Thermostable Basic Fibroblast Growth Factor Enhances the Production and Activity of Human Wharton's Jelly Mesenchymal Stem Cell-Derived Extracellular Vesicles.

Wharton's jelly-derived mesenchymal stem cell (WJ-MSC)-derived exosomes contain a diverse cargo and exhibit remarkable biological activity, rendering them suitable for regenerative and immune-modulating functions. However, the quantity of secretion is insufficient. A large body of prior work has investigated the use of various growth factors to enhance MSC-derived exosome production. In this study, we evaluated the utilization of thermostable basic fibroblast growth factor (TS-bFGF) with MSC culture and exosome production. MSCs cultured with TS-bFGF displayed superior proliferation, as evidenced by cell cycle analysis, compared with wild-type bFGF (WT-bFGF). Stemness was assessed through mRNA expression level and colony-forming unit (CFU) assays. Furthermore, nanoparticle tracking analysis (NTA) measurements revealed that MSCs cultured with TS-bFGF produced a greater quantity of exosomes, particularly under three-dimensional culture conditions. These produced exosomes demonstrated substantial anti-inflammatory and wound-healing effects, as confirmed by nitric oxide (NO) assays and scratch assays. Taken together, we demonstrate that utilization of TS-bFGF for WJ-MSC-derived exosome production not only increases exosome yield but also enhances the potential for various applications in inflammation regulation and wound healing.

Thermostable Human Basic Fibroblast Growth Factor (TS-bFGF) Engineered with a Disulfide Bond Demonstrates Superior Culture Outcomes in Human Pluripotent Stem Cell.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can differentiate into various tissues and are an essential source of various disease models and therapeutics. Various growth factors are required in order to culture pluripotent stem cells, among which basic fibroblast growth factor (bFGF) is essential for maintaining stem cell ability. However, bFGF has a short half-life (8 h) under normal mammalian cell culture conditions, and its activity decreases after 72 h, posing a serious problem in the production of high-quality stem cells. Here, we evaluated the various functions of pluripotent stem cells (PSCs) by utilizing an engineered thermostable bFGF (TS-bFGF) that is thermally stable and maintains activity longer under mammalian culture conditions. PSCs cultured with TS-bFGF showed better proliferation, stemness, morphology, and differentiation than cells cultured with wild-type bFGF. In light of the importance of stem cells in a wide range of applications in the medical and biotechnology fields, we anticipate that TS-bFGF, as a thermostable and long-acting bFGF, can play a key role in securing high-quality stem cells through various sets of stem cell culture processes.

A systematic review of computational approaches to understand cancer biology for informed drug repurposing.

Cancer is the second leading cause of death globally, trailing only heart disease. In the United States alone, 1.9 million new cancer cases and 609,360 deaths were recorded for 2022. Unfortunately, the success rate for new cancer drug development remains less than 10%, making the disease particularly challenging. This low success rate is largely attributed to the complex and poorly understood nature of cancer etiology. Therefore, it is critical to find alternative approaches to understanding cancer biology and developing effective treatments. One such approach is drug repurposing, which offers a shorter drug development timeline and lower costs while increasing the likelihood of success. In this review, we provide a comprehensive analysis of computational approaches for understanding cancer biology, including systems biology, multi-omics, and pathway analysis. Additionally, we examine the use of these methods for drug repurposing in cancer, including the databases and tools that are used for cancer research. Finally, we present case studies of drug repurposing, discussing their limitations and offering recommendations for future research in this area.

Rapid production method with increased yield of high-purity extracellular vesicles obtained using extended mitochondrial targeting domain peptide.

Extracellular vesicles (EVs) are nano-sized membranous structures involved in intercellular communication and various physiological and pathological processes. Here, we present a novel method for rapid (within 15 min), large-scale production of high-purity EVs using eMTDΔ4, a peptide derived from Noxa. The treatment of mesenchymal stem cells derived from human Wharton's jelly after trypsinization and subsequent eMTDΔ4 stimulation in a chemically defined sucrose buffer with orbital shaking led to a substantial increase (approximately 30-fold) in EV production with markedly high purity (approximately 45-fold). These EVs (TS-eEVs) showed higher regenerative and immunomodulatory potential than natural EVs obtained from the culture media after 48 h. The calcium chelator BAPTA-AM and calpain inhibitor ALLM, but not the natural EV biogenesis inhibitor GW4869, blocked the TS-eEV production induced by eMTDΔ4, indicating that the eMTDΔ4-mediated regulation of intracellular calcium levels and calpain activity are closely associated with the rapid, mass production of TS-eEVs. The present study may lead to considerable advances in EV-based drug development and production of stem cell-derived EVs for cell therapy.

Modulation of Osteogenic Differentiation of Adipose-Derived Stromal Cells by Co-Treatment with 3, 4'-Dihydroxyflavone, U0126, and N-Acetyl Cysteine.

Background and Objectives: Flavonoids form the largest group of plant phenols and have various biological and pharmacological activities. In this study, we investigated the effect of a flavonoid, 3, 4'-dihydroxyflavone (3, 4'-DHF) on osteogenic differentiation of equine adipose-derived stromal cells (eADSCs). Methods and Results: Treatment of 3, 4'-DHF led to increased osteogenic differentiation of eADSCs by increasing phosphorylation of ERK and modulating Reactive Oxygen Species (ROS) generation. Although PD98059, an ERK inhibitor, suppressed osteogenic differentiation, another ERK inhibitor, U0126, apparently increased osteogenic differentiation of the 3, 4'-DHF-treated eADSCs, which may indicate that the effect of U0126 on bone morphogenetic protein signaling is involved in the regulation of 3, 4'-DHF in osteogenic differentiation of eADSCs. We revealed that 3, 4'-DHF could induce osteogenic differentiation of eADSCs by suppressing ROS generation and co-treatment of 3, 4'-DHF, U0126, and/or N-acetyl cysteine (NAC) resulted in the additive enhancement of osteogenic differentiation of eADSCs. Conclusions: Our results showed that co-treatment of 3, 4'-DHF, U0126, and/or NAC cumulatively regulated osteogenesis in eADSCs, suggesting that 3, 4'-DHF, a flavonoid, can provide a novel approach to the treatment of osteoporosis and can provide potential therapeutic applications in therapeutics and regenerative medicine for human and companion animals.

Superior therapeutic activity of TGF-ß-induced extracellular vesicles against interstitial cystitis.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic disease characterized by incapacitating pelvic pain. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are considered key mediators of the paracrine action of MSCs and show better biological activities than the parent MSCs, especially in the bladder tissue, which may be unfavorable for MSC survival. Here, we produced MSC-EVs using advanced three-dimensional (a3D) culture with exogenous transforming growth factor-ß3 (TGF-ß3) (T-a3D-EVs). Treatment with T-a3D-EVs led to significantly enhanced wound healing and anti-inflammatory capacities. Moreover, submucosal layer injection of T-a3D-EVs in chronic IC/BPS animal model resulted in restoration of bladder function, superior anti-inflammatory activity, and recovery of damaged urothelium compared to MSCs. Interestingly, we detected increased TGF-ß1 level in T-a3D-EVs, which might be involved in the anti-inflammatory activity of these EVs. Taken together, we demonstrate the excellent immune-modulatory and regenerative abilities of T-a3D-EVs as observed by recovery from urothelial denudation and dysfunction, which could be a promising therapeutic strategy for IC/BPS.

Analysis of the Region of Interest According to CNN Structure in Hierarchical Pattern Surface Inspection Using CAM.

A convolutional neural network (CNN), which exhibits excellent performance in solving image-based problem, has been widely applied to various industrial problems. In general, the CNN model was applied to defect inspection on the surface of raw materials or final products, and its accuracy also showed better performance compared to human inspection. However, surfaces with heterogeneous and complex backgrounds have difficulties in separating defects region from the background, which is a typical challenge in this field. In this study, the CNN model was applied to detect surface defects on a hierarchical patterned surface, one of the representative complex background surfaces. In order to optimize the CNN structure, the change in inspection performance was analyzed according to the number of layers and kernel size of the model using evaluation metrics. In addition, the change of the CNN's decision criteria according to the change of the model structure was analyzed using a class activation map (CAM) technique, which can highlight the most important region recognized by the CNN in performing classification. As a result, we were able to accurately understand the classification manner of the CNN for the hierarchical pattern surface, and an accuracy of 93.7% was achieved using the optimized model.

Estimating the phase volume fraction of multi-phase steel via unsupervised deep learning.

Advanced high strength steel (AHSS) is a steel of multi-phase microstructure that is processed under several conditions to meet the current high-performance requirements from the industry. Deep neural network (DNN) has emerged as a promising tool in materials science for the task of estimating the phase volume fraction of these steels. Despite its advantages, one of its major drawbacks is its requirement of a sufficient amount of training data with correct labels to the network. This often comes as a challenge in many areas where obtaining data and labeling it is extremely labor-intensive. To overcome this challenge, an unsupervised way of learning DNN, which does not require any manual labeling, is proposed. Information maximizing generative adversarial network (InfoGAN) is used to learn the underlying probability distribution of each phase and generate realistic sample points with class labels. Then, the generated data is used for training an MLP classifier, which in turn predicts the labels for the original dataset. The result shows a mean relative error of 4.53% at most, while it can be as low as 0.73%, which implies the estimated phase fraction closely matches the true phase fraction. This presents the high feasibility of using the proposed methodology for fast and precise estimation of phase volume fraction in both industry and academia.

Low-intensity ultrasound attenuates paw edema formation and decreases vascular permeability induced by carrageenan injection in rats.

BACKGROUND: Therapeutic potential of low-intensity ultrasound (LIUS) has become evident in various musculoskeletal diseases. We have previously shown that LIUS has an inhibitory effect on local edema in various diseases including the arthritis and brain injury. In this study, we examined whether LIUS can attenuate paw edema formation vis-à-vis vascular permeability and inflammation in rats induced by carrageenan. LIUS with a frequency of 1 MHz and the intensities of 50, 100, or 200 mW/cm2 were exposed on rat paws for 10 min immediately after carrageenan injection. RESULTS: Carrageenan injection induced paw edema which was peaked at 6 h and gradually decreased nearly to the initial baseline value after 72 h. LIUS showed a significant reduction of paw edema formation at 2 and 6 h at all intensities tested. The highest reduction was observed at the intensity of 50 mW/cm2. Histological analyses confirmed that LIUS clearly decreased the carrageenan-induced swelling of interstitial space under the paw skin and infiltration of polymorphonuclear leukocytes. Moreover, Evans Blue extravasation analyses exhibited a significant decreases of vascular permeability by LIUS. Finally, immunohistochemical staining showed that expression of pro-inflammatory proteins, namely, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) induced by carrageenan injection was reduced back to the normal level after LIUS stimulation. CONCLUSIONS: These results provide a new supporting evidence for LIUS as a therapeutic alternative for the treatment of edema in inflammatory diseases such as cellulitis.

Identification of indothiazinone as a natural antiplatelet agent.

Cardiovascular disease, which is caused by unregulated platelet aggregation, is one of the main causes of deaths worldwide. Many studies have focused on natural products with antiplatelet effects as a safe alternative therapy to prevent the disease. In this context, an in-house chemical library was screened to find natural products capable of inhibiting the interaction between platelet integrin αIIbß3 and fibrinogen, which is an essential step in platelet aggregation. On the basis of the screening results, indothiazinone, an alkaloid found in microbial cultures, was identified as a potential antiplatelet agent. Specifically, indothiazinone treatment significantly inhibited the binding of fibrinogen to Chinese hamster ovary cells expressing integrin αIIbß3. It also restricted thrombin- and adenosine diphosphate-dependent spreading of human platelets on a fibrinogen matrix. More importantly, surface plasmon resonance and molecular dynamics studies suggested that indothiazinone suppressed talin-induced activation of integrin αIIbß3 presumably by inhibiting talin-integrin interaction. In conclusion, these results suggest that indothiazinone can be used as a lead compound for the development of antiplatelet drugs with a novel mode of action.

Vimentin filaments regulate integrin-ligand interactions by binding to the cytoplasmic tail of integrin ß3.

Vimentin, an intermediate filament protein induced during epithelial-to-mesenchymal transition, is known to regulate cell migration and invasion. However, it is still unclear how vimentin controls such behaviors. In this study, we aimed to find a new integrin regulator by investigating the H-Ras-mediated integrin suppression mechanism. Through a proteomic screen using the integrin ß3 cytoplasmic tail protein, we found that vimentin might work as an effector of H-Ras signaling. H-Ras converted filamentous vimentin into aggregates near the nucleus, where no integrin binding can occur. In addition, an increase in the amount of vimentin filaments accessible to the integrin ß3 tail enhanced talin-induced integrin binding to its ligands by inducing integrin clustering. In contrast, the vimentin head domain, which was found to bind directly to the integrin ß3 tail and compete with endogenous vimentin filaments for integrin binding, induced nuclear accumulation of vimentin filaments and reduced the amount of integrin-ligand binding. Finally, we found that expression of the vimentin head domain can reduce cell migration and metastasis. From these data, we suggest that filamentous vimentin underneath the plasma membrane is involved in increasing integrin adhesiveness, and thus regulation of the vimentin-integrin interaction might control cell adhesion.

Intermolecular transmembrane domain interactions activate integrin αIIbß3.

Integrins are the major cell adhesion molecules responsible for cell attachment to the extracellular matrix. The strength of integrin-mediated adhesion is controlled by the affinity of individual integrins (integrin activation) as well as by the number of integrins involved in such adhesion. The positive correlation between integrin activation and integrin clustering had been suggested previously, but several trials to induce integrin clustering by dimerization of the transmembrane domain or tail region of integrin α subunits failed to demonstrate any change in integrin activation. Here, using platelet integrin αIIbß3 as a model system, we showed that there is intermolecular lateral interaction between integrins through the transmembrane domains, and this interaction can enhance the affinity state of integrins. In addition, when integrin clustering was induced through heteromeric lateral interactions using bimolecular fluorescence complementation, we could observe a significant increase in the number of active integrin molecules. Because the possibility of intermolecular interaction would be increased by a higher local concentration of integrins, we propose that integrin clustering can shift the equilibrium in favor of integrin activation.

Electric-field-controlled flow in nanoscale-thin wetting films.

A novel nanofluidic system based on electroosmotic flow in nanoscale-thin aqueous wetting films is reported. The water films formed spontaneously on mica substrates in a saturation humidity environment. The film thickness was determined to be a few tens of nanometers by optical interference and fluorescence intensity measurements and was consistent with a theoretical evaluation of the thickness of a film based on the competing forces of electrostatic repulsion and capillary pressure. Lateral flow was induced by applying a dc electric field tangential to the film and characterized by tracking the position of a fluorescent probe. The mobilities of the thin fluid layer and the flow marker were lower than the predictions of the electrokinetic theory, which may be a result of adsorption of the fluorescent molecules to the mica. Confinement of the film to two-dimensional "channels" was achieved by microcontact printing of patterned hydrophobic monolayers onto the substrate. This system has the advantage of simple and inexpensive fabrication in comparison to nanofluidic devices made by traditional lithography techniques.

Dielectrophoretic assembly of grain-boundary-free 2D colloidal single crystals.

Dielectrophoresis (DEP) force-assisted assembly of a colloidal single photonic-crystal monolayer in a microfluidic chamber was demonstrated. Negative DEP force with a high-frequency AC electric field induced the compression of colloidal microspheres to form a colloidal crystal domain at the center of a hexapolar-shaped electrode. While typical assembly by monotonic DEP force forms multicrystalline domains containing crystal defects, repetitions of the DEP/relaxation cycle significantly facilitated crystal growth of 10µm monodispersed polystyrene microspheres, allowing a grain-boundary-free single-crystal monolayer domain of ca. 200µm in size. Microsphere size as well as size distribution affected the formation of the single-crystal domain. A simple method was used to immobilize the single-crystal domain on the glass substrate without losing its crystallinity.

Electric-field-assisted convective assembly of colloidal crystal coatings.

A new technique that combines evaporative convective deposition of colloidal crystal coatings with an electric field to achieve more rapid assembly and reduce the defects in the crystal structure is reported. When an ac voltage is applied across the particle suspension and the substrate in the convective assembly process, a longer film spreads from the meniscus by the electrowetting-on-dielectric (EWOD) effect. The data suggest that the EWOD-increased liquid surface area results in increased evaporation-driven particle flux and crystal assembly that is up to five times more rapid. The extended drying film also provides more time for particle rearrangement before the structure becomes fixed, resulting in formation of crystal domains an order of magnitude larger than those deposited by convective assembly alone. The results demonstrate that EWOD is a facile tool for controlling particle assembly processes in wetting films. The technique could be used in improved large-scale colloidal crystal coating processes.

Long-term stabilization of foams and emulsions with in-situ formed microparticles from hydrophobic cellulose.

We report a simple method to produce foams and emulsions of extraordinary stability by using hydrophobic cellulose microparticles, which are formed in situ by a liquid-liquid dispersion technique. The hydrophobic cellulose derivative, hypromellose phthalate (HP), was initially dissolved in water-miscible solvents such as acetone and ethanol/water mixtures. As these HP stock solutions were sheared in aqueous media, micron sized cellulose particles formed by the solvent attrition. We also designed and investigated an effective and simple process for making HP particles without any organic solvents, where both the solvent and antisolvent were aqueous buffer solutions at different pH. Consequently, the HP particles adsorbed onto the water/air or water/oil interfaces created during shear blending, resulting in highly stable foams or foam/emulsions. The formation of HP particles and their ability for short-term and long-term stabilization of interfaces strongly depended on the HP concentration in stock solutions, as well as the solvent chemistry of both stock solutions and continuous phase media. Some foams and emulsion samples formed in the presence of ca. 1 wt% HP were stable for months. This new class of nontoxic inexpensive cellulose-based particle stabilizers has the potential to substitute conventional synthetic surfactants, especially in edible, pharmaceutical and biodegradable products.

Combined effects of swim training and ginseng supplementation on exercise performance time, ROS, lymphocyte proliferation, and DNA damage following exhaustive exercise stress.

The purpose of this study was to analyze the combined effects of regular exercise and ginseng supplementation on peritoneal exudate ROS (reactive oxygen species), lymphocyte proliferation by splenocytes, and DNA damage following exhaustive exercise stress. Thirty-six female BALB/c mice were randomly divided into control (UT, n = 12), trained (TR, n = 12), and ginseng supplemented and trained (GT, n = 12) groups. Each group was divided into two equal subgroups where mice were studied at rest (UTre, TRre, and GTre) or immediately after exhaustive exercise stress (UTex, TRex, and GTex). Animals were bred in the animal facility, where they were housed at 22-24 degrees C and relative humidity (RH) 50-60% in a controlled environment with a 12-hour photoperiod, and provided food and water ad libitum. The trained mice underwent 10 weeks of endurance swim training (5 times/week) in water at 27-30 degrees C for 60 minutes. The analytical items examined were weight, proliferative activity, the production of ROS from peritoneal exudate cells, and DNA damage following exhaustive exercise stress (2 h exercise stress). Significant level was set at p < 0.05. The results obtained showed that the trained group had a significantly lower mean body weight than the untrained group (p < 0.05). However, there was no significant difference between UT and GT. Swim training increased swim survival time in TRex and GTex, and TRex showed the highest swim survival time. With regard to mitogenic activities of splenocytes in response to exhaustive exercise stress, all groups showed much lower lymphocyte proliferative activity when stimulated with media (Med), concanavalin A (ConA), or lipopolysaccharide (LPS) after exhaustive exercise stress. However, GTex had a higher proliferative activity than the other groups. Trained and ginseng-supplemented groups showed lower peritoneal ROS responses and lymphocyte DNA damage levels after exhaustive exercise. These findings suggest that the combined effect of swim training and ginseng supplementation sustain lymphocyte function in the presence of reduced ROS production and DNA damage following acute exercise stress.

Length separation of Zwitterion-functionalized single wall carbon nanotubes by GPC.

The length-fractionation of shortened (250 to 25 nm), zwitterion-functionalized, single wall carbon nanotubes (SWNTs) has been demonstrated via gel permeation chromatography (GPC). The UV-Vis spectrum of each fraction indicates an apparent "solubilization", as evident by the direct observation of all predicted optically allowed interband transitions between the mirror image spikes in the density of states of both metallic and semiconducting SWNTs with various tube diameters. As evident by the presence or absence of the 270 nm, pi-plasmon absorption, this "solubilization" is a dynamic process and leads to re-aggregation if left undisturbed for a couple of weeks or upon dissociation of the pendant octadecylamine groups. This non-destructive and highly versatile separation methodology opens up an array of possible applications for shortened SWNTs in nanostructured devices.

Ceramide increases Fas-mediated apoptosis in glioblastoma cells through FLIP down-regulation.

Ceramide is a physiologic regulator of growth and differentiation in mammalian cells. In this study, the relationship between ceramide and FLICE inhibitory protein (FLIP) in the induction of apoptosis in glioblastoma cell lines was investigated. We found that LN215 cells were slightly more sensitive to Fas-mediated apoptosis than LN319 cells, which were more sensitive to ceramide than LN215 cells. FLIP was expressed in LN319 and LN215 cells constitutively, and this expression decreased with treatment of ceramide in LN215 cells, which might cause LN215 cells to be more sensitive to Fas-mediated apoptosis at lower level stimulation. In LN319 cells FLIP levels were not modified by ceramide treatment and the level of cell death induced by anti-Fas antibody was not affected. Our results suggest that FLIP may be down-regulated by low levels of ceramide in LN215 cells, which causes LN215 cells to be more sensitive to Fas-mediated apoptosis, whereas LN319 cells remain resistant.