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Cell-laden structures are widely applied for a variety of tissue engineering applications, including tissue restoration. Cell-to-cell interactions in bioprinted structures are important for successful tissue restoration, because cell-cell signaling pathways can regulate tissue development and stem cell fate. However, the low degree of cell-cell interaction in conventional cell-laden bioprinted structures is challenging for the therapeutic application of this modality. Herein, a microfluidic device with cell-laden methacrylated gelatin (GelMa) bioink and alginate as a matrix hydrogel is used to fabricate a functional hybrid structure laden with cell-aggregated microbeads. This approach effectively increases the degree of cell-to-cell interaction to a level comparable to cell spheroids. The hybrid structure is obtained using a one-step process without the exhausting procedure. It consists of cell bead fabrication and an extrusion process for the cell-bead laden structure. Different flow rates are appropriately selected to develop cell-laden struts with homogeneously distributed cell beads for each hydrogel in the process. The hybrid struts exhibit significantly higher cellular activities than those of conventional alginate/GelMa struts, which are bioprinted using similar cell densities and bioink formulations. Furthermore, hybrid struts with adipose stem cells are implanted into mice, resulting in significantly higher myogenesis in comparison to normally bioprinted struts.
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Hidrogeles , Ingeniería de Tejidos , Alginatos , Animales , Dispositivos Laboratorio en un Chip , Ratones , Impresión Tridimensional , Andamios del TejidoRESUMEN
Optimizing appropriate signal peptides in mammalian cell-based protein production is crucial given that most recombinant proteins produced in mammalian cells are thought to be secreted proteins. Until now, most studies on signal peptide in mammalian cells have replaced native signal peptides with well-known heterologous signal peptides and bioinformatics-based signal peptides. In the present study, we successfully established an in vitro screening system for synthetic signal peptide in CHO cells by combining a degenerate codon-based oligonucleotides library, a site-specific integration system, and a FACS-based antibody detection assay. Three new signal peptides were screened using this new screening system, confirming to have structural properties as signal peptides by the SignalP web server, a neural network-based algorithm that quantifies the signal peptide-ness of amino acid sequences. The novel signal peptides selected in this study increased Fc-fusion protein production in CHO cells by increasing specific protein productivity, whereas they did not negatively affect cell growth. Particularly, the SP-#149 clone showed the highest qp, 0.73 ± 0.01 pg/cell/day from day 1 to day 4, representing a 1.47-fold increase over the native signal peptide in a serum-free suspension culture mode. In addition, replacing native signal peptide with the novel signal peptides did not significantly affect sialylated N-glycan formation, N-terminal cleavage pattern, and biological function of Fc-fusion protein produced in CHO cells. The overall results indicate the utility of a novel in vitro screening system for synthetic signal peptide for mammalian cell-based protein production. KEY POINTS: ⢠An in vitro screening system for synthetic signal peptide in mammalian cells was established ⢠This system combined a degenerate codon-based library, site-specific integration, and a FACS-based detection assay ⢠The novel signal peptides selected in this study could increase Fc-fusion protein production in mammalian cells.
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Péptidos , Señales de Clasificación de Proteína , Animales , Células CHO , Cricetinae , Cricetulus , Péptidos/química , Péptidos/genética , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
ErbB3-binding protein 1 (EBP1) is implicated in diverse cellular functions, including apoptosis, cell proliferation, and differentiation. Here, by generating genetic inactivation of Ebp1 mice, we identified the physiological roles of EBP1 in vivo. Loss of Ebp1 in mice caused aberrant organogenesis, including brain malformation, and death between E13.5 and 15.5 owing to severe hemorrhages, with massive apoptosis and cessation of cell proliferation. Specific ablation of Ebp1 in neurons caused structural abnormalities of brain with neuron loss in [Nestin-Cre; Ebp1flox/flox ] mice. Notably, global methylation increased with high levels of the gene-silencing unit Suv39H1/DNMT1 in Ebp1-deficient mice. EBP1 repressed the transcription of Dnmt1 by binding to its promoter region and interrupted DNMT1-mediated methylation at its target gene, Survivin promoter region. Reinstatement of EBP1 into embryo brain relived gene repression and rescued neuron death. Our findings uncover an essential role for EBP1 in embryonic development and implicate its function in transcriptional regulation.
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Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Silenciador del Gen/fisiología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Animales , Apoptosis , Ciclo Celular , Proliferación Celular , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Transcripción GenéticaRESUMEN
Akt signaling is an important regulator of neural development, but the distinctive function of Akt isoforms in brain development presents a challenge. Here we show Siah1 as an ubiquitin ligase that preferentially interacts with Akt3 and facilitates ubiquitination and degradation of Akt3. Akt3 is enriched in the axonal shaft and branches but not growth cone tips, where Siah1 is prominently present. Depletion of Siah1 enhanced Akt3 levels in the soma and axonal tips, eliciting multiple branching. Brain-specific somatic mutation in Akt3-E17K escapes from Siah1-mediated degradation and causes improper neural development with dysmorphic neurons. Remarkably, coexpression of Siah1 with Akt3-WT restricted disorganization of neural development is caused by Akt3 overexpression, whereas forced expression of Siah1 with the Akt3-E17K mutant fails to cope with malformation of neural development. These findings demonstrate that Siah1 limits Akt3 turnover during brain development and that this event is essential for normal organization of the neural network.
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Encéfalo/crecimiento & desarrollo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Axones/metabolismo , Encéfalo/metabolismo , Ratones , Neurogénesis , Neuronas/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-akt/genética , Ratas , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
B23, also known as nucleophosmin (NPM), is multifunctional protein directly implicated in cell proliferation, cell cycle progression, and cell survival. In the current study, in addition to confirming its anti-apoptotic function in neuronal survival, we demonstrated that the spatial-temporal expression profile of B23 during development of hippocampal neurons is high in the embryonic stage, down-regulated after birth, and preferentially localized at the tips of growing neuritis and branching points. Overexpression of B23 promotes axon growth with abundant branching points in growing hippocampal neurons, but depletion of B23 impairs axon growth, leading to neuronal death. Following injury to the trisynaptic path in hippocampal slice, overexpression of B23 remarkably increased the number and length of regenerative fibers in the mossy fiber path. Our study suggests that B23 expression in developing neurons is essential for neuritogenesis and axon growth and that up-regulation of B23 may be a strategy for enhancing the reconstitution of synaptic paths after injury to hippocampal synapses.
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Hipocampo/lesiones , Hipocampo/metabolismo , Proteínas Nucleares/metabolismo , Sinapsis/metabolismo , Animales , Axones/metabolismo , Muerte Celular , Ratones , Fibras Musgosas del Hipocampo/metabolismo , Fibras Musgosas del Hipocampo/patología , Regeneración Nerviosa , Nucleofosmina , RatasRESUMEN
[Purpose] This study aimed to investigate the effects of McKenzie exercise, Kinesio taping, and myofascial release (MFR) on forward head posture (FHP). [Participants and Methods] Twenty-eight individuals with an FHP were enrolled. participants were randomly assigned to one of three groups: Group A, Group B and Group C. Group A underwent McKenzie exercise and MFR. Group B underwent McKenzie exercise and Kinesio taping. Group C underwent McKenzie exercise, MFR, and Kinesio taping. Interventions for all three groups were provided three times per week for four weeks. The acromion and tragus of ear (A-T length), craniovertebral angle (CVA), Cranial rotation angle (CRA), and neck disability index (NDI) were measured. [Results] The A-T length significantly differed after intervention in all three groups. CVA only significantly differed after intervention in group C. All three groups showed an increase in CRA after intervention; however, these increases were not statistically significant. [Conclusion] Considering these results, these three types of exercise may be recommended as effective exercises to improve posture in adults with FHP.
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The experience of loneliness is universal and may have an adverse effect on neurocognitive functioning even at a younger age. Using a comprehensive neurocognitive functioning test (NCFT) battery, we examined the possible negative effects of loneliness on neurocognitive functioning in young adults. The high-loneliness and low-loneliness groups were screened using the UCLA Loneliness Scale v. 3, and measures pertaining to the domains of intelligence, attention, memory, executive function, and psychomotor functioning were tested and compared. As depression and anxiety were significantly higher in the high-loneliness group, an analysis of covariance was conducted. As a result, the high-loneliness group showed significantly poor performance on measures of executive function and attention prior to controlling for depression and anxiety, and executive function retained its significance even after controlling for these variables. Additional analysis showed that depression and anxiety did not significantly mediate the relationship between loneliness and neurocognitive functioning. Such results suggest that loneliness is likely to negatively affect executive functioning and attention in early adulthood and then progressively spread to other domains of cognitive functioning, as reported in the older adult population. The limitations and implications of the present study were considered and addressed.
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Depresión , Función Ejecutiva , Soledad , Humanos , Soledad/psicología , Masculino , Femenino , Adulto Joven , Depresión/psicología , Atención , Ansiedad/psicología , Adulto , Pruebas Neuropsicológicas , AdolescenteRESUMEN
Cardiovascular diseases (CVDs) and metabolic disorders stand as formidable challenges that significantly impact the clinical outcomes and living quality for afflicted individuals. An intricate comprehension of the underlying mechanisms is paramount for the development of efficacious therapeutic strategies. Protein arginine methyltransferases (PRMTs), a class of enzymes responsible for the precise regulation of protein methylation, have ascended to pivotal roles and emerged as crucial regulators within the intrinsic pathophysiology of these diseases. Herein, we review recent advancements in research elucidating on the multifaceted involvements of PRMTs in cardiovascular system and metabolic diseases, contributing significantly to deepen our understanding of the pathogenesis and progression of these maladies. In addition, this review provides a comprehensive analysis to unveil the distinctive roles of PRMTs across diverse cell types implicated in cardiovascular and metabolic disorders, which holds great potential to reveal novel therapeutic interventions targeting PRMTs, thus presenting promising perspectives to effectively address the substantial global burden imposed by CVDs and metabolic disorders.
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Enfermedades Cardiovasculares , Enfermedades Metabólicas , Proteína-Arginina N-Metiltransferasas , Humanos , Enfermedades Cardiovasculares/metabolismo , Enfermedades Metabólicas/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Animales , MetilaciónRESUMEN
Therapeutic communication, of which nonverbal communication is a vital component, is an essential skill for professional nurses. The aim of this study is to assess the possibility of incorporating computer analysis programs into nursing education programs to improve the nonverbal communication skills of those preparing to become professional nurses. In this pilot observational study, the research team developed a computer program for nonverbal communication analysis including facial expressions and poses. The video clip data captured during nursing simulation practice by 10 3rd- and 4th-grade nursing students at a university in South Korea involved two scenarios of communication with a child's mother regarding the child's pre- and post-catheterization care. The dominant facial expressions varied, with sadness (30.73%), surprise (30.14%), and fear (24.11%) being the most prevalent, while happiness (7.96%) and disgust (6.79%) were less common. The participants generally made eye contact with the mother, but there were no instances of light touch by hand and the physical distance for nonverbal communication situations was outside the typical range. These results confirm the potential use of facial expression and pose analysis programs for communication education in nursing practice.
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Three-dimensional (3D) bioprinting is a highly effective technique for fabricating cell-loaded constructs in tissue engineering. However, the versatility of fabricating precise and complex cell-loaded hydrogels is limited owing to the poor crosslinking ability of cell-containing hydrogels. Herein, we propose an optic-fiber-assisted bioprinting (OAB) process to efficiently crosslink methacrylated hydrogels. By selecting appropriate processing conditions for the photo-crosslinking technique, we fabricated biofunctional cell-laden structures including methacrylated gelatin (Gelma), collagen, and decellularized extracellular matrix. To apply the method to skeletal muscle regeneration, cell-laden Gelma constructs were processed with a functional nozzle having a topographical cue and an OAB process that could induce a uniaxial alignment of C2C12 and human adipose stem cells (hASCs). Significantly higher degrees of cell alignment and myogenic activities in the cell-laden Gelma structure were observed compared with those in the cell construct that was printed using a conventional crosslinking method. Moreover, an in vivo regenerative potential was observed in volumetric muscle defects in a mouse model. The hASC-laden construct significantly induced greater muscle regeneration than the cell construct without topographical cues. Based on the results, the newly designed bioprinting process can prove to be highly effective in fabricating biofunctional cell-laden constructs for various tissue engineering applications.
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In the present study, to determine the efficacy of oral supplementation of ginseng berry extracts in augmenting exercise performance and exercise-associated metabolism, male mice were given orally 200 and 400 mg/kg of body weight (BW) of GBC for nine weeks. Although there are no differences in pre-exercise blood lactate levels among (1) the control group that received neither exercise nor GBC, (2) the group that performed only twice-weekly endurance exercise, and (3) and (4) the groups that combined twice-weekly endurance exercise with either 200 or 400 mg/kg GBC, statistically significant reductions in post-exercise blood lactate levels were observed in the groups that combined twice-weekly endurance exercise with oral administration of either 200 or 400 mg/kg GBC. Histological analysis showed no muscle hypertrophy, but transcriptome analysis revealed changes in gene sets related to lactate metabolism and mitochondrial function. GBC intake increased nicotinamide adenine dinucleotide levels in the gastrocnemius, possibly enhancing the mitochondrial electron transport system and lactate metabolism. Further molecular mechanisms are needed to confirm this hypothesis. [BMB Reports 2023; 56(6): 353-358].
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Panax , Condicionamiento Físico Animal , Ratones , Masculino , Animales , Frutas , Músculo Esquelético/metabolismo , Administración Oral , Lactatos/metabolismoRESUMEN
This study investigates the relationship between cancer cachexia and the gut microbiota, focusing on the influence of cancer on microbial composition. Lewis lung cancer cell allografts were used to induce cachexia in mice, and body and muscle weight changes were monitored. Fecal samples were collected for targeted metabolomic analysis for short chain fatty acids and microbiome analysis. The cachexia group exhibited lower alpha diversity and distinct beta diversity in gut microbiota, compared to the control group. Differential abundance analysis revealed higher Bifidobacterium and Romboutsia, but lower Streptococcus abundance in the cachexia group. Additionally, lower proportions of acetate and butyrate were observed in the cachexia group. The study observed that the impact of cancer cachexia on gut microbiota and their generated metabolites was significant, indicating a host-to-gut microbiota axis. [BMB Reports 2023; 56(7): 404-409].
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Microbioma Gastrointestinal , Neoplasias , Animales , Ratones , Microbioma Gastrointestinal/fisiología , Caquexia , Modelos Animales de Enfermedad , Ácidos Grasos Volátiles/análisis , Butiratos , Neoplasias/complicacionesRESUMEN
Neuromuscular dysfunction is tightly associated with muscle wasting that occurs with age or due to degenerative diseases. However, the molecular mechanisms underlying neuromuscular dysfunction are currently unclear. Recent studies have proposed important roles of Protein arginine methyltransferase 1 (Prmt1) in muscle stem cell function and muscle maintenance. In the current study, we set out to determine the role of Prmt1 in neuromuscular function by generating mice with motor neuron-specific ablation of Prmt1 (mnKO) using Hb9-Cre. mnKO exhibited age-related motor neuron degeneration and neuromuscular dysfunction leading to premature muscle loss and lethality. Prmt1 deficiency also impaired motor function recovery and muscle reinnervation after sciatic nerve injury. The transcriptome analysis of aged mnKO lumbar spinal cords revealed alterations in genes related to inflammation, cell death, oxidative stress, and mitochondria. Consistently, mnKO lumbar spinal cords of sciatic nerve injury model or aged mice exhibited elevated cellular stress response in motor neurons. Furthermore, Prmt1 inhibition in motor neurons elicited mitochondrial dysfunction. Our findings demonstrate that Prmt1 ablation in motor neurons causes age-related motor neuron degeneration attributing to muscle loss. Thus, Prmt1 is a potential target for the prevention or intervention of sarcopenia and neuromuscular dysfunction related to aging.
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Volumetric muscle loss (VML) is associated with a severe loss of muscle tissue that overwhelms the regenerative potential of skeletal muscles. Tissue engineering has shown promise for the treatment of VML injuries, as evidenced by various preclinical trials. The present study describes the fabrication of a cell-laden GelMa muscle construct using an in situ crosslinking (ISC) strategy to improve muscle functionality. To obtain optimal biophysical properties of the muscle construct, two UV exposure sources, UV exposure dose, and wall shear stress were evaluated using C2C12 myoblasts. Additionally, the ISC system showed a significantly higher degree of uniaxial alignment and myogenesis compared to the conventional crosslinking strategy (post-crosslinking). To evaluate the in vivo regenerative potential, muscle constructs laden with human adipose stem cells were used. The VML defect group implanted with the bio-printed muscle construct showed significant restoration of functionality and muscular volume. The data presented in this study suggest that stem cell-based therapies combined with the modified bioprinting process could potentially be effective against VML injuries.
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Purpose: The purpose of this study was to explore the association between breastfeeding and the prevalence of breast, thyroid, and cervical cancer among Korean adult women. Methods: The study was a secondary analysis of data from the Korea Genome and Epidemiology Study. The final samples were 113,944 Korean women among 173,205 urban-based cohort participants collected between 2004 and 2013 for adults aged forty and over. To determine the association between female cancers and breastfeeding experience, the number of childbirth, and total breastfeeding duration, logistic regression analysis was done. The demographic characteristics, health behavior, and female history were adjusted. Results: The prevalence of breast cancer was 1.37 times higher in the non-breastfeeding group than in the breastfeeding group. Compared to having breastfed for more than 36 months, the prevalence of thyroid cancer was 1.68 times higher at breastfeeding for 13 to 36 months, 1.67 times higher at breastfeeding for 6 to 12 months, and 2.06 times higher at breastfeeding less than 6 months. Also, the prevalence of cervical cancer was 1.54 times higher at breastfeeding for 13 to 36 months, compared to breastfeeding for more than 36 months. Conclusion: This study found that breastfeeding experience and a longer breastfeeding duration are associated with reduced risk of breast, thyroid, and cervical cancer in Korean women. It can be used as a basis for encouraging breastfeeding, and suggests further research on modifiable factors that reduce cancer risks.
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Neurite growth is controlled by a complex molecular signaling network that regulates filamentous actin (F-actin) dynamics at the growth cone. The evolutionarily conserved ezrin, radixin, and moesin family of proteins tether F-actin to the cell membrane when phosphorylated at a conserved threonine residue and modulate neurite outgrowth. Here we show that Akt binds to and phosphorylates a threonine 573 residue on radixin. Akt-mediated phosphorylation protects radixin from ubiquitin-dependent proteasomal degradation, thereby enhancing radixin protein stability, which permits proper neurite outgrowth and growth cone formation. Conversely, the inhibition of Akt kinase or disruption of Akt-dependent phosphorylation reduces the binding affinity of radixin to F-actin as well as lowers radixin protein levels, resulting in decreased neurite outgrowth and growth cone formation. Our findings suggest that Akt signaling regulates neurite outgrowth by stabilizing radixin interactions with F-actin, thus facilitating local F-actin dynamics.
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Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/metabolismo , Proyección Neuronal/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Actinas/metabolismo , Animales , Conos de Crecimiento/fisiología , Células HEK293 , Humanos , Ratones , Neurogénesis , Proyección Neuronal/genética , Células PC12 , Fosforilación , Unión Proteica , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-akt/genética , Ratas , Transducción de SeñalRESUMEN
Mechanistic studies of axon growth during development are beneficial to the search for neuron-intrinsic regulators of axon regeneration. Here, we discovered that, in the developing neuron from rat, Akt signaling regulates axon growth and growth cone formation through phosphorylation of serine 14 (S14) on Inhibitor of DNA binding 2 (Id2). This enhances Id2 protein stability by means of escape from proteasomal degradation, and steers its localization to the growth cone, where Id2 interacts with radixin that is critical for growth cone formation. Knockdown of Id2, or abrogation of Id2 phosphorylation at S14, greatly impairs axon growth and the architecture of growth cone. Intriguingly, reinstatement of Akt/Id2 signaling after injury in mouse hippocampal slices redeemed growth promoting ability, leading to obvious axon regeneration. Our results suggest that Akt/Id2 signaling is a key module for growth cone formation and axon growth, and its augmentation plays a potential role in CNS axonal regeneration.