammonia and carbon dioxide in a bicarbonate-dependent reaction. The structures of cyanase complexed with dianion inhibitors, in conjunction with biochemical studies, suggest putative binding sites for substrates. However, the substrate-recognition and reaction mechanisms of cyanase remain unclear. Here, crystal structures of cyanase from Escherichia coli were determined in the native form and in complexes with cyanate, bicarbonate and intermediates at 1.5-1.9â Å resolution using synchrotron X-rays and an X-ray free-electron laser. Cyanate and bicarbonate interact with the highly conserved Arg96, Ser122 and Ala123 in the active site. In the presence of a mixture of cyanate and bicarbonate, three different electron densities for intermediates were observed in the cyanase structures. Moreover, the observed electron density could explain the dynamics of the substrate or product. In addition to conformational changes in the substrate-binding pocket, dynamic movement of Leu151 was observed, which functions as a gate for the passage of substrates or products. These findings provide a structural mechanism for the substrate-binding and reaction process of cyanase.
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Bicarbonatos , Escherichia coli , Bicarbonatos/metabolismo , Bicarbonatos/farmacologia , Carbono-Nitrogênio Liases/química , Cianatos/metabolismo , Cianatos/farmacologiaRESUMO
X-ray structural science is undergoing a revolution driven by the emergence of X-ray Free-electron Laser (XFEL) facilities. The structures of crystalline solids can now be studied on the picosecond time scale relevant to phonons, atomic vibrations which travel at acoustic velocities. In the work presented here, X-ray diffuse scattering is employed to characterize the time dependence of the liquid phase emerging from femtosecond laser-induced melting of polycrystalline gold thin films using an XFEL. In a previous analysis of Bragg peak profiles, we showed the supersonic disappearance of the solid phase and presented a model of pumped hot electrons carrying energy from the gold surface to scatter at internal grain boundaries. This generates melt fronts propagating relatively slowly into the crystal grains. By conversion of diffuse scattering to a partial X-ray pair distribution function, we demonstrate that it has the characteristic shape obtained by Fourier transformation of the measured F(Q). The diffuse signal fraction increases with a characteristic rise-time of 13â ps, roughly independent of the incident pump fluence and consequent final liquid fraction. This suggests the role of further melt-front nucleation processes beyond grain boundaries.